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+anno_start	anno_end	anno_text	entity_type	sentence	section
+17	34	Crystal Structure	evidence	Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily	TITLE
+38	54	Ectoine Synthase	protein_type	Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily	TITLE
+58	74	Metal-Containing	protein_state	Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily	TITLE
+89	106	Cupin Superfamily	protein_type	Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily	TITLE
+0	7	Ectoine	chemical	Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth.	ABSTRACT
+84	92	Bacteria	taxonomy_domain	Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth.	ABSTRACT
+103	110	Archaea	taxonomy_domain	Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth.	ABSTRACT
+0	16	Ectoine synthase	protein_type	Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction.	ABSTRACT
+18	22	EctC	protein_type	Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction.	ABSTRACT
+51	58	ectoine	chemical	Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction.	ABSTRACT
+117	157	N-gamma-acetyl-L-2,4-diaminobutyric acid	chemical	Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction.	ABSTRACT
+168	173	water	chemical	Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction.	ABSTRACT
+13	30	crystal structure	evidence	However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking.	ABSTRACT
+34	50	ectoine synthase	protein_type	However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking.	ABSTRACT
+10	26	ectoine synthase	protein_type	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
+49	65	marine bacterium	taxonomy_domain	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
+66	89	Sphingopyxis alaskensis	species	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
+91	93	Sa	species	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
+163	167	EctC	protein	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
+199	216	crystal structure	evidence	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
+224	227	apo	protein_state	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
+0	19	Structural analysis	experimental_method	Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily.	ABSTRACT
+36	38	Sa	species	Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily.	ABSTRACT
+39	43	EctC	protein	Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily.	ABSTRACT
+71	88	cupin superfamily	protein_type	Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily.	ABSTRACT
+0	4	EctC	protein	EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure.	ABSTRACT
+13	18	dimer	oligomeric_state	EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure.	ABSTRACT
+26	38	head-to-tail	protein_state	EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure.	ABSTRACT
+80	97	crystal structure	evidence	EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure.	ABSTRACT
+4	13	interface	site	The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer.	ABSTRACT
+21	26	dimer	oligomeric_state	The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer.	ABSTRACT
+81	105	hydrophobic interactions	bond_interaction	The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer.	ABSTRACT
+122	133	beta-sheets	structure_element	The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer.	ABSTRACT
+146	153	monomer	oligomeric_state	The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer.	ABSTRACT
+32	48	ectoine synthase	protein_type	We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme.	ABSTRACT
+83	88	metal	chemical	We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme.	ABSTRACT
+100	146	metal depletion and reconstitution experiments	experimental_method	We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme.	ABSTRACT
+160	164	EctC	protein	We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme.	ABSTRACT
+180	194	iron-dependent	protein_state	We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme.	ABSTRACT
+14	18	EctC	protein	We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency.	ABSTRACT
+71	111	N-gamma-acetyl-L-2,4-diaminobutyric acid	chemical	We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency.	ABSTRACT
+117	124	ectoine	chemical	We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency.	ABSTRACT
+199	239	N-alpha-acetyl-L-2,4-diaminobutyric acid	chemical	We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency.	ABSTRACT
+292	312	catalytic efficiency	evidence	We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency.	ABSTRACT
+0	42	Structure-guided site-directed mutagenesis	experimental_method	Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants.	ABSTRACT
+94	125	evolutionarily highly conserved	protein_state	Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants.	ABSTRACT
+145	164	EctC protein family	protein_type	Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants.	ABSTRACT
+206	223	iron-binding site	site	Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants.	ABSTRACT
+296	300	EctC	protein	Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants.	ABSTRACT
+37	41	iron	chemical	An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.	ABSTRACT
+130	141	active site	site	An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.	ABSTRACT
+176	180	EctC	protein	An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.	ABSTRACT
+181	193	cupin barrel	structure_element	An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.	ABSTRACT
+0	7	Ectoine	chemical	Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes.	INTRO
+9	68	(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid	chemical	Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes.	INTRO
+89	105	5-hydroxyectoine	chemical	Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes.	INTRO
+107	180	(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid	chemical	Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes.	INTRO
+5	42	marine and terrestrial microorganisms	taxonomy_domain	Both marine and terrestrial microorganisms produce them widely in response to osmotic or temperature stress.	INTRO
+13	20	ectoine	chemical	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
+61	87	L-aspartate-ß-semialdehyde	chemical	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
+146	180	L-2,4-diaminobutyrate transaminase	protein_type	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
+182	186	EctB	protein_type	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
+202	239	2,4-diaminobutyrate acetyltransferase	protein_type	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
+241	245	EctA	protein_type	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
+266	282	ectoine synthase	protein_type	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
+284	288	EctC	protein_type	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
+4	11	ectoine	chemical	The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers.	INTRO
+23	39	5-hydroxyectoine	chemical	The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers.	INTRO
+144	151	ectoine	chemical	The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers.	INTRO
+45	52	ectoine	chemical	This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily.	INTRO
+81	100	ectoine hydroxylase	protein_type	This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily.	INTRO
+102	106	EctD	protein_type	This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily.	INTRO
+138	219	non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily	protein_type	This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily.	INTRO
+46	54	ectoines	chemical	The remarkable function preserving effects of ectoines for macromolecules and cells, frequently also addressed as chemical chaperones, led to a substantial interest in exploiting these compounds for biotechnological purposes and medical applications.	INTRO
+24	31	ectoine	chemical	Biosynthetic routes for ectoine and 5-hydroxyectoine.	FIG
+36	52	5-hydroxyectoine	chemical	Biosynthetic routes for ectoine and 5-hydroxyectoine.	FIG
+14	21	ectoine	chemical	Scheme of the ectoine and 5-hydroxyectoine biosynthetic pathway.	FIG
+26	42	5-hydroxyectoine	chemical	Scheme of the ectoine and 5-hydroxyectoine biosynthetic pathway.	FIG
+17	33	ectoine synthase	protein_type	Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1).	INTRO
+35	39	EctC	protein	Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1).	INTRO
+64	71	ectoine	chemical	Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1).	INTRO
+12	29	characterizations	experimental_method	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
+33	50	ectoine synthases	protein_type	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
+60	73	extremophiles	taxonomy_domain	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
+74	92	Halomonas elongata	species	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
+94	123	Methylomicrobium alcaliphilum	species	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
+129	149	Acidiphilium cryptum	species	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
+164	183	nitrifying archaeon	taxonomy_domain	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
+184	208	Nitrosopumilus maritimus	species	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
+74	110	N-γ-acetyl-L-2,4-diaminobutyric acid	chemical	Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).	INTRO
+112	121	N-γ-ADABA	chemical	Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).	INTRO
+152	189	2,4-diaminobutyrate acetyltransferase	protein_type	Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).	INTRO
+191	195	EctA	protein_type	Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).	INTRO
+201	208	ectoine	chemical	Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).	INTRO
+243	248	water	chemical	Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).	INTRO
+19	23	EctC	protein	In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.	INTRO
+85	129	5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate	chemical	In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.	INTRO
+131	135	ADPC	chemical	In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.	INTRO
+176	185	glutamine	chemical	In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.	INTRO
+250	257	ectoine	chemical	In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.	INTRO
+272	279	ectoine	chemical	In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.	INTRO
+84	100	ectoine synthase	protein_type	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
+135	144	structure	evidence	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
+248	275	biochemically characterized	experimental_method	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
+280	296	ectoine synthase	protein_type	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
+319	335	marine bacterium	taxonomy_domain	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
+336	359	Sphingopyxis alaskensis	species	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
+361	363	Sa	species	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
+48	64	ectoine synthase	protein_type	We demonstrate here for the first time that the ectoine synthase is a metal-dependent enzyme, with iron as the most likely physiologically relevant co-factor.	INTRO
+70	75	metal	chemical	We demonstrate here for the first time that the ectoine synthase is a metal-dependent enzyme, with iron as the most likely physiologically relevant co-factor.	INTRO
+99	103	iron	chemical	We demonstrate here for the first time that the ectoine synthase is a metal-dependent enzyme, with iron as the most likely physiologically relevant co-factor.	INTRO
+4	8	EctC	protein	The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily.	INTRO
+25	30	dimer	oligomeric_state	The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily.	INTRO
+51	70	structural analysis	experimental_method	The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily.	INTRO
+104	121	cupin superfamily	protein_type	The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily.	INTRO
+8	26	crystal structures	evidence	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
+56	58	Sa	species	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
+59	63	EctC	protein	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
+157	182	site-directed mutagenesis	experimental_method	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
+205	236	evolutionarily highly conserved	protein_state	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
+266	278	EctC protein	protein_type	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
+337	351	catalytic core	site	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
+359	375	ectoine synthase	protein_type	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
+0	14	Overproduction	experimental_method	Overproduction, purification and oligomeric state of the ectoine synthase in solution	RESULTS
+16	28	purification	experimental_method	Overproduction, purification and oligomeric state of the ectoine synthase in solution	RESULTS
+57	73	ectoine synthase	protein_type	Overproduction, purification and oligomeric state of the ectoine synthase in solution	RESULTS
+15	49	biochemical and structural studies	experimental_method	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
+57	73	ectoine synthase	protein_type	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
+79	92	S. alaskensis	species	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
+95	97	Sa	species	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
+98	102	EctC	protein	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
+120	147	marine ultra-microbacterium	taxonomy_domain	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
+192	209	crystal structure	evidence	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
+217	236	ectoine hydroxylase	protein_type	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
+238	242	EctD	protein_type	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
+244	259	in complex with	protein_state	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
+46	59	S. alaskensis	species	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
+60	64	ectC	gene	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
+73	80	E. coli	species	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
+149	178	Strep-tag II affinity peptide	experimental_method	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
+209	211	Sa	species	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
+212	216	EctC	protein	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
+217	229	Strep-Tag-II	experimental_method	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
+241	264	affinity chromatography	experimental_method	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
+5	7	Sa	species	The (Sa)EctC protein was overproduced and isolated with good yields (30–40 mg L-1 of culture) and purity (S2a Fig).	RESULTS
+8	12	EctC	protein	The (Sa)EctC protein was overproduced and isolated with good yields (30–40 mg L-1 of culture) and purity (S2a Fig).	RESULTS
+13	42	size-exclusion chromatography	experimental_method	Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution.	RESULTS
+44	47	SEC	experimental_method	Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution.	RESULTS
+73	75	Sa	species	Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution.	RESULTS
+76	80	EctC	protein	Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution.	RESULTS
+162	168	dimers	oligomeric_state	Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution.	RESULTS
+0	38	High performance liquid chromatography	experimental_method	High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig).	RESULTS
+52	90	multi-angle light-scattering detection	experimental_method	High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig).	RESULTS
+92	101	HPLC-MALS	experimental_method	High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig).	RESULTS
+161	163	Sa	species	High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig).	RESULTS
+164	168	EctC	protein	High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig).	RESULTS
+89	91	Sa	species	This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa).	RESULTS
+92	96	EctC	protein	This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa).	RESULTS
+97	102	dimer	oligomeric_state	This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa).	RESULTS
+126	133	monomer	oligomeric_state	This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa).	RESULTS
+149	178	Strep-tag II affinity peptide	experimental_method	This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa).	RESULTS
+30	35	dimer	oligomeric_state	Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus.	RESULTS
+67	80	EctC proteins	protein_type	Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus.	RESULTS
+86	97	H. elongata	species	Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus.	RESULTS
+102	114	N. maritimus	species	Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus.	RESULTS
+30	46	ectoine synthase	protein_type	Biochemical properties of the ectoine synthase	RESULTS
+4	8	EctA	protein	The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available.	RESULTS
+35	51	ectoine synthase	protein_type	The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available.	RESULTS
+53	89	N-γ-acetyl-L-2,4-diaminobutyric acid	chemical	The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available.	RESULTS
+91	100	N-γ-ADABA	chemical	The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available.	RESULTS
+31	38	ectoine	chemical	We used alkaline hydrolysis of ectoine and subsequent chromatography on silica gel columns to obtain N-γ-ADABA in chemically highly purified form (S1a Fig).	RESULTS
+101	110	N-γ-ADABA	chemical	We used alkaline hydrolysis of ectoine and subsequent chromatography on silica gel columns to obtain N-γ-ADABA in chemically highly purified form (S1a Fig).	RESULTS
+42	51	N-γ-ADABA	chemical	This procedure also yielded the isomer of N-γ-ADABA, N-α-acetyl-L-2,4-diaminobutyric acid (N-α-ADABA) (S1b Fig).	RESULTS
+53	89	N-α-acetyl-L-2,4-diaminobutyric acid	chemical	This procedure also yielded the isomer of N-γ-ADABA, N-α-acetyl-L-2,4-diaminobutyric acid (N-α-ADABA) (S1b Fig).	RESULTS
+91	100	N-α-ADABA	chemical	This procedure also yielded the isomer of N-γ-ADABA, N-α-acetyl-L-2,4-diaminobutyric acid (N-α-ADABA) (S1b Fig).	RESULTS
+0	9	N-α-ADABA	chemical	N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism.	RESULTS
+60	64	EctC	protein	N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism.	RESULTS
+70	84	microorganisms	taxonomy_domain	N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism.	RESULTS
+94	101	ectoine	chemical	N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism.	RESULTS
+6	15	N-γ-ADABA	chemical	Using N-γ-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein.	RESULTS
+109	111	Sa	species	Using N-γ-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein.	RESULTS
+112	116	EctC	protein	Using N-γ-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein.	RESULTS
+0	13	S. alaskensis	species	S. alaskensis, from which the studied ectoine synthase was originally derived, is a microorganism that is well-adapted to a life in permanently cold ocean waters.	RESULTS
+38	54	ectoine synthase	protein_type	S. alaskensis, from which the studied ectoine synthase was originally derived, is a microorganism that is well-adapted to a life in permanently cold ocean waters.	RESULTS
+84	97	microorganism	taxonomy_domain	S. alaskensis, from which the studied ectoine synthase was originally derived, is a microorganism that is well-adapted to a life in permanently cold ocean waters.	RESULTS
+69	71	Sa	species	Consistent with the physicochemical attributes of this habitat, the (Sa)EctC protein was already enzymatically active at 5°C, had a temperature optimum of 15°C and was able to function over a broad range of temperatures (S3a Fig).	RESULTS
+72	76	EctC	protein	Consistent with the physicochemical attributes of this habitat, the (Sa)EctC protein was already enzymatically active at 5°C, had a temperature optimum of 15°C and was able to function over a broad range of temperatures (S3a Fig).	RESULTS
+97	117	enzymatically active	protein_state	Consistent with the physicochemical attributes of this habitat, the (Sa)EctC protein was already enzymatically active at 5°C, had a temperature optimum of 15°C and was able to function over a broad range of temperatures (S3a Fig).	RESULTS
+16	24	alkaline	protein_state	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
+77	94	ectoine synthases	protein_type	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
+104	117	halo-tolerant	protein_state	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
+118	129	H. elongata	species	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
+162	173	alkaliphile	taxonomy_domain	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
+174	189	M. alcaliphilum	species	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
+219	229	acidophile	taxonomy_domain	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
+230	250	Acidiphilium cryptum	species	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
+291	295	EctC	protein	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
+309	321	N. maritimus	species	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
+328	338	neutral pH	protein_state	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
+100	102	Sa	species	The salinity of the assay buffer had a significant influence on the maximal enzyme activity of the (Sa)EctC protein.	RESULTS
+103	107	EctC	protein	The salinity of the assay buffer had a significant influence on the maximal enzyme activity of the (Sa)EctC protein.	RESULTS
+26	30	NaCl	chemical	An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity.	RESULTS
+38	41	KCl	chemical	An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity.	RESULTS
+106	122	ectoine synthase	protein_type	An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity.	RESULTS
+32	34	Sa	species	The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively.	RESULTS
+35	39	EctC	protein	The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively.	RESULTS
+63	67	NaCl	chemical	The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively.	RESULTS
+71	74	KCl	chemical	The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively.	RESULTS
+1	3	Sa	species	(Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig).	RESULTS
+4	8	EctC	protein	(Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig).	RESULTS
+97	101	NaCl	chemical	(Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig).	RESULTS
+106	109	KCl	chemical	(Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig).	RESULTS
+19	23	EctC	protein	The stimulation of EctC enzyme activity by salts has previously also been observed for other ectoine synthases.	RESULTS
+93	110	ectoine synthases	protein_type	The stimulation of EctC enzyme activity by salts has previously also been observed for other ectoine synthases.	RESULTS
+4	20	ectoine synthase	protein_type	The ectoine synthase is a metal-containing protein	RESULTS
+26	50	metal-containing protein	protein_type	The ectoine synthase is a metal-containing protein	RESULTS
+53	57	EctC	protein	Considerations based on bioinformatics suggests that EctC belongs to the cupin superfamily.	RESULTS
+73	90	cupin superfamily	protein_type	Considerations based on bioinformatics suggests that EctC belongs to the cupin superfamily.	RESULTS
+87	91	iron	chemical	Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.	RESULTS
+93	99	copper	chemical	Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.	RESULTS
+101	105	zinc	chemical	Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.	RESULTS
+107	116	manganese	chemical	Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.	RESULTS
+118	124	cobalt	chemical	Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.	RESULTS
+129	135	nickel	chemical	Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.	RESULTS
+0	6	Cupins	protein_type	Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal).	RESULTS
+19	28	conserved	protein_state	Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal).	RESULTS
+37	57	G(X)5HXH(X)3,4E(X)6G	structure_element	Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal).	RESULTS
+62	80	G(X)5PXG(X)2H(X)3N	structure_element	Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal).	RESULTS
+153	158	metal	chemical	Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal).	RESULTS
+25	62	alignment of the amino acid sequences	experimental_method	Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.	RESULTS
+70	88	EctC-type proteins	protein_type	Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.	RESULTS
+117	136	metal-binding motif	structure_element	Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.	RESULTS
+143	162	cupin-type proteins	protein_type	Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.	RESULTS
+166	179	not conserved	protein_state	Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.	RESULTS
+210	241	ectoine synthase protein family	protein_type	Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.	RESULTS
+15	51	alignment of the amino acid sequence	experimental_method	An abbreviated alignment of the amino acid sequence of EctC-type proteins is shown in Fig 2.	RESULTS
+55	73	EctC-type proteins	protein_type	An abbreviated alignment of the amino acid sequence of EctC-type proteins is shown in Fig 2.	RESULTS
+12	21	alignment	experimental_method	Abbreviated alignment of EctC-type proteins.	FIG
+25	43	EctC-type proteins	protein_type	Abbreviated alignment of EctC-type proteins.	FIG
+40	58	EctC-type proteins	protein_type	The amino acid sequences of 20 selected EctC-type proteins are compared.	FIG
+0	18	Strictly conserved	protein_state	Strictly conserved amino acid residues are shown in yellow.	FIG
+22	24	Sa	species	Dots shown above the (Sa)EctC protein sequence indicate residues likely to be involved in iron-binding (red), ligand-binding (green) and stabilization of the loop-architecture (blue).	FIG
+25	29	EctC	protein	Dots shown above the (Sa)EctC protein sequence indicate residues likely to be involved in iron-binding (red), ligand-binding (green) and stabilization of the loop-architecture (blue).	FIG
+90	94	iron	chemical	Dots shown above the (Sa)EctC protein sequence indicate residues likely to be involved in iron-binding (red), ligand-binding (green) and stabilization of the loop-architecture (blue).	FIG
+4	13	conserved	protein_state	The conserved residue Tyr-52 with so-far undefined functions is indicated by a green dot circled in red.	FIG
+22	28	Tyr-52	residue_name_number	The conserved residue Tyr-52 with so-far undefined functions is indicated by a green dot circled in red.	FIG
+31	40	α-helices	structure_element	Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.	FIG
+45	53	β-sheets	structure_element	Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.	FIG
+69	71	Sa	species	Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.	FIG
+72	76	EctC	protein	Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.	FIG
+77	94	crystal structure	evidence	Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.	FIG
+142	160	EctC-type proteins	protein_type	Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.	FIG
+40	59	metal-binding motif	structure_element	Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).	RESULTS
+140	145	metal	chemical	Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).	RESULTS
+178	180	Sa	species	Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).	RESULTS
+181	185	EctC	protein	Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).	RESULTS
+197	239	inductive-coupled plasma mass spectrometry	experimental_method	Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).	RESULTS
+241	247	ICP-MS	experimental_method	Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).	RESULTS
+39	41	Sa	species	For this analysis we used recombinant (Sa)EctC preparations from three independent protein overproduction and purification experiments.	RESULTS
+42	46	EctC	protein	For this analysis we used recombinant (Sa)EctC preparations from three independent protein overproduction and purification experiments.	RESULTS
+4	10	ICP-MS	experimental_method	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
+31	35	iron	chemical	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
+63	67	iron	chemical	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
+101	103	Sa	species	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
+104	108	EctC	protein	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
+163	167	zinc	chemical	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
+178	182	zinc	chemical	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
+26	32	copper	chemical	All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively).	RESULTS
+37	43	nickel	chemical	All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively).	RESULTS
+90	95	metal	chemical	All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively).	RESULTS
+16	20	iron	chemical	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
+31	33	Sa	species	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
+34	38	EctC	protein	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
+87	106	colorimetric method	experimental_method	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
+127	131	iron	chemical	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
+178	182	iron	chemical	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
+222	224	Sa	species	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
+225	229	EctC	protein	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
+12	18	ICP-MS	experimental_method	Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein.	RESULTS
+27	46	colorimetric method	experimental_method	Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein.	RESULTS
+99	115	ectoine synthase	protein_type	Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein.	RESULTS
+121	134	S. alaskensis	species	Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein.	RESULTS
+141	145	iron	chemical	Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein.	RESULTS
+58	62	iron	chemical	We note in this context, that the values obtained for the iron content of the (Sa)EctC proteins varied by approximately 10 to 20% between the two methods.	RESULTS
+79	81	Sa	species	We note in this context, that the values obtained for the iron content of the (Sa)EctC proteins varied by approximately 10 to 20% between the two methods.	RESULTS
+82	86	EctC	protein	We note in this context, that the values obtained for the iron content of the (Sa)EctC proteins varied by approximately 10 to 20% between the two methods.	RESULTS
+85	103	colorimetric assay	experimental_method	The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree.	RESULTS
+131	135	iron	chemical	The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree.	RESULTS
+148	150	Sa	species	The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree.	RESULTS
+151	155	EctC	protein	The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree.	RESULTS
+2	7	metal	chemical	A metal cofactor is important for the catalytic activity of EctC	RESULTS
+60	64	EctC	protein	A metal cofactor is important for the catalytic activity of EctC	RESULTS
+4	8	iron	chemical	The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily.	RESULTS
+26	28	Sa	species	The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily.	RESULTS
+29	33	EctC	protein	The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily.	RESULTS
+180	197	cupin superfamily	protein_type	The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily.	RESULTS
+31	40	incubated	experimental_method	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
+46	48	Sa	species	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
+49	53	EctC	protein	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
+61	91	with increasing concentrations	experimental_method	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
+99	104	metal	chemical	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
+114	147	ethylene-diamine-tetraacetic-acid	chemical	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
+149	153	EDTA	chemical	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
+180	196	ectoine synthase	protein_type	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
+43	47	EDTA	chemical	The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a).	RESULTS
+65	69	EctC	protein	The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a).	RESULTS
+123	139	ectoine synthase	protein_type	The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a).	RESULTS
+174	178	EDTA	chemical	The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a).	RESULTS
+18	34	ectoine synthase	protein_type	Dependency of the ectoine synthase activity on metals.	FIG
+18	22	iron	chemical	(a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein.	FIG
+32	36	EDTA	chemical	(a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein.	FIG
+77	79	Sa	species	(a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein.	FIG
+80	84	EctC	protein	(a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein.	FIG
+0	46	Metal depletion and reconstitution experiments	experimental_method	Metal depletion and reconstitution experiments with (b) stoichiometric and (c) excess amounts of metals.	FIG
+5	7	Sa	species	The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added.	FIG
+8	12	EctC	protein	The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added.	FIG
+139	155	ectoine synthase	protein_type	The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added.	FIG
+156	169	enzyme assays	experimental_method	The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added.	FIG
+184	189	FeCl2	chemical	The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added.	FIG
+21	32	inactivated	protein_state	We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme.	RESULTS
+65	69	EDTA	chemical	We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme.	RESULTS
+73	81	dialysis	experimental_method	We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme.	RESULTS
+150	152	Sa	species	We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme.	RESULTS
+153	157	EctC	protein	We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme.	RESULTS
+16	21	FeCl2	chemical	The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.	RESULTS
+29	41	enzyme assay	experimental_method	The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.	RESULTS
+105	110	ZnCl2	chemical	The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.	RESULTS
+114	119	CoCl2	chemical	The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.	RESULTS
+129	131	Sa	species	The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.	RESULTS
+132	136	EctC	protein	The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.	RESULTS
+35	39	Fe3+	chemical	All other tested metals, including Fe3+, were unable to restore activity (Fig 3b).	RESULTS
+52	64	enzyme assay	experimental_method	When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c).	RESULTS
+89	93	Fe2+	chemical	When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c).	RESULTS
+233	235	Sa	species	When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c).	RESULTS
+236	240	EctC	protein	When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c).	RESULTS
+300	304	EDTA	chemical	When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c).	RESULTS
+64	68	zinc	chemical	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
+70	76	cobalt	chemical	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
+78	84	nickel	chemical	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
+86	92	copper	chemical	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
+98	107	manganese	chemical	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
+143	160	cupin superfamily	protein_type	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
+191	207	ectoine synthase	protein_type	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
+50	68	cupin-type enzymes	protein_type	This is in line with literature data showing that cupin-type enzymes are often promiscuous with respect to the use of the catalytically important metal.	RESULTS
+146	151	metal	chemical	This is in line with literature data showing that cupin-type enzymes are often promiscuous with respect to the use of the catalytically important metal.	RESULTS
+22	26	EctC	protein	Kinetic parameters of EctC for N-γ-ADABA and N-α-ADABA	RESULTS
+31	40	N-γ-ADABA	chemical	Kinetic parameters of EctC for N-γ-ADABA and N-α-ADABA	RESULTS
+45	54	N-α-ADABA	chemical	Kinetic parameters of EctC for N-γ-ADABA and N-α-ADABA	RESULTS
+66	80	activity assay	experimental_method	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
+89	105	ectoine synthase	protein_type	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
+109	122	S. alaskensis	species	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
+181	183	Sa	species	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
+184	188	EctC	protein	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
+227	236	N-γ-ADABA	chemical	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
+252	261	N-α-ADABA	chemical	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
+4	8	EctC	protein	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
+35	44	N-γ-ADABA	chemical	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
+53	60	ectoine	chemical	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
+71	96	Michaelis-Menten-kinetics	experimental_method	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
+114	116	Km	evidence	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
+136	140	vmax	evidence	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
+166	170	kcat	evidence	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
+34	43	N-α-ADABA	chemical	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
+70	79	N-γ-ADABA	chemical	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
+88	104	ectoine synthase	protein_type	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
+145	147	Sa	species	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
+148	152	EctC	protein	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
+168	177	N-α-ADABA	chemical	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
+189	196	ectoine	chemical	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
+1	3	Sa	species	(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).	RESULTS
+4	8	EctC	protein	(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).	RESULTS
+38	63	Michaelis-Menten-kinetics	experimental_method	(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).	RESULTS
+87	89	Km	evidence	(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).	RESULTS
+110	114	vmax	evidence	(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).	RESULTS
+140	144	kcat	evidence	(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).	RESULTS
+7	16	N-α-ADABA	chemical	Hence, N-α-ADABA is a newly recognized substrate for ectoine synthase.	RESULTS
+53	69	ectoine synthase	protein_type	Hence, N-α-ADABA is a newly recognized substrate for ectoine synthase.	RESULTS
+18	26	affinity	evidence	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
+28	30	Km	evidence	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
+40	42	Sa	species	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
+43	47	EctC	protein	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
+64	84	catalytic efficiency	evidence	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
+86	93	kcat/Km	evidence	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
+136	145	N-γ-ADABA	chemical	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
+4	6	Km	evidence	The Km dropped fife-fold from 4.9 ± 0.5 mM to 25.4 ± 2.9 mM, and the catalytic efficiency was reduced from 1.47 mM-1 s-1 to 0.02 mM-1 s-1, a 73-fold decrease.	RESULTS
+69	89	catalytic efficiency	evidence	The Km dropped fife-fold from 4.9 ± 0.5 mM to 25.4 ± 2.9 mM, and the catalytic efficiency was reduced from 1.47 mM-1 s-1 to 0.02 mM-1 s-1, a 73-fold decrease.	RESULTS
+5	14	N-γ-ADABA	chemical	Both N-γ-ADABA and N-α-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism.	RESULTS
+19	28	N-α-ADABA	chemical	Both N-γ-ADABA and N-α-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism.	RESULTS
+93	100	ectoine	chemical	Both N-γ-ADABA and N-α-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism.	RESULTS
+17	26	N-α-ADABA	chemical	Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine.	RESULTS
+46	62	ectoine synthase	protein_type	Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine.	RESULTS
+124	138	microorganisms	taxonomy_domain	Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine.	RESULTS
+179	186	ectoine	chemical	Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine.	RESULTS
+24	38	microorganisms	taxonomy_domain	However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).	RESULTS
+102	110	affinity	evidence	However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).	RESULTS
+114	130	ectoine synthase	protein_type	However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).	RESULTS
+135	144	N-γ-ADABA	chemical	However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).	RESULTS
+149	158	N-α-ADABA	chemical	However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).	RESULTS
+168	188	catalytic efficiency	evidence	However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).	RESULTS
+0	15	Crystallization	experimental_method	Crystallization of the (Sa)EctC protein	RESULTS
+24	26	Sa	species	Crystallization of the (Sa)EctC protein	RESULTS
+27	31	EctC	protein	Crystallization of the (Sa)EctC protein	RESULTS
+9	26	crystal structure	evidence	Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein.	RESULTS
+30	46	ectoine synthase	protein_type	Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein.	RESULTS
+80	91	crystallize	experimental_method	Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein.	RESULTS
+97	99	Sa	species	Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein.	RESULTS
+100	104	EctC	protein	Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein.	RESULTS
+19	27	crystals	evidence	Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful.	RESULTS
+32	34	Sa	species	Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful.	RESULTS
+35	39	EctC	protein	Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful.	RESULTS
+40	50	in complex	protein_state	Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful.	RESULTS
+77	86	N-γ-ADABA	chemical	Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful.	RESULTS
+111	118	ectoine	chemical	Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful.	RESULTS
+13	26	crystal forms	evidence	However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained.	RESULTS
+35	37	Sa	species	However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained.	RESULTS
+38	42	EctC	protein	However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained.	RESULTS
+58	68	absence of	protein_state	However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained.	RESULTS
+22	39	crystal structure	evidence	Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed.	RESULTS
+48	50	Sa	species	Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed.	RESULTS
+51	55	EctC	protein	Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed.	RESULTS
+67	88	molecular replacement	experimental_method	Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed.	RESULTS
+32	40	crystals	evidence	However, we were able to obtain crystals of form B that were derivatized with mercury and these diffracted up to 2.8 Å (S1 Table).	RESULTS
+78	85	mercury	chemical	However, we were able to obtain crystals of form B that were derivatized with mercury and these diffracted up to 2.8 Å (S1 Table).	RESULTS
+43	59	structural model	evidence	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
+68	70	Sa	species	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
+71	75	EctC	protein	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
+130	151	molecular replacement	experimental_method	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
+274	282	monomers	oligomeric_state	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
+287	289	Sa	species	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
+290	294	EctC	protein	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
+319	336	crystal structure	evidence	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
+360	366	Rcryst	evidence	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
+383	388	Rfree	evidence	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
+11	18	monomer	oligomeric_state	Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table).	RESULTS
+27	36	structure	evidence	Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table).	RESULTS
+64	85	molecular replacement	experimental_method	Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table).	RESULTS
+192	198	Rcryst	evidence	Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table).	RESULTS
+215	220	Rfree	evidence	Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table).	RESULTS
+21	23	Sa	species	Overall fold of the (Sa)EctC protein	RESULTS
+24	28	EctC	protein	Overall fold of the (Sa)EctC protein	RESULTS
+8	12	EctC	protein	The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c).	RESULTS
+13	23	structures	evidence	The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c).	RESULTS
+61	77	ectoine synthase	protein_type	The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c).	RESULTS
+93	110	cupin superfamily	protein_type	The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c).	RESULTS
+52	67	137 amino acids	residue_range	However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
+80	82	Sa	species	However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
+83	87	EctC	protein	However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
+17	26	structure	evidence	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
+69	71	Sa	species	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
+72	76	EctC	protein	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
+109	125	Met-1 to Glu-115	residue_range	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
+137	142	lacks	protein_state	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
+143	157	22 amino acids	residue_range	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
+165	181	carboxy-terminus	structure_element	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
+200	202	Sa	species	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
+203	207	EctC	protein	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
+5	14	structure	evidence	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
+25	29	open	protein_state	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
+79	92	cupin barrels	structure_element	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
+139	143	open	protein_state	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
+146	148	Sa	species	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
+149	153	EctC	protein	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
+154	163	structure	evidence	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
+8	17	structure	evidence	In this structure no metal co-factor was identified.	RESULTS
+21	26	metal	chemical	In this structure no metal co-factor was identified.	RESULTS
+11	28	crystal structure	evidence	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
+37	39	Sa	species	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
+40	44	EctC	protein	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
+57	63	solved	experimental_method	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
+165	173	protomer	oligomeric_state	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
+174	175	A	structure_element	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
+197	213	Met-1 to Gly-121	residue_range	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
+227	233	closed	protein_state	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
+16	21	lacks	protein_state	Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
+22	35	16 amino acid	residue_range	Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
+52	68	carboxy-terminus	structure_element	Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
+86	101	137 amino acids	residue_range	Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
+114	116	Sa	species	Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
+117	121	EctC	protein	Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
+38	55	crystal structure	evidence	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
+79	91	fully closed	protein_state	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
+105	121	ectoine synthase	protein_type	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
+167	178	semi-closed	protein_state	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
+181	183	Sa	species	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
+184	188	EctC	protein	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
+189	198	structure	evidence	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
+31	39	monomers	oligomeric_state	Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure.	RESULTS
+86	102	Met-1 to Glu-115	residue_range	Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure.	RESULTS
+144	148	open	protein_state	Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure.	RESULTS
+150	154	EctC	protein	Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure.	RESULTS
+155	164	structure	evidence	Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure.	RESULTS
+8	17	structure	evidence	Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC.	FIG
+26	30	open	protein_state	Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC.	FIG
+37	48	semi-closed	protein_state	Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC.	FIG
+50	68	crystal structures	evidence	Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC.	FIG
+73	75	Sa	species	Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC.	FIG
+76	80	EctC	protein	Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC.	FIG
+16	25	structure	evidence	(a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation.	FIG
+34	45	semi-closed	protein_state	(a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation.	FIG
+48	50	Sa	species	(a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation.	FIG
+51	55	EctC	protein	(a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation.	FIG
+4	13	β-strands	structure_element	The β-strands are numbered β1-β11 and the helices α-I to α-II.	FIG
+27	33	β1-β11	structure_element	The β-strands are numbered β1-β11 and the helices α-I to α-II.	FIG
+42	49	helices	structure_element	The β-strands are numbered β1-β11 and the helices α-I to α-II.	FIG
+50	61	α-I to α-II	structure_element	The β-strands are numbered β1-β11 and the helices α-I to α-II.	FIG
+16	25	structure	evidence	(b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation.	FIG
+34	38	open	protein_state	(b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation.	FIG
+41	43	Sa	species	(b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation.	FIG
+44	48	EctC	protein	(b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation.	FIG
+20	31	active site	site	The entrance to the active site of the ectoine synthase is marked.	FIG
+39	55	ectoine synthase	protein_type	The entrance to the active site of the ectoine synthase is marked.	FIG
+4	11	Overlay	experimental_method	(c) Overlay of the “semi-closed” and “open” (Sa)EctC structures.	FIG
+20	31	semi-closed	protein_state	(c) Overlay of the “semi-closed” and “open” (Sa)EctC structures.	FIG
+38	42	open	protein_state	(c) Overlay of the “semi-closed” and “open” (Sa)EctC structures.	FIG
+45	47	Sa	species	(c) Overlay of the “semi-closed” and “open” (Sa)EctC structures.	FIG
+48	52	EctC	protein	(c) Overlay of the “semi-closed” and “open” (Sa)EctC structures.	FIG
+53	63	structures	evidence	(c) Overlay of the “semi-closed” and “open” (Sa)EctC structures.	FIG
+12	21	structure	evidence	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
+26	28	Sa	species	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
+29	33	EctC	protein	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
+64	72	crystals	evidence	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
+88	104	carboxy-terminus	structure_element	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
+148	160	cupin barrel	structure_element	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
+186	193	monomer	oligomeric_state	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
+194	195	A	structure_element	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
+204	215	semi-closed	protein_state	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
+217	226	structure	evidence	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
+36	62	root mean square deviation	evidence	This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other.	RESULTS
+64	68	RMSD	evidence	This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other.	RESULTS
+143	147	open	protein_state	This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other.	RESULTS
+149	157	monomers	oligomeric_state	This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other.	RESULTS
+14	25	semi-closed	protein_state	However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure.	RESULTS
+27	34	monomer	oligomeric_state	However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure.	RESULTS
+57	61	RMSD	evidence	However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure.	RESULTS
+115	119	open	protein_state	However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure.	RESULTS
+127	136	structure	evidence	However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure.	RESULTS
+52	61	structure	evidence	Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail.	RESULTS
+71	82	semi-closed	protein_state	Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail.	RESULTS
+97	99	Sa	species	Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail.	RESULTS
+100	104	EctC	protein	Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail.	RESULTS
+180	184	open	protein_state	Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail.	RESULTS
+191	202	semi-closed	protein_state	Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail.	RESULTS
+4	13	structure	evidence	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
+22	33	semi-closed	protein_state	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
+36	38	Sa	species	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
+39	43	EctC	protein	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
+67	76	β-strands	structure_element	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
+78	84	β1-β11	structure_element	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
+94	103	α-helices	structure_element	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
+105	108	α-I	structure_element	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
+113	117	α-II	structure_element	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
+4	13	β-strands	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
+23	45	anti-parallel β-sheets	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
+47	49	β2	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
+50	52	β3	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
+54	56	β4	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
+58	61	β11	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
+63	65	β6	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
+71	73	β9	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
+89	111	three-stranded β-sheet	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
+113	115	β7	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
+117	119	β8	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
+125	128	β10	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
+10	18	β-sheets	structure_element	These two β-sheets pack against each other, forming a cup-shaped β-sandwich with a topology characteristic for the cupin-fold.	RESULTS
+54	75	cup-shaped β-sandwich	structure_element	These two β-sheets pack against each other, forming a cup-shaped β-sandwich with a topology characteristic for the cupin-fold.	RESULTS
+115	125	cupin-fold	structure_element	These two β-sheets pack against each other, forming a cup-shaped β-sandwich with a topology characteristic for the cupin-fold.	RESULTS
+8	10	Sa	species	Hence, (Sa)EctC adopts an overall bowl shape in which one side is opened towards the solvent (Fig 4a to 4c).	RESULTS
+11	15	EctC	protein	Hence, (Sa)EctC adopts an overall bowl shape in which one side is opened towards the solvent (Fig 4a to 4c).	RESULTS
+8	19	semi-closed	protein_state	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
+21	30	structure	evidence	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
+41	62	carboxy-terminal tail	structure_element	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
+81	97	electron density	evidence	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
+114	125	small helix	structure_element	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
+127	131	α-II	structure_element	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
+149	160	active site	site	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
+169	171	Sa	species	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
+172	176	EctC	protein	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
+22	32	α-II helix	structure_element	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+77	89	unstructured	protein_state	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+90	94	loop	structure_element	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+116	120	open	protein_state	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+122	131	structure	evidence	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+144	146	β4	structure_element	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+151	153	β6	structure_element	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+189	195	stable	protein_state	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+196	204	β-strand	structure_element	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+205	207	β5	structure_element	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+228	239	semi-closed	protein_state	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+254	256	Sa	species	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+257	261	EctC	protein	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
+0	30	Structural comparison analyses	experimental_method	Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily.	RESULTS
+41	52	DALI server	experimental_method	Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily.	RESULTS
+68	70	Sa	species	Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily.	RESULTS
+71	75	EctC	protein	Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily.	RESULTS
+122	139	cupin superfamily	protein_type	Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily.	RESULTS
+57	96	Cupin 2 conserved barrel domain protein	protein	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+98	109	YP_751781.1	protein	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+116	140	Shewanella frigidimarina	species	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+175	182	Z-score	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+198	202	RMSD	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+332	341	structure	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+369	395	manganese-containing cupin	protein	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+397	403	TM1459	protein	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+410	429	Thermotoga maritima	species	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+464	471	Z-score	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+487	491	RMSD	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+524	531	cyclase	protein_type	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+532	536	RemF	protein	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+542	571	Streptomyces resistomycificus	species	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+605	612	Z-score	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+628	632	RMSD	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+669	692	auxin-binding protein 1	protein	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+698	706	Zea mays	species	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+742	749	Z-score	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+765	769	RMSD	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
+18	22	EctC	protein	Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction.	RESULTS
+43	61	polyketide cyclase	protein_type	Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction.	RESULTS
+62	66	RemF	protein	Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction.	RESULTS
+88	101	cupin-related	protein_type	Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction.	RESULTS
+8	12	RemF	protein	Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.	RESULTS
+21	46	aldos-2-ulose dehydratase	protein_type	Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.	RESULTS
+47	56	isomerase	protein_type	Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.	RESULTS
+62	78	ectoine synthase	protein_type	Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.	RESULTS
+111	122	dehydratase	protein_type	Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.	RESULTS
+134	151	cupin superfamily	protein_type	Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.	RESULTS
+16	20	EctC	protein	Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure	RESULTS
+21	36	dimer interface	site	Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure	RESULTS
+57	59	Sa	species	Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure	RESULTS
+60	64	EctC	protein	Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure	RESULTS
+65	82	crystal structure	evidence	Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure	RESULTS
+9	12	SEC	experimental_method	Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution.	RESULTS
+30	39	HPLC-MALS	experimental_method	Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution.	RESULTS
+82	98	ectoine synthase	protein_type	Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution.	RESULTS
+104	117	S. alaskensis	species	Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution.	RESULTS
+123	128	dimer	oligomeric_state	Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution.	RESULTS
+4	21	crystal structure	evidence	The crystal structure of this protein reflects this quaternary arrangement.	RESULTS
+8	19	semi-closed	protein_state	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
+21	38	crystal structure	evidence	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
+41	43	Sa	species	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
+44	48	EctC	protein	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
+53	65	crystallized	experimental_method	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
+71	76	dimer	oligomeric_state	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
+80	86	dimers	oligomeric_state	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
+5	10	dimer	oligomeric_state	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+46	54	monomers	oligomeric_state	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+69	81	head-to-tail	protein_state	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+152	174	antiparallel β-strands	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+176	184	β-strand	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+185	187	β1	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+198	205	1MIVRN5	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+212	219	monomer	oligomeric_state	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+220	221	A	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+226	234	β-strand	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+235	237	β8	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+243	250	monomer	oligomeric_state	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+251	252	B	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+263	273	82GVMYAL87	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
+38	47	β-strands	structure_element	The strong interactions between these β-strands rely primarily on backbone contacts.	RESULTS
+47	71	hydrophobic interactions	bond_interaction	In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands.	RESULTS
+106	114	monomers	oligomeric_state	In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands.	RESULTS
+123	128	loops	structure_element	In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands.	RESULTS
+144	153	β-strands	structure_element	In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands.	RESULTS
+19	27	PDBePISA	experimental_method	As calculated with PDBePISA, the surface area buried upon dimer formation is 1462 Å2, which is 20.5% of the total accessible surface of a monomer of this protein.	RESULTS
+58	63	dimer	oligomeric_state	As calculated with PDBePISA, the surface area buried upon dimer formation is 1462 Å2, which is 20.5% of the total accessible surface of a monomer of this protein.	RESULTS
+138	145	monomer	oligomeric_state	As calculated with PDBePISA, the surface area buried upon dimer formation is 1462 Å2, which is 20.5% of the total accessible surface of a monomer of this protein.	RESULTS
+55	61	dimers	oligomeric_state	Both values fall within the range for known functional dimers.	RESULTS
+0	17	Crystal structure	evidence	Crystal structure of (Sa)EctC.	FIG
+22	24	Sa	species	Crystal structure of (Sa)EctC.	FIG
+25	29	EctC	protein	Crystal structure of (Sa)EctC.	FIG
+20	25	dimer	oligomeric_state	(a) Top-view of the dimer of the (Sa)EctC protein.	FIG
+34	36	Sa	species	(a) Top-view of the dimer of the (Sa)EctC protein.	FIG
+37	41	EctC	protein	(a) Top-view of the dimer of the (Sa)EctC protein.	FIG
+20	25	water	chemical	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
+92	100	monomers	oligomeric_state	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
+142	144	Sa	species	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
+145	149	EctC	protein	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
+150	155	dimer	oligomeric_state	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
+186	201	dimer interface	site	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
+216	225	β-strands	structure_element	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
+234	241	monomer	oligomeric_state	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
+35	50	dimer interface	site	(c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6.	FIG
+63	74	beta-strand	structure_element	(c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6.	FIG
+75	77	β1	structure_element	(c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6.	FIG
+82	84	β6	structure_element	(c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6.	FIG
+8	12	open	protein_state	In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit.	RESULTS
+15	17	Sa	species	In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit.	RESULTS
+18	22	EctC	protein	In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit.	RESULTS
+23	32	structure	evidence	In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit.	RESULTS
+38	45	monomer	oligomeric_state	In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit.	RESULTS
+35	42	packing	experimental_method	We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well.	RESULTS
+60	67	monomer	oligomeric_state	We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well.	RESULTS
+68	75	monomer	oligomeric_state	We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well.	RESULTS
+169	174	dimer	oligomeric_state	We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well.	RESULTS
+202	214	crystal form	evidence	We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well.	RESULTS
+18	23	dimer	oligomeric_state	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
+68	79	semi-closed	protein_state	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
+82	84	Sa	species	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
+85	89	EctC	protein	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
+90	99	structure	evidence	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
+126	133	monomer	oligomeric_state	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
+134	141	monomer	oligomeric_state	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
+175	183	β-sheets	structure_element	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
+109	117	monomers	oligomeric_state	The crystallographic two-fold axis present within the crystal symmetry is located exactly in between the two monomers, resulting in a monomer within the asymmetric unit.	RESULTS
+134	141	monomer	oligomeric_state	The crystallographic two-fold axis present within the crystal symmetry is located exactly in between the two monomers, resulting in a monomer within the asymmetric unit.	RESULTS
+16	21	dimer	oligomeric_state	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
+39	50	semi-closed	protein_state	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
+52	61	structure	evidence	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
+66	68	Sa	species	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
+69	73	EctC	protein	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
+103	107	open	protein_state	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
+109	118	structure	evidence	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
+62	73	DALI search	experimental_method	Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).	RESULTS
+105	109	EctC	protein	Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).	RESULTS
+135	141	dimers	oligomeric_state	Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).	RESULTS
+161	168	monomer	oligomeric_state	Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).	RESULTS
+169	176	monomer	oligomeric_state	Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).	RESULTS
+201	206	dimer	oligomeric_state	Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).	RESULTS
+33	41	flexible	protein_state	Structural rearrangements of the flexible (Sa)EctC carboxy-terminus	RESULTS
+43	45	Sa	species	Structural rearrangements of the flexible (Sa)EctC carboxy-terminus	RESULTS
+46	50	EctC	protein	Structural rearrangements of the flexible (Sa)EctC carboxy-terminus	RESULTS
+51	67	carboxy-terminus	structure_element	Structural rearrangements of the flexible (Sa)EctC carboxy-terminus	RESULTS
+54	70	ectoine synthase	protein_type	The cupin core represents the structural framework of ectoine synthase (Figs 4 and 5).	RESULTS
+32	50	crystal structures	evidence	The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus.	RESULTS
+59	61	Sa	species	The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus.	RESULTS
+62	66	EctC	protein	The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus.	RESULTS
+115	131	carboxy-terminus	structure_element	The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus.	RESULTS
+32	55	carboxy-terminal region	structure_element	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
+63	78	137 amino acids	residue_range	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
+91	93	Sa	species	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
+94	98	EctC	protein	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
+111	127	highly conserved	protein_state	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
+147	155	extended	protein_state	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
+156	168	EctC protein	protein_type	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
+14	22	β-strand	structure_element	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
+23	26	β11	structure_element	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
+44	53	conserved	protein_state	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
+54	61	proline	residue_name	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
+72	79	Pro-109	residue_name_number	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
+84	91	Pro-110	residue_name_number	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
+163	165	Sa	species	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
+166	170	EctC	protein	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
+8	19	semi-closed	protein_state	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
+22	24	Sa	species	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
+25	29	EctC	protein	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
+30	39	structure	evidence	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
+53	69	electron density	evidence	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
+77	93	carboxy-terminus	structure_element	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
+109	130	7 amino acid residues	residue_range	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
+152	159	Gly-121	residue_name_number	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
+41	52	small helix	structure_element	These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).	RESULTS
+70	74	open	protein_state	These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).	RESULTS
+75	81	cavity	site	These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).	RESULTS
+89	99	cupin-fold	structure_element	These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).	RESULTS
+108	110	Sa	species	These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).	RESULTS
+111	115	EctC	protein	These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).	RESULTS
+18	23	helix	structure_element	Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement.	RESULTS
+64	75	loop region	structure_element	Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement.	RESULTS
+84	93	β-strands	structure_element	Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement.	RESULTS
+94	96	β4	structure_element	Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement.	RESULTS
+101	103	β6	structure_element	Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement.	RESULTS
+30	38	β-strand	structure_element	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
+39	41	β5	structure_element	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
+73	95	small C-terminal helix	structure_element	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
+99	105	absent	protein_state	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
+126	130	open	protein_state	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
+133	135	Sa	species	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
+136	140	EctC	protein	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
+141	150	structure	evidence	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
+30	38	β-strand	structure_element	As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c).	RESULTS
+39	41	β5	structure_element	As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c).	RESULTS
+87	98	semi-closed	protein_state	As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c).	RESULTS
+101	103	Sa	species	As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c).	RESULTS
+104	108	EctC	protein	As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c).	RESULTS
+109	118	structure	evidence	As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c).	RESULTS
+28	36	β-strand	structure_element	It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below).	RESULTS
+37	39	β5	structure_element	It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below).	RESULTS
+59	65	His-93	residue_name_number	It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below).	RESULTS
+103	108	metal	chemical	It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below).	RESULTS
+21	24	His	residue_name	The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5.	RESULTS
+62	64	Sa	species	The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5.	RESULTS
+65	69	EctC	protein	The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5.	RESULTS
+70	80	structures	evidence	The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5.	RESULTS
+120	128	β-strand	structure_element	The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5.	RESULTS
+129	131	β5	structure_element	The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5.	RESULTS
+29	39	cupin fold	structure_element	Therefore the sealing of the cupin fold, as described above, seem to have an indirect influence on the architecture of the postulated iron-binding site.	RESULTS
+134	151	iron-binding site	site	Therefore the sealing of the cupin fold, as described above, seem to have an indirect influence on the architecture of the postulated iron-binding site.	RESULTS
+16	23	Pro-109	residue_name_number	The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2).	RESULTS
+28	35	Pro-110	residue_name_number	The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2).	RESULTS
+65	73	β-strand	structure_element	The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2).	RESULTS
+74	77	β11	structure_element	The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2).	RESULTS
+81	97	highly conserved	protein_state	The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2).	RESULTS
+101	119	EctC-type proteins	protein_type	The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2).	RESULTS
+69	85	carboxy-terminus	structure_element	They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold.	RESULTS
+87	109	27 amino acid residues	residue_range	They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold.	RESULTS
+115	117	Sa	species	They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold.	RESULTS
+118	122	EctC	protein	They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold.	RESULTS
+136	146	cupin fold	structure_element	They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold.	RESULTS
+8	19	semi-closed	protein_state	In the “semi-closed” structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c).	RESULTS
+21	30	structure	evidence	In the “semi-closed” structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c).	RESULTS
+86	98	cupin barrel	structure_element	In the “semi-closed” structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c).	RESULTS
+8	12	open	protein_state	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
+15	17	Sa	species	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
+18	22	EctC	protein	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
+23	32	structure	evidence	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
+39	46	proline	residue_name	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
+75	91	electron density	evidence	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
+124	131	Pro-110	residue_name_number	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
+137	153	electron density	evidence	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
+213	229	carboxy-terminus	structure_element	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
+39	46	Pro-109	residue_name_number	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+121	127	His-55	residue_name_number	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+152	156	open	protein_state	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+163	174	semi-closed	protein_state	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+177	179	Sa	species	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+180	184	EctC	protein	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+185	195	structures	evidence	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+4	11	Pro-109	residue_name_number	The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11.	RESULTS
+12	18	His-55	residue_name_number	The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11.	RESULTS
+43	49	stable	protein_state	The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11.	RESULTS
+70	77	proline	residue_name	The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11.	RESULTS
+101	109	β-strand	structure_element	The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11.	RESULTS
+110	113	β11	structure_element	The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11.	RESULTS
+12	19	proline	residue_name	Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.	RESULTS
+49	72	carboxy-terminal region	structure_element	Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.	RESULTS
+81	83	Sa	species	Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.	RESULTS
+84	88	EctC	protein	Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.	RESULTS
+117	123	His-55	residue_name_number	Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.	RESULTS
+129	136	Pro-109	residue_name_number	Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.	RESULTS
+40	47	Pro-109	residue_name_number	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
+52	58	His-55	residue_name_number	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
+64	87	carboxy-terminal region	structure_element	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
+92	94	Sa	species	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
+95	99	EctC	protein	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
+142	149	Glu-115	residue_name_number	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
+155	161	His-55	residue_name_number	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
+204	215	small helix	structure_element	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
+223	239	carboxy-terminus	structure_element	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
+24	31	Glu-115	residue_name_number	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
+36	42	His-55	residue_name_number	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
+67	78	semi-closed	protein_state	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
+80	89	structure	evidence	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
+100	118	partially extended	protein_state	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
+119	135	carboxy-terminus	structure_element	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
+155	171	electron density	evidence	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
+8	12	open	protein_state	In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b).	RESULTS
+14	23	structure	evidence	In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b).	RESULTS
+32	34	Sa	species	In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b).	RESULTS
+35	39	EctC	protein	In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b).	RESULTS
+87	94	Glu-115	residue_name_number	In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b).	RESULTS
+105	121	carboxy-terminus	structure_element	Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region.	RESULTS
+130	134	open	protein_state	Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region.	RESULTS
+137	139	Sa	species	Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region.	RESULTS
+140	144	EctC	protein	Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region.	RESULTS
+145	154	structure	evidence	Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region.	RESULTS
+201	217	electron density	evidence	Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region.	RESULTS
+29	47	metal-binding site	site	Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus.	FIG
+56	58	Sa	species	Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus.	FIG
+59	63	EctC	protein	Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus.	FIG
+80	88	flexible	protein_state	Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus.	FIG
+89	105	carboxy-terminus	structure_element	Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus.	FIG
+18	23	water	chemical	(a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93.	FIG
+111	117	Glu-57	residue_name_number	(a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93.	FIG
+119	125	Tyr-85	residue_name_number	(a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93.	FIG
+131	137	His-93	residue_name_number	(a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93.	FIG
+30	35	water	chemical	The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase.	FIG
+85	89	Fe2+	chemical	The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase.	FIG
+106	117	active side	site	The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase.	FIG
+125	141	ectoine synthase	protein_type	The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase.	FIG
+0	6	His-55	residue_name_number	His-55 interacts with the double proline motif (Pro-109 and Pro-110).	FIG
+26	46	double proline motif	structure_element	His-55 interacts with the double proline motif (Pro-109 and Pro-110).	FIG
+48	55	Pro-109	residue_name_number	His-55 interacts with the double proline motif (Pro-109 and Pro-110).	FIG
+60	67	Pro-110	residue_name_number	His-55 interacts with the double proline motif (Pro-109 and Pro-110).	FIG
+67	74	Glu-115	residue_name_number	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
+101	109	flexible	protein_state	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
+110	126	carboxy-terminus	structure_element	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
+151	153	Sa	species	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
+154	158	EctC	protein	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
+183	194	semi-closed	protein_state	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
+197	199	Sa	species	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
+200	204	EctC	protein	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
+205	214	structure	evidence	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
+7	14	overlay	experimental_method	(b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein.	FIG
+23	27	open	protein_state	(b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein.	FIG
+62	73	semi-closed	protein_state	(b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein.	FIG
+94	103	structure	evidence	(b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein.	FIG
+112	114	Sa	species	(b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein.	FIG
+115	119	EctC	protein	(b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein.	FIG
+13	30	iron binding site	site	The putative iron binding site of (Sa)EctC	RESULTS
+35	37	Sa	species	The putative iron binding site of (Sa)EctC	RESULTS
+38	42	EctC	protein	The putative iron binding site of (Sa)EctC	RESULTS
+8	19	semi-closed	protein_state	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
+21	30	structure	evidence	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
+35	37	Sa	species	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
+38	42	EctC	protein	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
+61	69	monomers	oligomeric_state	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
+120	136	electron density	evidence	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
+159	171	cupin barrel	structure_element	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
+7	9	Sa	species	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
+10	14	EctC	protein	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
+20	25	metal	chemical	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
+77	81	Fe2+	chemical	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
+86	90	Zn2+	chemical	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
+106	113	density	evidence	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
+123	140	refined occupancy	experimental_method	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
+23	27	Fe2+	chemical	Only the refinement of Fe2+ resulted in a visibly improved electron density, however with a low degree of occupancy.	RESULTS
+59	75	electron density	evidence	Only the refinement of Fe2+ resulted in a visibly improved electron density, however with a low degree of occupancy.	RESULTS
+14	18	iron	chemical	This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b).	RESULTS
+59	65	Glu-57	residue_name_number	This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b).	RESULTS
+67	73	Tyr-85	residue_name_number	This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b).	RESULTS
+78	84	His-93	residue_name_number	This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b).	RESULTS
+74	78	iron	chemical	The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively.	RESULTS
+102	108	Glu-57	residue_name_number	The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively.	RESULTS
+120	126	Tyr-85	residue_name_number	The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively.	RESULTS
+142	148	His-93	residue_name_number	The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively.	RESULTS
+51	69	iron binding sites	site	These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure.	RESULTS
+106	116	absence of	protein_state	These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure.	RESULTS
+146	148	Sa	species	These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure.	RESULTS
+149	153	EctC	protein	These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure.	RESULTS
+154	171	crystal structure	evidence	These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure.	RESULTS
+71	75	iron	chemical	Since both the refinement and the distance did not clearly identify an iron molecule, we decided to conservatively place a water molecule at this position.	RESULTS
+123	128	water	chemical	Since both the refinement and the distance did not clearly identify an iron molecule, we decided to conservatively place a water molecule at this position.	RESULTS
+21	26	water	chemical	The position of this water molecule is described in more detail below and is highlighted in Figs 5a and 5b and 6a and 6b as a sphere.	RESULTS
+55	60	water	chemical	Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2).	RESULTS
+74	92	strictly conserved	protein_state	Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2).	RESULTS
+103	112	alignment	experimental_method	Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2).	RESULTS
+135	147	EctC protein	protein_type	Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2).	RESULTS
+188	206	EctC-type proteins	protein_type	Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2).	RESULTS
+8	12	open	protein_state	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
+14	23	structure	evidence	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
+32	34	Sa	species	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
+35	39	EctC	protein	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
+49	65	electron density	evidence	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
+99	103	iron	chemical	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
+126	137	semi-closed	protein_state	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
+139	148	structure	evidence	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
+14	30	electron density	evidence	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
+51	56	water	chemical	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
+80	84	iron	chemical	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
+94	99	water	chemical	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
+190	194	open	protein_state	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
+197	199	Sa	species	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
+200	204	EctC	protein	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
+205	214	structure	evidence	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
+5	20	superimposition	experimental_method	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+30	32	Sa	species	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+33	37	EctC	protein	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+38	56	crystal structures	evidence	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+128	134	Glu-57	residue_name_number	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+136	142	Tyr-85	residue_name_number	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+148	154	His-93	residue_name_number	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+178	182	iron	chemical	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+191	202	semi-closed	protein_state	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+204	213	structure	evidence	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+276	285	iron-free	protein_state	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+288	292	open	protein_state	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+294	303	structure	evidence	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
+5	11	His-93	residue_name_number	Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above.	RESULTS
+48	59	semi-closed	protein_state	Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above.	RESULTS
+61	70	structure	evidence	Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above.	RESULTS
+104	112	β-strand	structure_element	Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above.	RESULTS
+113	115	β5	structure_element	Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above.	RESULTS
+85	102	iron-binding site	site	Taken together, this observations indicate, that the architecture of the presumptive iron-binding site is pre-set for the binding of the catalytically important metal by the ectoine synthase.	RESULTS
+161	166	metal	chemical	Taken together, this observations indicate, that the architecture of the presumptive iron-binding site is pre-set for the binding of the catalytically important metal by the ectoine synthase.	RESULTS
+174	190	ectoine synthase	protein_type	Taken together, this observations indicate, that the architecture of the presumptive iron-binding site is pre-set for the binding of the catalytically important metal by the ectoine synthase.	RESULTS
+66	72	Tyr-52	residue_name_number	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+87	91	loop	structure_element	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+109	117	β-strand	structure_element	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+118	120	β5	structure_element	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+130	134	open	protein_state	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+141	152	semi-closed	protein_state	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+155	157	Sa	species	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+158	162	EctC	protein	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+163	173	structures	evidence	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
+8	19	semi-closed	protein_state	In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding.	RESULTS
+21	30	structure	evidence	In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding.	RESULTS
+72	78	Tyr-52	residue_name_number	In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding.	RESULTS
+98	102	iron	chemical	In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding.	RESULTS
+189	195	Tyr-52	residue_name_number	In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding.	RESULTS
+220	225	metal	chemical	In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding.	RESULTS
+18	30	substitution	experimental_method	Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1).	RESULTS
+37	40	Ala	residue_name	Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1).	RESULTS
+77	81	iron	chemical	Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1).	RESULTS
+117	123	mutant	protein_state	Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1).	RESULTS
+28	35	overlay	experimental_method	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
+44	48	open	protein_state	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
+55	66	semi-closed	protein_state	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
+69	71	Sa	species	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
+72	76	EctC	protein	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
+77	95	crystal structures	evidence	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
+119	125	Tyr-52	residue_name_number	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
+176	180	iron	chemical	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
+253	258	metal	chemical	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
+269	275	Glu-57	residue_name_number	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
+277	283	Tyr-85	residue_name_number	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
+289	295	His-93	residue_name_number	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
+6	12	Tyr-52	residue_name_number	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
+16	34	strictly conserved	protein_state	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
+41	50	alignment	experimental_method	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
+58	76	EctC-type proteins	protein_type	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
+160	176	ectoine synthase	protein_type	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
+190	200	absence of	protein_state	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
+201	210	N-γ-ADABA	chemical	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
+219	221	Sa	species	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
+222	226	EctC	protein	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
+227	245	crystal structures	evidence	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
+276	282	Tyr-52	residue_name_number	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
+32	49	iron binding site	site	To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity.	RESULTS
+73	115	structure-guided site-directed mutagenesis	experimental_method	To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity.	RESULTS
+144	146	Sa	species	To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity.	RESULTS
+147	151	EctC	protein	To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity.	RESULTS
+171	175	iron	chemical	To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity.	RESULTS
+27	33	Glu-57	residue_name_number	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
+35	41	Tyr-85	residue_name_number	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
+43	49	His-93	residue_name_number	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
+72	96	mono-nuclear iron center	site	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
+105	107	Sa	species	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
+108	112	EctC	protein	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
+113	130	crystal structure	evidence	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
+149	157	replaced	experimental_method	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
+164	167	Ala	residue_name	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
+213	217	iron	chemical	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
+233	239	mutant	protein_state	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
+28	54	iron-coordinating residues	site	For some of the presumptive iron-coordinating residues, additional site-directed mutagenesis experiments were carried out.	RESULTS
+67	92	site-directed mutagenesis	experimental_method	For some of the presumptive iron-coordinating residues, additional site-directed mutagenesis experiments were carried out.	RESULTS
+67	73	Glu-57	residue_name_number	To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant.	RESULTS
+89	92	Asp	residue_name	To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant.	RESULTS
+93	100	variant	protein_state	To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant.	RESULTS
+5	11	mutant	protein_state	This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1).	RESULTS
+52	56	iron	chemical	This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1).	RESULTS
+72	75	Ala	residue_name	This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1).	RESULTS
+76	88	substitution	experimental_method	This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1).	RESULTS
+8	16	replaced	experimental_method	We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.	RESULTS
+17	23	Tyr-85	residue_name_number	We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.	RESULTS
+38	41	Phe	residue_name	We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.	RESULTS
+47	50	Trp	residue_name	We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.	RESULTS
+68	74	mutant	protein_state	We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.	RESULTS
+126	130	iron	chemical	We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.	RESULTS
+64	70	Tyr-85	residue_name_number	Collectively, these data suggest that the hydroxyl group of the Tyr-85 side chain is needed for the binding of the iron (Fig 6a).	RESULTS
+115	119	iron	chemical	Collectively, these data suggest that the hydroxyl group of the Tyr-85 side chain is needed for the binding of the iron (Fig 6a).	RESULTS
+8	16	replaced	experimental_method	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
+33	53	iron-binding residue	site	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
+54	60	His-93	residue_name_number	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
+67	70	Asn	residue_name	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
+92	94	Sa	species	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
+95	99	EctC	protein	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
+161	165	iron	chemical	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
+210	219	wild-type	protein_state	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
+68	94	iron-coordinating residues	site	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
+96	102	Glu-57	residue_name_number	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
+104	110	Tyr-85	residue_name_number	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
+112	118	His-93	residue_name_number	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
+149	153	Fe2+	chemical	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
+170	172	Sa	species	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
+173	177	EctC	protein	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
+38	40	Sa	species	A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate	RESULTS
+41	45	EctC	protein	A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate	RESULTS
+46	55	structure	evidence	A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate	RESULTS
+94	103	N-γ-ADABA	chemical	A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate	RESULTS
+47	65	co-crystallization	experimental_method	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
+69	88	soaking experiments	experimental_method	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
+120	122	Sa	species	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
+123	127	EctC	protein	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
+128	146	crystal structures	evidence	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
+183	192	N-γ-ADABA	chemical	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
+197	204	ectoine	chemical	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
+230	246	ectoine synthase	protein_type	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
+17	28	semi-closed	protein_state	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
+31	33	Sa	species	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
+34	38	EctC	protein	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
+39	48	structure	evidence	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
+59	80	carboxy-terminal loop	structure_element	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
+119	135	electron density	evidence	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
+174	185	active site	site	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
+227	254	crystallographic refinement	experimental_method	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
+44	48	open	protein_state	This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement.	RESULTS
+50	59	structure	evidence	This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement.	RESULTS
+68	70	Sa	species	This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement.	RESULTS
+71	75	EctC	protein	This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement.	RESULTS
+104	120	electron density	evidence	This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement.	RESULTS
+57	69	purification	experimental_method	We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched.	RESULTS
+74	89	crystallization	experimental_method	We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched.	RESULTS
+108	124	electron density	evidence	We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched.	RESULTS
+91	93	Sa	species	This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization.	RESULTS
+94	98	EctC	protein	This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization.	RESULTS
+145	152	E. coli	species	This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization.	RESULTS
+163	178	crystallization	experimental_method	This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization.	RESULTS
+14	17	PEG	chemical	Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments.	RESULTS
+76	83	density	evidence	Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments.	RESULTS
+121	124	PEG	chemical	Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments.	RESULTS
+159	162	PEG	chemical	Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments.	RESULTS
+207	210	PEG	chemical	Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments.	RESULTS
+38	62	electron density feature	evidence	Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density.	RESULTS
+125	127	Sa	species	Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density.	RESULTS
+128	132	EctC	protein	Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density.	RESULTS
+146	161	hexane-1,6-diol	chemical	Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density.	RESULTS
+218	234	electron density	evidence	Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density.	RESULTS
+39	54	hexane-1,6-diol	chemical	However, to the best of our knowledge, hexane-1,6-diol is not part of the E. coli metabolome.	RESULTS
+74	81	E. coli	species	However, to the best of our knowledge, hexane-1,6-diol is not part of the E. coli metabolome.	RESULTS
+179	183	EctC	protein	Despite these notable limitations, we considered the serendipitously trapped compound as a mock ligand that might provide useful insights into the spatial positioning of the true EctC substrate and those residues that coordinate it within the ectoine synthase active site.	RESULTS
+243	259	ectoine synthase	protein_type	Despite these notable limitations, we considered the serendipitously trapped compound as a mock ligand that might provide useful insights into the spatial positioning of the true EctC substrate and those residues that coordinate it within the ectoine synthase active site.	RESULTS
+260	271	active site	site	Despite these notable limitations, we considered the serendipitously trapped compound as a mock ligand that might provide useful insights into the spatial positioning of the true EctC substrate and those residues that coordinate it within the ectoine synthase active site.	RESULTS
+18	27	N-γ-ADABA	chemical	We note that both N-γ-ADABA and hexane-1,6-diol are both C6-compounds and display similar length (Fig 7a).	RESULTS
+32	47	hexane-1,6-diol	chemical	We note that both N-γ-ADABA and hexane-1,6-diol are both C6-compounds and display similar length (Fig 7a).	RESULTS
+49	60	active site	site	A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure.	FIG
+69	80	semi-closed	protein_state	A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure.	FIG
+83	85	Sa	species	A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure.	FIG
+86	90	EctC	protein	A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure.	FIG
+91	108	crystal structure	evidence	A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure.	FIG
+17	33	electron density	evidence	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
+41	52	active site	site	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
+61	72	semi-closed	protein_state	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
+74	83	structure	evidence	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
+88	90	Sa	species	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
+91	95	EctC	protein	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
+112	127	hexane-1,6-diol	chemical	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
+159	175	electron density	evidence	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
+183	192	N-γ-ADABA	chemical	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
+210	226	ectoine synthase	protein_type	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
+19	31	binding site	site	(b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted.	FIG
+39	43	iron	chemical	(b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted.	FIG
+73	88	hexane-1,6-diol	chemical	(b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted.	FIG
+39	43	iron	chemical	The amino acid side chains involved in iron-ligand binding are colored in blue and those involved in the binding of the chemically undefined ligand are colored in green using a ball and stick representation.	FIG
+4	12	flexible	protein_state	The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange.	FIG
+13	34	carboxy-terminal loop	structure_element	The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange.	FIG
+39	41	Sa	species	The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange.	FIG
+42	46	EctC	protein	The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange.	FIG
+4	20	electron density	evidence	The electron density was calculated as an omit map and contoured at 1.0 σ.	FIG
+42	50	omit map	evidence	The electron density was calculated as an omit map and contoured at 1.0 σ.	FIG
+3	10	refined	experimental_method	We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%.	RESULTS
+16	18	Sa	species	We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%.	RESULTS
+19	23	EctC	protein	We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%.	RESULTS
+24	33	structure	evidence	We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%.	RESULTS
+116	135	R- and Rfree-factor	evidence	We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%.	RESULTS
+22	30	omit map	evidence	We also calculated an omit map and the electron density reappeared (Fig 7b).	RESULTS
+39	55	electron density	evidence	We also calculated an omit map and the electron density reappeared (Fig 7b).	RESULTS
+61	63	Sa	species	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+64	68	EctC	protein	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+98	103	bound	protein_state	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+126	132	Trp-21	residue_name_number	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+137	143	Ser-23	residue_name_number	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+147	154	β-sheet	structure_element	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+155	157	β3	structure_element	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+159	165	Thr-40	residue_name_number	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+177	184	β-sheet	structure_element	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+185	187	β4	structure_element	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+193	200	Cys-105	residue_name_number	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+205	212	Phe-107	residue_name_number	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+237	244	β-sheet	structure_element	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+245	248	β11	structure_element	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
+38	54	highly conserved	protein_state	Remarkably, all of these residues are highly conserved throughout the extended EctC protein family (Fig 2).	RESULTS
+79	91	EctC protein	protein_type	Remarkably, all of these residues are highly conserved throughout the extended EctC protein family (Fig 2).	RESULTS
+0	42	Structure-guided site-directed mutagenesis	experimental_method	Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase	RESULTS
+50	64	catalytic core	site	Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase	RESULTS
+72	88	ectoine synthase	protein_type	Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase	RESULTS
+14	51	alignment of the amino acid sequences	experimental_method	In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues.	RESULTS
+59	77	EctC-type proteins	protein_type	In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues.	RESULTS
+113	131	strictly conserved	protein_state	In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues.	RESULTS
+32	38	Thr-40	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+40	46	Tyr-52	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+48	54	His-55	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+56	62	Glu-57	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+64	70	Gly-64	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+72	78	Tyr-85	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+80	86	Leu-87	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+88	94	His-93	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+96	103	Phe-107	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+105	112	Pro-109	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+114	121	Gly-113	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+123	130	Glu-115	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+136	143	His-117	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+152	154	Sa	species	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+155	159	EctC	protein	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
+20	26	Gly-64	residue_name_number	Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices.	RESULTS
+28	35	Pro-109	residue_name_number	Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices.	RESULTS
+41	48	Gly-113	residue_name_number	Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices.	RESULTS
+148	157	β-strands	structure_element	Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices.	RESULTS
+162	171	α-helices	structure_element	Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices.	RESULTS
+147	155	flexible	protein_state	We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein.	RESULTS
+156	172	carboxy-terminus	structure_element	We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein.	RESULTS
+181	183	Sa	species	We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein.	RESULTS
+184	188	EctC	protein	We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein.	RESULTS
+39	45	Glu-57	residue_name_number	As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a).	RESULTS
+47	53	Tyr-85	residue_name_number	As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a).	RESULTS
+59	65	His-93	residue_name_number	As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a).	RESULTS
+91	95	iron	chemical	As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a).	RESULTS
+16	18	Sa	species	In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1).	RESULTS
+19	23	EctC	protein	In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1).	RESULTS
+24	33	structure	evidence	In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1).	RESULTS
+172	214	structure-guided site-directed mutagenesis	experimental_method	In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1).	RESULTS
+14	20	mutant	protein_state	Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations.	RESULTS
+22	24	Sa	species	Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations.	RESULTS
+25	29	EctC	protein	Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations.	RESULTS
+59	66	E. coli	species	Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations.	RESULTS
+83	106	affinity chromatography	experimental_method	Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations.	RESULTS
+36	38	Sa	species	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
+39	43	EctC	protein	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
+58	88	single time-point enzyme assay	experimental_method	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
+125	134	wild-type	protein_state	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
+136	138	Sa	species	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
+139	143	EctC	protein	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
+199	208	N-γ-ADABA	chemical	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
+230	237	ectoine	chemical	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
+31	35	iron	chemical	In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1).	RESULTS
+59	65	mutant	protein_state	In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1).	RESULTS
+67	69	Sa	species	In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1).	RESULTS
+70	74	EctC	protein	In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1).	RESULTS
+88	106	colorimetric assay	experimental_method	In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1).	RESULTS
+23	47	evolutionarily conserved	protein_state	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
+48	54	Trp-21	residue_name_number	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
+56	62	Ser-23	residue_name_number	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
+64	70	Thr-40	residue_name_number	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
+72	79	Cys-105	residue_name_number	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
+85	92	Phe-107	residue_name_number	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
+186	197	semi-closed	protein_state	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
+200	202	Sa	species	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
+203	207	EctC	protein	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
+208	217	structure	evidence	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
+3	11	replaced	experimental_method	We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins.	RESULTS
+43	46	Ala	residue_name	We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins.	RESULTS
+107	111	iron	chemical	We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins.	RESULTS
+127	133	mutant	protein_state	We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins.	RESULTS
+0	6	Thr-40	residue_name_number	Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).	RESULTS
+24	32	β-strand	structure_element	Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).	RESULTS
+33	35	β5	structure_element	Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).	RESULTS
+87	99	cupin barrel	structure_element	Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).	RESULTS
+115	117	Sa	species	Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).	RESULTS
+118	122	EctC	protein	Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).	RESULTS
+8	16	replaced	experimental_method	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
+17	24	Phe-107	residue_name_number	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
+40	43	Tyr	residue_name	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
+50	53	Trp	residue_name	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
+67	78	Phe-107/Tyr	mutant	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
+79	91	substitution	experimental_method	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
+107	116	wild-type	protein_state	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
+158	162	iron	chemical	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
+180	191	Phe-107/Trp	mutant	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
+192	204	substitution	experimental_method	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
+248	252	iron	chemical	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
+277	286	wild-type	protein_state	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
+24	30	mutant	protein_state	The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.	RESULTS
+90	93	107	residue_number	The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.	RESULTS
+98	100	Sa	species	The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.	RESULTS
+101	105	EctC	protein	The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.	RESULTS
+134	146	substitution	experimental_method	The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.	RESULTS
+260	264	iron	chemical	The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.	RESULTS
+0	11	Replacement	experimental_method	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+24	27	Cys	residue_name	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+40	42	Sa	species	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+43	47	EctC	protein	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+49	56	Cys-105	residue_name_number	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+70	73	Ser	residue_name	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+130	143	EctC proteins	protein_type	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+197	199	Sa	species	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+200	204	EctC	protein	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+205	212	variant	protein_state	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+222	231	wild-type	protein_state	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+248	252	iron	chemical	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+284	293	wild-type	protein_state	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
+13	24	Cys-105/Ala	mutant	However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1).	RESULTS
+25	32	variant	protein_state	However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1).	RESULTS
+49	71	catalytically inactive	protein_state	However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1).	RESULTS
+102	106	iron	chemical	However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1).	RESULTS
+25	28	Cys	residue_name	Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase.	RESULTS
+105	112	Cys-105	residue_name_number	Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase.	RESULTS
+117	124	Ser-105	residue_name_number	Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase.	RESULTS
+154	170	ectoine synthase	protein_type	Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase.	RESULTS
+16	40	amino acid substitutions	experimental_method	We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).	RESULTS
+99	103	iron	chemical	We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).	RESULTS
+128	138	Tyr-52/Ala	mutant	We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).	RESULTS
+147	157	His-55/Ala	mutant	We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).	RESULTS
+159	161	Sa	species	We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).	RESULTS
+162	166	EctC	protein	We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).	RESULTS
+14	16	Sa	species	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
+17	21	EctC	protein	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
+22	40	crystal structures	evidence	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
+110	121	replacement	experimental_method	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
+125	131	Tyr-52	residue_name_number	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
+135	138	Ala	residue_name	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
+167	171	iron	chemical	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
+26	36	His-55/Ala	mutant	This is different for the His-55/Ala substitution.	RESULTS
+4	27	carboxy-terminal region	structure_element	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
+36	38	Sa	species	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
+39	43	EctC	protein	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
+98	105	Glu-115	residue_name_number	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
+111	117	His-55	residue_name_number	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
+125	131	His-55	residue_name_number	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
+155	162	Pro-110	residue_name_number	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
+26	57	evolutionarily highly conserved	protein_state	Each of these residues is evolutionarily highly conserved.	RESULTS
+15	27	substitution	experimental_method	The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity.	RESULTS
+38	45	Glu-115	residue_name_number	The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity.	RESULTS
+49	55	His-55	residue_name_number	The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity.	RESULTS
+62	65	Ala	residue_name	The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity.	RESULTS
+103	122	interactive network	site	The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity.	RESULTS
+12	23	Glu-115/Ala	mutant	Indeed, the Glu-115/Ala and the His-55/Ala substitutions possessed only 21% and 16% activity of the wild-type protein, respectively (Table 1).	RESULTS
+32	42	His-55/Ala	mutant	Indeed, the Glu-115/Ala and the His-55/Ala substitutions possessed only 21% and 16% activity of the wild-type protein, respectively (Table 1).	RESULTS
+100	109	wild-type	protein_state	Indeed, the Glu-115/Ala and the His-55/Ala substitutions possessed only 21% and 16% activity of the wild-type protein, respectively (Table 1).	RESULTS
+4	15	Glu-115/Ala	mutant	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
+16	22	mutant	protein_state	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
+33	42	wild-type	protein_state	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
+53	57	iron	chemical	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
+71	75	iron	chemical	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
+91	101	His-55/Ala	mutant	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
+138	147	wild-type	protein_state	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
+8	16	replaced	experimental_method	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
+17	24	Glu-115	residue_name_number	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
+60	63	Asp	residue_name	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
+72	74	Sa	species	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
+75	79	EctC	protein	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
+98	107	wild-type	protein_state	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
+118	122	iron	chemical	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
+150	159	wild-type	protein_state	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
+69	85	carboxy-terminus	structure_element	Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase.	RESULTS
+94	96	Sa	species	Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase.	RESULTS
+97	101	EctC	protein	Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase.	RESULTS
+177	193	ectoine synthase	protein_type	Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase.	RESULTS
+9	15	Leu-87	residue_name_number	Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family.	RESULTS
+20	26	Asp-91	residue_name_number	Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family.	RESULTS
+31	47	highly conserved	protein_state	Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family.	RESULTS
+55	71	ectoine synthase	protein_type	Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family.	RESULTS
+4	15	replacement	experimental_method	The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1).	RESULTS
+19	25	Leu-87	residue_name_number	The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1).	RESULTS
+29	32	Ala	residue_name	The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1).	RESULTS
+16	27	replacement	experimental_method	Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).	RESULTS
+31	37	Asp-91	residue_name_number	Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).	RESULTS
+41	44	Ala	residue_name	Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).	RESULTS
+49	52	Glu	residue_name	Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).	RESULTS
+67	69	Sa	species	Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).	RESULTS
+70	74	EctC	protein	Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).	RESULTS
+56	62	Asp-91	residue_name_number	We currently cannot comment on possible functional role Asp-91.	RESULTS
+9	15	Leu-87	residue_name_number	However, Leu-87 is positioned at the end of one of the β-sheets that form the dimer interface (Fig 5c) and it might therefore possess a structural role.	RESULTS
+55	63	β-sheets	structure_element	However, Leu-87 is positioned at the end of one of the β-sheets that form the dimer interface (Fig 5c) and it might therefore possess a structural role.	RESULTS
+78	93	dimer interface	site	However, Leu-87 is positioned at the end of one of the β-sheets that form the dimer interface (Fig 5c) and it might therefore possess a structural role.	RESULTS
+24	30	Tyr-85	residue_name_number	It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects.	RESULTS
+81	85	iron	chemical	It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects.	RESULTS
+108	110	Sa	species	It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects.	RESULTS
+111	115	EctC	protein	It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects.	RESULTS
+116	127	active site	site	It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects.	RESULTS
+0	7	His-117	residue_name_number	His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1).	RESULTS
+13	31	strictly conserved	protein_state	His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1).	RESULTS
+48	60	substitution	experimental_method	His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1).	RESULTS
+67	70	Ala	residue_name	His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1).	RESULTS
+137	141	iron	chemical	His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1).	RESULTS
+13	20	His-117	residue_name_number	We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase.	RESULTS
+81	83	Sa	species	We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase.	RESULTS
+84	88	EctC	protein	We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase.	RESULTS
+89	98	structure	evidence	We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase.	RESULTS
+178	194	ectoine synthase	protein_type	We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase.	RESULTS
+31	54	mutagenesis experiments	experimental_method	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
+64	75	substituted	experimental_method	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
+76	82	Thr-41	residue_name_number	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
+87	93	His-51	residue_name_number	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
+117	145	not evolutionarily conserved	protein_state	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
+149	167	EctC-type proteins	protein_type	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
+173	176	Ala	residue_name	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
+6	8	Sa	species	Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1).	RESULTS
+9	13	EctC	protein	Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1).	RESULTS
+41	50	wild-type	protein_state	Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1).	RESULTS
+91	95	iron	chemical	Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1).	RESULTS
+125	134	wild-type	protein_state	Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1).	RESULTS
+64	66	Sa	species	This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content.	RESULTS
+67	71	EctC	protein	This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content.	RESULTS
+141	145	iron	chemical	This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content.	RESULTS
+4	25	crystallographic data	evidence	The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.	DISCUSS
+57	73	ectoine synthase	protein_type	The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.	DISCUSS
+75	79	EctC	protein	The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.	DISCUSS
+127	136	microbial	taxonomy_domain	The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.	DISCUSS
+175	182	ectoine	chemical	The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.	DISCUSS
+207	224	cupin superfamily	protein_type	The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.	DISCUSS
+40	42	Sa	species	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
+43	47	EctC	protein	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
+83	92	β-strands	structure_element	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
+94	100	β1-β11	structure_element	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
+110	119	α-helices	structure_element	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (��-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
+121	124	α-I	structure_element	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
+129	133	α-II	structure_element	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
+195	213	crystal structures	evidence	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
+217	223	cupins	protein_type	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
+19	35	ectoine synthase	protein_type	In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.	DISCUSS
+41	59	polyketide cyclase	protein_type	In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.	DISCUSS
+60	64	RemF	protein	In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.	DISCUSS
+99	112	cupin-related	protein_type	In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.	DISCUSS
+190	194	EctC	protein	In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.	DISCUSS
+199	203	RemF	protein	In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.	DISCUSS
+50	54	EctC	protein	As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins.	DISCUSS
+59	63	RemF	protein	As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins.	DISCUSS
+162	180	EctC-type proteins	protein_type	As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins.	DISCUSS
+215	224	microbial	taxonomy_domain	As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins.	DISCUSS
+246	255	RemF-like	protein_type	As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins.	DISCUSS
+4	8	pro-	taxonomy_domain	The pro- and eukaryotic members of the cupin superfamily perform a variety of both enzymatic and non-enzymatic functions that are built upon a common structural scaffold.	DISCUSS
+13	23	eukaryotic	taxonomy_domain	The pro- and eukaryotic members of the cupin superfamily perform a variety of both enzymatic and non-enzymatic functions that are built upon a common structural scaffold.	DISCUSS
+39	56	cupin superfamily	protein_type	The pro- and eukaryotic members of the cupin superfamily perform a variety of both enzymatic and non-enzymatic functions that are built upon a common structural scaffold.	DISCUSS
+5	11	cupins	protein_type	Most cupins contain transition state metals that can promote different types of chemical reactions.	DISCUSS
+16	38	cupin-related proteins	protein_type	Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites.	DISCUSS
+64	82	metallo-chaperones	protein_type	Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites.	DISCUSS
+88	93	bound	protein_state	Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites.	DISCUSS
+94	99	metal	chemical	Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites.	DISCUSS
+138	150	active sites	site	Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites.	DISCUSS
+43	59	ectoine synthase	protein_type	We report here for the first time that the ectoine synthase is a metal-dependent enzyme.	DISCUSS
+65	70	metal	chemical	We report here for the first time that the ectoine synthase is a metal-dependent enzyme.	DISCUSS
+0	6	ICP-MS	experimental_method	ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction.	DISCUSS
+8	54	metal-depletion and reconstitution experiments	experimental_method	ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction.	DISCUSS
+85	89	iron	chemical	ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction.	DISCUSS
+124	129	metal	chemical	ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction.	DISCUSS
+138	142	EctC	protein	ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction.	DISCUSS
+32	38	cupins	protein_type	However, as observed with other cupins, EctC is a somewhat promiscuous enzyme as far as the catalytically important metal is concerned when they are provided in large molar excess (Fig 3c).	DISCUSS
+40	44	EctC	protein	However, as observed with other cupins, EctC is a somewhat promiscuous enzyme as far as the catalytically important metal is concerned when they are provided in large molar excess (Fig 3c).	DISCUSS
+116	121	metal	chemical	However, as observed with other cupins, EctC is a somewhat promiscuous enzyme as far as the catalytically important metal is concerned when they are provided in large molar excess (Fig 3c).	DISCUSS
+114	119	metal	chemical	Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1).	DISCUSS
+132	134	Sa	species	Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1).	DISCUSS
+135	139	EctC	protein	Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1).	DISCUSS
+145	187	structure-guided site-directed mutagenesis	experimental_method	Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1).	DISCUSS
+226	247	iron-binding residues	site	Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1).	DISCUSS
+24	36	metal center	site	The architecture of the metal center of ectoine synthase seems to be subjected to considerable evolutionary constraints.	DISCUSS
+40	56	ectoine synthase	protein_type	The architecture of the metal center of ectoine synthase seems to be subjected to considerable evolutionary constraints.	DISCUSS
+20	26	Glu-57	residue_name_number	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
+28	34	Tyr-85	residue_name_number	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
+36	42	His-93	residue_name_number	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
+88	106	strictly conserved	protein_state	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
+132	150	EctC-type proteins	protein_type	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
+171	180	bacterial	taxonomy_domain	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
+191	199	archaeal	taxonomy_domain	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
+80	89	N-γ-ADABA	chemical	We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig).	DISCUSS
+91	95	EctC	protein	We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig).	DISCUSS
+121	130	N-α-ADABA	chemical	We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig).	DISCUSS
+136	143	ectoine	chemical	We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig).	DISCUSS
+175	195	catalytic efficiency	evidence	We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig).	DISCUSS
+11	22	active site	site	Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules.	DISCUSS
+26	42	ectoine synthase	protein_type	Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules.	DISCUSS
+147	151	EctC	protein	Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules.	DISCUSS
+207	211	ADPC	chemical	Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules.	DISCUSS
+251	260	glutamine	chemical	Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules.	DISCUSS
+17	26	N-α-ADABA	chemical	Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route.	DISCUSS
+53	69	ectoine synthase	protein_type	Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route.	DISCUSS
+123	137	microorganisms	taxonomy_domain	Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route.	DISCUSS
+178	185	ectoine	chemical	Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route.	DISCUSS
+263	272	N-α-ADABA	chemical	Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route.	DISCUSS
+60	62	C6	chemical	Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a).	DISCUSS
+97	99	Sa	species	Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a).	DISCUSS
+100	104	EctC	protein	Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a).	DISCUSS
+105	114	structure	evidence	Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a).	DISCUSS
+204	220	ectoine synthase	protein_type	Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a).	DISCUSS
+122	131	N-γ-ADABA	chemical	We assumed that its location and mode of binding gives, in all likelihood, clues as to the position of the true substrate N-γ-ADABA within the EctC active site.	DISCUSS
+143	147	EctC	protein	We assumed that its location and mode of binding gives, in all likelihood, clues as to the position of the true substrate N-γ-ADABA within the EctC active site.	DISCUSS
+148	159	active site	site	We assumed that its location and mode of binding gives, in all likelihood, clues as to the position of the true substrate N-γ-ADABA within the EctC active site.	DISCUSS
+8	33	site-directed mutagenesis	experimental_method	Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1).	DISCUSS
+112	114	Sa	species	Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1).	DISCUSS
+115	119	EctC	protein	Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1).	DISCUSS
+177	186	wild-type	protein_state	Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1).	DISCUSS
+197	201	iron	chemical	Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1).	DISCUSS
+61	79	strongly conserved	protein_state	This set of data and the fact that the targeted residues are strongly conserved among EctC-type proteins (Fig 2) is consistent with their potential role in N-γ-ADABA binding or enzyme catalysis.	DISCUSS
+86	104	EctC-type proteins	protein_type	This set of data and the fact that the targeted residues are strongly conserved among EctC-type proteins (Fig 2) is consistent with their potential role in N-γ-ADABA binding or enzyme catalysis.	DISCUSS
+156	165	N-γ-ADABA	chemical	This set of data and the fact that the targeted residues are strongly conserved among EctC-type proteins (Fig 2) is consistent with their potential role in N-γ-ADABA binding or enzyme catalysis.	DISCUSS
+30	51	crystallographic data	evidence	We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b).	DISCUSS
+60	91	site-directed mutagenesis study	experimental_method	We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b).	DISCUSS
+176	180	EctC	protein	We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b).	DISCUSS
+181	192	active site	site	We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b).	DISCUSS
+4	20	ectoine synthase	protein_type	The ectoine synthase from the cold-adapted marine bacterium S. alaskensis can be considered as a psychrophilic enzyme (S3a Fig), types of proteins with a considerable structural flexibility.	DISCUSS
+43	59	marine bacterium	taxonomy_domain	The ectoine synthase from the cold-adapted marine bacterium S. alaskensis can be considered as a psychrophilic enzyme (S3a Fig), types of proteins with a considerable structural flexibility.	DISCUSS
+60	73	S. alaskensis	species	The ectoine synthase from the cold-adapted marine bacterium S. alaskensis can be considered as a psychrophilic enzyme (S3a Fig), types of proteins with a considerable structural flexibility.	DISCUSS
+64	82	crystal structures	evidence	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
+90	101	full-length	protein_state	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
+103	105	Sa	species	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
+106	110	EctC	protein	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
+119	134	in complex with	protein_state	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
+142	151	N-γ-ADABA	chemical	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
+155	162	ectoine	chemical	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
+8	17	microbial	taxonomy_domain	Because microbial ectoine producers can colonize ecological niches with rather different physicochemical attributes, it seems promising to exploit this considerable biodiversity to identify EctC proteins with enhanced protein stability.	DISCUSS
+18	25	ectoine	chemical	Because microbial ectoine producers can colonize ecological niches with rather different physicochemical attributes, it seems promising to exploit this considerable biodiversity to identify EctC proteins with enhanced protein stability.	DISCUSS
+190	203	EctC proteins	protein_type	Because microbial ectoine producers can colonize ecological niches with rather different physicochemical attributes, it seems promising to exploit this considerable biodiversity to identify EctC proteins with enhanced protein stability.	DISCUSS
+57	61	EctC	protein	It is hoped that these can be further employed to obtain EctC crystal structures with either the substrate or the reaction product.	DISCUSS
+62	80	crystal structures	evidence	It is hoped that these can be further employed to obtain EctC crystal structures with either the substrate or the reaction product.	DISCUSS
+31	47	ectoine synthase	protein_type	Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction.	DISCUSS
+51	66	metal dependent	protein_state	Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction.	DISCUSS
+74	92	crystal structures	evidence	Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction.	DISCUSS
+168	172	EctC	protein	Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction.	DISCUSS