PMC 20201215 pmc.key 4795551 CC BY no 0 0 Functional and Structural Characterization of Ectoine Synthase 10.1371/journal.pone.0151285 4795551 26986827 PONE-D-15-52796 e0151285 3 This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. surname:Widderich;given-names:Nils surname:Kobus;given-names:Stefanie surname:Höppner;given-names:Astrid surname:Riclea;given-names:Ramona surname:Seubert;given-names:Andreas surname:Dickschat;given-names:Jeroen S. surname:Heider;given-names:Johann surname:Smits;given-names:Sander H. J. surname:Bremer;given-names:Erhard surname:Hofmann;given-names:Andreas All relevant data are within the paper and its Supporting Information files. TITLE Data Availability front 11 2016 0 Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily 0.99961436 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: Crystal Structure 0.9995517 protein_type cleaner0 2023-07-20T10:07:27Z MESH: Ectoine Synthase protein_state DUMMY: cleaner0 2023-07-20T10:06:42Z Metal-Containing 0.9991269 protein_type cleaner0 2023-07-20T10:07:20Z MESH: Cupin Superfamily ABSTRACT abstract 107 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. 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. 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. 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. Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. 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. 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. 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. 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. 0.9922638 chemical cleaner0 2023-07-20T10:07:51Z CHEBI: Ectoine 0.9993475 taxonomy_domain cleaner0 2023-07-20T10:09:01Z DUMMY: Bacteria 0.9993168 taxonomy_domain cleaner0 2023-07-20T10:09:07Z DUMMY: Archaea 0.9973253 protein_type cleaner0 2023-07-20T10:07:29Z MESH: Ectoine synthase 0.9420252 protein_type cleaner0 2023-07-20T10:09:58Z MESH: EctC 0.9979506 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: ectoine 0.999782 chemical cleaner0 2023-07-20T10:08:34Z CHEBI: N-gamma-acetyl-L-2,4-diaminobutyric acid 0.99200696 chemical cleaner0 2023-07-20T14:18:25Z CHEBI: water 0.99961984 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure 0.9996322 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.9996338 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase taxonomy_domain DUMMY: cleaner0 2023-07-20T10:11:50Z marine bacterium 0.9991011 species cleaner0 2023-07-20T10:06:48Z MESH: Sphingopyxis alaskensis 0.9982413 species cleaner0 2023-07-20T10:06:55Z MESH: Sa 0.99890184 protein cleaner0 2023-07-20T10:09:39Z PR: EctC 0.99962497 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure 0.9996493 protein_state cleaner0 2023-07-20T10:12:17Z DUMMY: apo 0.99915904 experimental_method cleaner0 2023-07-20T14:28:34Z MESH: Structural analysis species MESH: cleaner0 2023-07-20T10:10:28Z Sa 0.50138265 protein cleaner0 2023-07-20T10:09:38Z PR: EctC 0.98862517 protein_type cleaner0 2023-07-20T10:07:21Z MESH: cupin superfamily 0.911944 protein cleaner0 2023-07-20T10:09:39Z PR: EctC 0.9992981 oligomeric_state cleaner0 2023-07-20T10:12:02Z DUMMY: dimer 0.99934924 protein_state cleaner0 2023-07-20T10:12:22Z DUMMY: head-to-tail 0.9996296 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure 0.9992674 site cleaner0 2023-07-20T14:38:53Z SO: interface 0.99930906 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:19Z hydrophobic interactions 0.999515 structure_element cleaner0 2023-07-20T14:39:50Z SO: beta-sheets 0.99926466 oligomeric_state cleaner0 2023-07-20T10:12:08Z DUMMY: monomer 0.99964887 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.99949694 experimental_method cleaner0 2023-07-20T14:18:33Z MESH: metal depletion and reconstitution experiments 0.8798571 protein cleaner0 2023-07-20T10:09:39Z PR: EctC protein_state DUMMY: cleaner0 2023-07-20T10:11:01Z iron-dependent 0.8113488 protein cleaner0 2023-07-20T10:09:39Z PR: EctC 0.99978054 chemical cleaner0 2023-07-20T10:08:36Z CHEBI: N-gamma-acetyl-L-2,4-diaminobutyric acid 0.99935395 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: ectoine 0.9997851 chemical cleaner0 2023-07-20T14:19:09Z CHEBI: N-alpha-acetyl-L-2,4-diaminobutyric acid evidence DUMMY: cleaner0 2023-07-20T11:31:32Z catalytic efficiency 0.99946934 experimental_method cleaner0 2023-07-20T14:18:52Z MESH: Structure-guided site-directed mutagenesis 0.99933404 protein_state cleaner0 2023-07-20T14:46:26Z DUMMY: evolutionarily highly conserved protein_type MESH: cleaner0 2023-07-20T10:11:19Z EctC protein family 0.9996268 site cleaner0 2023-07-20T13:28:16Z SO: iron-binding site 0.77332145 protein cleaner0 2023-07-20T10:09:39Z PR: EctC chemical CHEBI: cleaner0 2023-07-20T11:22:00Z iron 0.9996016 site cleaner0 2023-07-20T13:43:14Z SO: active site 0.9987109 protein cleaner0 2023-07-20T10:09:39Z PR: EctC 0.9990552 structure_element cleaner0 2023-07-20T13:33:43Z SO: cupin barrel INTRO title_1 2268 Introduction INTRO paragraph 2281 Compatible solutes are exploited by members of all three domains of life as versatile cyto-protectants, in particular against cellular stress elicited by high osmolarity environments. They are especially useful for this latter purpose since their benign nature allows their amassing to exceedingly high cellular concentrations. As a result of compatible solute accumulation, dehydration of the cytoplasm of osmotically stressed cells is counteracted, and concomitantly, its solvent properties are optimized for the functioning of vital biochemical and physiological processes. INTRO paragraph 2858 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. Both marine and terrestrial microorganisms produce them widely in response to osmotic or temperature stress. 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). The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers. 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. 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. 0.99967897 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: Ectoine 0.99959415 chemical cleaner0 2023-07-20T10:12:52Z CHEBI: (S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid 0.99977165 chemical cleaner0 2023-07-20T10:13:04Z CHEBI: 5-hydroxyectoine 0.99902165 chemical cleaner0 2023-07-20T10:13:09Z CHEBI: (4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid taxonomy_domain DUMMY: cleaner0 2023-07-20T10:19:43Z marine and terrestrial microorganisms 0.99904186 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: ectoine 0.9997767 chemical cleaner0 2023-07-20T10:13:13Z CHEBI: L-aspartate-ß-semialdehyde 0.99959826 protein_type cleaner0 2023-07-20T10:13:35Z MESH: L-2,4-diaminobutyrate transaminase 0.9996469 protein_type cleaner0 2023-07-20T10:16:02Z MESH: EctB 0.999636 protein_type cleaner0 2023-07-20T10:13:38Z MESH: 2,4-diaminobutyrate acetyltransferase 0.9996762 protein_type cleaner0 2023-07-20T10:16:26Z MESH: EctA protein_type MESH: cleaner0 2023-07-20T10:07:29Z ectoine synthase 0.99961436 protein_type cleaner0 2023-07-20T10:16:38Z MESH: EctC 0.99946684 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: ectoine 0.99977547 chemical cleaner0 2023-07-20T10:13:18Z CHEBI: 5-hydroxyectoine 0.5892295 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: ectoine 0.99958915 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: ectoine 0.99327844 protein_type cleaner0 2023-07-20T14:16:45Z MESH: ectoine hydroxylase 0.99969697 protein_type cleaner0 2023-07-20T10:16:49Z MESH: EctD 0.9819392 protein_type cleaner0 2023-07-20T10:16:54Z MESH: non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily 0.9726849 chemical cleaner0 2023-07-20T10:16:59Z CHEBI: ectoines pone.0151285.g001.jpg pone.0151285.g001 FIG fig_title_caption 4127 Biosynthetic routes for ectoine and 5-hydroxyectoine. 0.99978477 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: ectoine 0.9997826 chemical cleaner0 2023-07-20T10:13:18Z CHEBI: 5-hydroxyectoine pone.0151285.g001.jpg pone.0151285.g001 FIG fig_caption 4181 Scheme of the ectoine and 5-hydroxyectoine biosynthetic pathway. 0.9997751 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: ectoine 0.99978995 chemical cleaner0 2023-07-20T10:13:18Z CHEBI: 5-hydroxyectoine INTRO paragraph 4246 Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1). 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. 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). 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. 0.9990746 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.9434349 protein cleaner0 2023-07-20T10:09:39Z PR: EctC 0.99730337 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: ectoine 0.9768098 experimental_method cleaner0 2023-07-20T14:28:42Z MESH: characterizations 0.9996544 protein_type cleaner0 2023-07-20T10:17:22Z MESH: ectoine synthases 0.9770065 taxonomy_domain cleaner0 2023-07-20T10:17:57Z DUMMY: extremophiles 0.9993757 species cleaner0 2023-07-20T10:17:30Z MESH: Halomonas elongata 0.99943984 species cleaner0 2023-07-20T10:17:36Z MESH: Methylomicrobium alcaliphilum 0.9993987 species cleaner0 2023-07-20T10:17:42Z MESH: Acidiphilium cryptum taxonomy_domain DUMMY: cleaner0 2023-07-20T10:18:19Z nitrifying archaeon 0.99944043 species cleaner0 2023-07-20T10:17:47Z MESH: Nitrosopumilus maritimus 0.99977577 chemical cleaner0 2023-07-20T10:18:25Z CHEBI: N-γ-acetyl-L-2,4-diaminobutyric acid 0.9997686 chemical cleaner0 2023-07-20T10:18:34Z CHEBI: N-γ-ADABA 0.9994516 protein_type cleaner0 2023-07-20T10:18:29Z MESH: 2,4-diaminobutyrate acetyltransferase 0.9980604 protein_type cleaner0 2023-07-20T10:19:02Z MESH: EctA 0.99930656 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: ectoine 0.9997781 chemical cleaner0 2023-07-20T14:19:14Z CHEBI: water 0.9992575 protein cleaner0 2023-07-20T10:09:39Z PR: EctC 0.9997859 chemical cleaner0 2023-07-20T10:18:43Z CHEBI: 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate 0.99978095 chemical cleaner0 2023-07-20T10:18:48Z CHEBI: ADPC 0.9978382 chemical cleaner0 2023-07-20T10:19:06Z CHEBI: glutamine 0.99942786 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: ectoine 0.9993905 chemical cleaner0 2023-07-20T10:07:52Z CHEBI: ectoine INTRO paragraph 5189 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). 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. The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily. 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. 0.99968475 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.7485945 evidence cleaner0 2023-07-20T14:10:36Z DUMMY: structure 0.8181996 experimental_method cleaner0 2023-07-20T14:28:46Z MESH: biochemically characterized 0.99963343 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase taxonomy_domain DUMMY: cleaner0 2023-07-20T10:19:51Z marine bacterium 0.9990964 species cleaner0 2023-07-20T10:06:50Z MESH: Sphingopyxis alaskensis 0.9987779 species cleaner0 2023-07-20T10:06:56Z MESH: Sa 0.9996583 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.99915826 chemical cleaner0 2023-07-20T11:22:00Z CHEBI: iron 0.9993026 protein cleaner0 2023-07-20T10:09:39Z PR: EctC 0.99935967 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer 0.99945956 experimental_method cleaner0 2023-07-20T14:28:50Z MESH: structural analysis 0.99953306 protein_type cleaner0 2023-07-20T10:07:21Z MESH: cupin superfamily 0.99961185 evidence cleaner0 2023-07-20T13:57:50Z DUMMY: crystal structures species MESH: cleaner0 2023-07-20T10:06:56Z Sa 0.9697854 protein cleaner0 2023-07-20T10:09:39Z PR: EctC 0.9992135 experimental_method cleaner0 2023-07-20T14:28:53Z MESH: site-directed mutagenesis 0.9993755 protein_state cleaner0 2023-07-20T14:40:00Z DUMMY: evolutionarily highly conserved 0.9995388 protein_type cleaner0 2023-07-20T10:20:35Z MESH: EctC protein 0.9978783 site cleaner0 2023-07-20T14:39:00Z SO: catalytic core 0.9996662 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase METHODS title_1 6216 Materials and Methods METHODS title_2 6238 Chemicals METHODS paragraph 6248 Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] was a kind gift from bitop AG (Witten, Germany). Anhydrotetracycline (AHT), desthiobiotine and the strepavidin affinity matrix for the purification of Strep-tag II labeled proteins was purchased from IBA GmbH (Göttingen, Germany). Hydroxylamine and phenanthroline for the photometric determination of the iron-content of the recombinant (Sa)EctC proteins were purchased from Sigma-Aldrich (München, Germany). METHODS title_2 6729 Synthesis of N-γ-acetyl-L-2,4-diaminobutyric acid and N-α-acetyl-L-2,4-diaminobutyric acid through hydrolysis of ectoine METHODS paragraph 6858 All chemicals used to synthesize the gamma and alpha forms of N-acetyl-l-2,4-diaminobutyric acid (ADABA) for EctC enzyme activity assays were purchased either from Sigma Aldrich (Steinheim, Germany), or Acros (Geel, Belgium). Alkaline hydrolysis of ectoine (284 mg, 2.0 mmol) was accomplished in aqueous KOH (50 mL, 0.1 M) for 20 h at 50°C. The reaction mixture was subsequently neutralized with perchloric acid (60% in water, 4 mL) and the precipitated potassium perchlorate was filtered off. Subsequently, the filtrate was concentrated under reduced pressure. Purification of the residue and separation of the formed compounds was then performed by repeated chromatography on a silica gel column (Merck silica gel 60) using a gradient of ethanol/25% ammonia/water 50:1:2–10:1:2 as eluent to yield pure N-γ-ADABA (192 mg, 1.20 mmol, 60%) and N-α-ADABA (32 mg, 0.20 mmol, 10%). The identity and purity of theses compounds was unequivocally established by thin-layer chromatography (TLC) and nuclear magnetic resonance (1H-NMR and 13C-NMR) spectroscopy (S1a and S1b Fig) as described on a Bruker AVIII-400 or DRX-500 NMR spectrometer. (i) Analytical data for N-γ-ADABA: TLC: Rf = 0.55 (ethanol/25% ammonia/water 7:1:2); 1H-NMR (400 MHz, D2O): δ = 3.71 (dd, 3J(H,H) = 7.6 Hz, 3J(H,H) = 5.6 Hz, 1H, CH), 3.41–3.24 (m, 2H, CH2), 2.15–2.01 (m, 2H, CH2), 1.99 (s, 3H, CH3) ppm; 13C-NMR (100 MHz, D2O): δ = 177.5 (CO), 177.0 (COOH), 55.3 (CH), 38.3 (CH2), 33.0 (CH2), 24.6 (CH3) ppm. (ii) Analytical data for N-α-ADABA: TLC: Rf = 0.38 (ethanol/25% ammonia/water 7:1:2); 1H-NMR (400 MHz, D2O): δ = 4.24 (dd, 3J(H,H) = 8.8 Hz, 3J(H,H) = 5.1 Hz, 1H, CH), 3.07–3.02 (m, 2H, CH2), 2.22–2.11 (m, 2H, CH2), 2.04 (s, 3H, CH3) ppm; 13C-NMR (100 MHz, D2O): δ = 180.1 (CO), 176.7 (COOH), 55.4 (CH), 39.6 (CH2), 32.5 (CH2), 24.7 (CH3) ppm. METHODS title_2 8711 Bacterial strains, plasmids and media METHODS paragraph 8749 The nucleotide sequence of the ectC gene from S. alaskensis (genome accession number: NC_008048) was used as a template to obtain a codon-optimized ectC DNA sequence (Life Technologies, Darmstadt, Germany) for its expression in E. coli. The nucleotide sequence of the synthetic ectC gene was deposited in the NCBI database under accession number KR002036. The synthetic ectC gene was used to construct an expression plasmid (pNW12) that is based on the pASG-IBA3 vector (IBA GmbH, Göttingen, Germany). In plasmid pNW12, the ectC gene is fused at its 3’ end to a short open reading frame encoding a Strep-tag II affinity peptide (NWSHPQFEK). It is transcribed from the TetR-controlled tet promoter carried by the backbone of the pASG-IBA3 expression vector. De-repression of tet promoter activity can be triggered by adding the synthetic inducer AHT for the TetR repressor to the growth medium. The details of the construction of pNW12 have been reported. METHODS paragraph 9707 Plasmids carrying ectC genes were routinely maintained in the Escherichia coli strain DH5α (Invitrogen, Karlsruhe, Germany) on LB agar plates containing ampicillin (100 μg ml-1). Plasmid DNA was isolated by routine procedures. Minimal medium A (MMA) containing 0.5% (w/v) glucose as the carbon source, 0.5% (w/v) casamino acids, 1 mM MgSO4, and 3 mM thiamine was used to cultivate the E. coli strain BL21 carrying pNW12 for the overproduction of the (Sa)EctC protein and its mutant derivatives. No additional metal solution was added to the components of the original recipe of MMA. METHODS title_2 10295 Site-directed mutagenesis of the ectC gene METHODS paragraph 10338 Variants of the codon-optimized ectC gene from S. alaskensis present on plasmid pNW12 were prepared by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, Waldbronn, Germany) with custom synthesized DNA primers purchased from Microsynth AG (Lindau, Germany). The DNA sequence of the entire coding region of each mutant ectC gene was determined by Eurofins MWG (Ebersberg, Germany) to ensure the presence of the desired mutation and the absence of unwanted alterations. Details on the genetic changes introduced into ectC genes are listed in Table 1. pone.0151285.t001.xml pone.0151285.t001 TABLE table_title_caption 10933 Conversion of N-γ-ADABA into ectoine by (Sa)EctC mutant derivatives and their iron-content. pone.0151285.t001.xml pone.0151285.t001 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><thead><tr><th align="center" rowspan="1" colspan="1">Mutation</th><th align="center" rowspan="1" colspan="1">Amino acid-substitution</th><th align="center" rowspan="1" colspan="1">Ectoine formed [mM]</th><th align="center" rowspan="1" colspan="1">Activity (%)</th><th align="center" rowspan="1" colspan="1">Iron content of the protein preparation (mol %)</th></tr></thead><tbody><tr><td align="center" rowspan="1" colspan="1">WT</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">9.33 ± 0.28</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">92.1 ± 3.4</td></tr><tr><td align="left" rowspan="1" colspan="1">TAT/GCT</td><td align="left" rowspan="1" colspan="1">Tyr-52/Ala</td><td align="center" rowspan="1" colspan="1">2.53 ± 0.18</td><td align="center" rowspan="1" colspan="1">27</td><td align="center" rowspan="1" colspan="1">19.8 ± 1.7</td></tr><tr><td align="center" rowspan="1" colspan="1">GAA/GCA</td><td align="left" rowspan="1" colspan="1">Glu-57/Ala</td><td align="center" rowspan="1" colspan="1">0.97 ± 0.16</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">4.3 ± 2.9</td></tr><tr><td align="center" rowspan="1" colspan="1">GAA/GAT</td><td align="left" rowspan="1" colspan="1">Glu-57/Asp</td><td align="center" rowspan="1" colspan="1">6.19 ± 0.42</td><td align="center" rowspan="1" colspan="1">66</td><td align="center" rowspan="1" colspan="1">68.7 ± 5.1</td></tr><tr><td align="left" rowspan="1" colspan="1">TAT/GCT</td><td align="left" rowspan="1" colspan="1">Tyr-85/Ala</td><td align="center" rowspan="1" colspan="1">2.15 ± 0.28</td><td align="center" rowspan="1" colspan="1">23</td><td align="center" rowspan="1" colspan="1">8.3 ± 2.3</td></tr><tr><td align="left" rowspan="1" colspan="1">TAT/TTT</td><td align="left" rowspan="1" colspan="1">Tyr-85/Phe</td><td align="center" rowspan="1" colspan="1">2.74± 0.50</td><td align="center" rowspan="1" colspan="1">29</td><td align="center" rowspan="1" colspan="1">9.4 ± 3.9</td></tr><tr><td align="left" rowspan="1" colspan="1">TAT/TGG</td><td align="left" rowspan="1" colspan="1">Tyr-85/Trp</td><td align="center" rowspan="1" colspan="1">0.95 ± 0.08</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">5.1 ± 1.7</td></tr><tr><td align="left" rowspan="1" colspan="1">CAT/GCT</td><td align="left" rowspan="1" colspan="1">His-93/Ala</td><td align="center" rowspan="1" colspan="1">0.72 ± 0.09</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">4.5 ± 0.8</td></tr><tr><td align="left" rowspan="1" colspan="1">CAT/AAT</td><td align="left" rowspan="1" colspan="1">His-93/Asn</td><td align="center" rowspan="1" colspan="1">2.14 ± 0.31</td><td align="center" rowspan="1" colspan="1">23</td><td align="center" rowspan="1" colspan="1">12.9 ± 2.6</td></tr><tr><td align="left" rowspan="1" colspan="1">TGG/GCG</td><td align="left" rowspan="1" colspan="1">Trp-21/Ala</td><td align="center" rowspan="1" colspan="1">2.41 ± 0.39</td><td align="center" rowspan="1" colspan="1">26</td><td align="center" rowspan="1" colspan="1">89.4 ± 4.5</td></tr><tr><td align="left" rowspan="1" colspan="1">ACG/GCC</td><td align="left" rowspan="1" colspan="1">Ser-23/Ala</td><td align="center" rowspan="1" colspan="1">1.98 ± 0.42</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">91.6 ± 2.8</td></tr><tr><td align="left" rowspan="1" colspan="1">ACC/GCC</td><td align="left" rowspan="1" colspan="1">Thr-40/Ala</td><td align="center" rowspan="1" colspan="1">1.12 ± 0.13</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">89.6 ± 2.2</td></tr><tr><td align="left" rowspan="1" colspan="1">TGT/GCT</td><td align="left" rowspan="1" colspan="1">Cys-105/Ala</td><td align="center" rowspan="1" colspan="1">0.96 ± 0.21</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">90.1 ± 1.6</td></tr><tr><td align="left" rowspan="1" colspan="1">TGT/TCT</td><td align="left" rowspan="1" colspan="1">Cys-105/Ser</td><td align="center" rowspan="1" colspan="1">7.81 ± 0.65</td><td align="center" rowspan="1" colspan="1">84</td><td align="center" rowspan="1" colspan="1">88.7 ± 3.1</td></tr><tr><td align="left" rowspan="1" colspan="1">TTT/GCT</td><td align="left" rowspan="1" colspan="1">Phe-107/Ala</td><td align="center" rowspan="1" colspan="1">4.77 ± 0.10</td><td align="center" rowspan="1" colspan="1">51</td><td align="center" rowspan="1" colspan="1">87.9 ± 2.2</td></tr><tr><td align="left" rowspan="1" colspan="1">TTT/TAT</td><td align="left" rowspan="1" colspan="1">Phe-107/Tyr</td><td align="center" rowspan="1" colspan="1">8.87 ± 0.62</td><td align="center" rowspan="1" colspan="1">95</td><td align="center" rowspan="1" colspan="1">90.9 ± 3.9</td></tr><tr><td align="left" rowspan="1" colspan="1">TTT/TGG</td><td align="left" rowspan="1" colspan="1">Phe-107/Trp</td><td align="center" rowspan="1" colspan="1">1.08 ± 0.27</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">72.6 ± 5.8</td></tr><tr><td align="left" rowspan="1" colspan="1">CAT/GCT</td><td align="left" rowspan="1" colspan="1">His-117/Ala</td><td align="center" rowspan="1" colspan="1">4.14 ± 0.27</td><td align="center" rowspan="1" colspan="1">44</td><td align="center" rowspan="1" colspan="1">82.9 ± 1.1</td></tr><tr><td align="left" rowspan="1" colspan="1">CAT/GCT</td><td align="left" rowspan="1" colspan="1">His-55/Ala</td><td align="center" rowspan="1" colspan="1">1.53 ± 0.19</td><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1">15.4 ± 4.3</td></tr><tr><td align="left" rowspan="1" colspan="1">GAA/GCA</td><td align="center" rowspan="1" colspan="1">Glu-115/Ala</td><td align="center" rowspan="1" colspan="1">1.92 ± 0.44</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">87.6 ± 4.4</td></tr><tr><td align="left" rowspan="1" colspan="1">GAA/GAT</td><td align="center" rowspan="1" colspan="1">Glu-115/Asp</td><td align="center" rowspan="1" colspan="1">7.15 ± 0.60</td><td align="center" rowspan="1" colspan="1">77</td><td align="center" rowspan="1" colspan="1">88.0 ± 3.2</td></tr><tr><td align="left" rowspan="1" colspan="1">CTG/GCG</td><td align="left" rowspan="1" colspan="1">Leu-87/Ala</td><td align="center" rowspan="1" colspan="1">5.81 ± 0.44</td><td align="center" rowspan="1" colspan="1">62</td><td align="center" rowspan="1" colspan="1">92.0 ± 1.3</td></tr><tr><td align="left" rowspan="1" colspan="1">GAT/GCT</td><td align="left" rowspan="1" colspan="1">Asp-91/Ala</td><td align="center" rowspan="1" colspan="1">7.48 ± 0.81</td><td align="center" rowspan="1" colspan="1">80</td><td align="center" rowspan="1" colspan="1">89.6 ± 2.2</td></tr><tr><td align="left" rowspan="1" colspan="1">GAT/GAA</td><td align="left" rowspan="1" colspan="1">Asp-91/Glu</td><td align="center" rowspan="1" colspan="1">9.13 ± 0.57</td><td align="center" rowspan="1" colspan="1">98</td><td align="center" rowspan="1" colspan="1">90.0 ± 1.9</td></tr><tr><td align="left" rowspan="1" colspan="1">ACC/GCC</td><td align="left" rowspan="1" colspan="1">Thr-41/Ala</td><td align="center" rowspan="1" colspan="1">8.84 ± 0.63</td><td align="center" rowspan="1" colspan="1">95</td><td align="center" rowspan="1" colspan="1">91.7 ± 1.9</td></tr><tr><td align="left" rowspan="1" colspan="1">CAT/GCT</td><td align="left" rowspan="1" colspan="1">His-51/Ala</td><td align="center" rowspan="1" colspan="1">8.94 ± 0.47</td><td align="center" rowspan="1" colspan="1">96</td><td align="center" rowspan="1" colspan="1">90.2 ± 2.6</td></tr></tbody></table> 11029 Mutation Amino acid-substitution Ectoine formed [mM] Activity (%) Iron content of the protein preparation (mol %) WT - 9.33 ± 0.28 100 92.1 ± 3.4 TAT/GCT Tyr-52/Ala 2.53 ± 0.18 27 19.8 ± 1.7 GAA/GCA Glu-57/Ala 0.97 ± 0.16 10 4.3 ± 2.9 GAA/GAT Glu-57/Asp 6.19 ± 0.42 66 68.7 ± 5.1 TAT/GCT Tyr-85/Ala 2.15 ± 0.28 23 8.3 ± 2.3 TAT/TTT Tyr-85/Phe 2.74± 0.50 29 9.4 ± 3.9 TAT/TGG Tyr-85/Trp 0.95 ± 0.08 10 5.1 ± 1.7 CAT/GCT His-93/Ala 0.72 ± 0.09 8 4.5 ± 0.8 CAT/AAT His-93/Asn 2.14 ± 0.31 23 12.9 ± 2.6 TGG/GCG Trp-21/Ala 2.41 ± 0.39 26 89.4 ± 4.5 ACG/GCC Ser-23/Ala 1.98 ± 0.42 21 91.6 ± 2.8 ACC/GCC Thr-40/Ala 1.12 ± 0.13 12 89.6 ± 2.2 TGT/GCT Cys-105/Ala 0.96 ± 0.21 10 90.1 ± 1.6 TGT/TCT Cys-105/Ser 7.81 ± 0.65 84 88.7 ± 3.1 TTT/GCT Phe-107/Ala 4.77 ± 0.10 51 87.9 ± 2.2 TTT/TAT Phe-107/Tyr 8.87 ± 0.62 95 90.9 ± 3.9 TTT/TGG Phe-107/Trp 1.08 ± 0.27 12 72.6 ± 5.8 CAT/GCT His-117/Ala 4.14 ± 0.27 44 82.9 ± 1.1 CAT/GCT His-55/Ala 1.53 ± 0.19 16 15.4 ± 4.3 GAA/GCA Glu-115/Ala 1.92 ± 0.44 21 87.6 ± 4.4 GAA/GAT Glu-115/Asp 7.15 ± 0.60 77 88.0 ± 3.2 CTG/GCG Leu-87/Ala 5.81 ± 0.44 62 92.0 ± 1.3 GAT/GCT Asp-91/Ala 7.48 ± 0.81 80 89.6 ± 2.2 GAT/GAA Asp-91/Glu 9.13 ± 0.57 98 90.0 ± 1.9 ACC/GCC Thr-41/Ala 8.84 ± 0.63 95 91.7 ± 1.9 CAT/GCT His-51/Ala 8.94 ± 0.47 96 90.2 ± 2.6 pone.0151285.t001.xml pone.0151285.t001 TABLE table_footnote 12408 The conversion of N-γ-ADABA into ectoine by the (Sa)EctC protein and its mutant derivatives was monitored in a reaction that contained 10 mM N-γ-ADABA as the substrate, 1 mM FeSO4 and 5 μg of the EctC protein under study. The amount of ectoine formed was measured after 20 min of incubation of the enzyme-substrate mixture by HPLC analysis. The iron-content of the investigated protein preparations was determined photometrically; note that in comparison with data obtained via ICP-MS, the colorimetric assay overestimates somewhat the iron content of the (Sa)EctC protein preparations. METHODS title_2 13004 Overproduction and purification of recombinant EctC proteins METHODS paragraph 13065 For the overproduction of the (Sa)EctC-Strep-tag II protein, an overnight culture of strain [BL21 (pNW12)] was prepared in MMA and used to inoculate 1 L of MMA (in a 2 L Erlenmeyer flask) to an OD578 of 0.05. The cells were grown on an aerial shaker (set to 180 rpm) at 37°C until the culture reached an OD578 of 0.5. At this time point, the growth temperature was lowered to 30°C and the speed of the shaker was reduced to 100 rpm. Growth of the culture was continued and when it reached an OD578 of 0.7, AHT was added to the growth medium at a final concentration of 0.2 mg ml-1 to boost expression of the recombinant ectC gene. After 2 h of further incubation of the culture, the E. coli cells were harvested by centrifugation and disrupted by passing them several times through a French Pressure cell; a cleared cell lysate was prepared by ultracentrifugation (100 000 g) at 4°C for 1 h. The supernatant of this cleared lysate was then passed through a column filled with 5 ml of Strep-Tactin Superflow material (IBA GmbH, Göttingen, Germany); the column had been equilibrated with a buffer containing 200 mM NaCl and 20 mM Tris-HCl (pH 8). The (Sa)EctC-Strep-tag II protein was eluted from the affinity matrix with three column volumes of the same buffer containing 2.5 mM desthiobiotin. The recombinant (Sa)EctC-Strep-tag II protein was then concentrated to either 5 mg ml-1 for enzymes assays or 10 mg ml-1 for crystallization trials with Vivaspin 6 columns (Satorius Stedim Biotech GmbH, Göttingen, Germany) in the same buffer as described above. Desthiobiotin was not removed by dialysis from these protein preparations. The purified and concentrated (Sa)EctC-Strep-tag II protein was either used immediately for enzymes assays or kept at 4°C since the flash-freezing of the protein with liquid nitrogen and its subsequent storage at -80°C resulted in a rapid inactivation of ectoine synthase activity. 25 Variants of the (Sa)EctC-Strep-tag II protein carrying singe amino acid substitutions (Table 1) were overproduced and purified using the same procedure employed for the isolation of the wild-type protein. These mutant proteins behaved like the wild-type (Sa)EctC-Strep-tag II protein during the overproduction and purification procedure. Protein concentrations were determined both with a Pierce BCA Protein Assay Kit (Thermo Scientific, Schwerte, Germany) using BSA as the standard protein and spectrophotometrically by using an extinction coefficient of 15 470 M-1 cm-1 for the (Sa)EctC-Strep-tag II protein at a wavelength of 280 nm. The purity and integrity of the isolated (Sa)EctC-Strep-tag II proteins was inspected by SDS-polyacrylamide (15%) gel electrophoresis (SDS-PAGE). Molecular mass marker proteins for SDS-PAGE were purchased from LifeTechnologies (Darmstadt, Germany). METHODS title_2 15873 Ectoine synthase enzyme activity assays METHODS paragraph 15913 The ectoine synthase activity of the (Sa)EctC protein was determined by HPLC-based enzyme assays. The initial enzyme activity assays were performed in a 30 μl-reaction volume for 20 min at 20°C. The used standard buffer (20 mM Tris, pH 8.0) contained 150 mM NaCl, 1 mM FeCl2, and 10 mM N-γ-ADABA. To determine optimal enzyme assay conditions for the (Sa)EctC-Strep-tag II protein, assay parameters and buffer conditions (e.g., the salt-concentrations, temperature, pH) were individually changed. The finally optimized assay buffer for ectoine synthase activity of the (Sa)EctC protein contained 20 mM Tris (pH 8.5), 200 mM NaCl, 1 mM FeCl2 and 10 mM N-γ-ADABA. Activity assays were run for 20 min at 15°C. Usually, 10 μg of the purified (Sa)EctC protein were added to start the enzyme assay. To assess the kinetic parameters of the ectoine synthase, varying concentrations of the substrates were used in the optimized assay buffer with a constant amount (10 μg) of the (Sa)EctC protein. The concentration of the natural EctC substrate N-γ-ADABA was varied between 0 and 40 mM, whereas that of N-α-ADABA was varied between 0 and 200 mM in the enzyme assays. Enzyme reactions were stopped by adding 30 μl of acetonitrile (100%) to the reaction vessel. The samples were centrifuged (13000 rpm, at room temperature for 5 min) to remove denatured proteins; the supernatant was subsequently analyzed for the formation of ectoine by HPLC analysis. Usually, 5- to 10-μl samples were injected into the HPLC system and the reaction product ectoine was analytically detected on a GROM-SIL Amino-1PR column (125 x 4 mm with a particle size of 3μm; purchased from GROM, Rottenburg-Hailfingen, Germany). Synthesis of ectoine by the purified (Sa)EctC-Strep-tag II protein and its mutant derivatives was monitored using a Infinity 1260 Diode Array Detector (DAD) (Agilent, Waldbronn, Germany) integrated into an Agilent 1260 Infinity LC system (Agilent). The ectoine content of the samples was quantified using the OpenLAB software suite (Agilent). The data shown for each ectC mutant (Table 1) were derived from two independent (Sa)EctC preparations, and each (Sa)EctC protein solution was assayed three times for its enzyme activity. METHODS title_2 18155 Metal depletion and reconstitution of the (Sa)EctC protein METHODS paragraph 18214 To assess the dependency of the ectoine synthase for its enzyme activity on iron and other metals, purified and concentrated (Sa)EctC protein preparations (10 μM) were treated with different concentrations of EDTA for 10 minutes. They were subsequently dialyzed to remove the EDTA and the remaining (Sa)EctC enzyme activity was analyzed. To determine metal ion specificity of the ectoine synthase, 500 μl of the (Sa)EctC protein (100 μM) were initially treated with 1 mM EDTA for 10 minutes to obtain apo-(Sa)EctC protein preparations and the EDTA was then removed by dialysis. Enzyme activity assays with 10 μM of such protein preparations were then performed in the presence of either stoichiometric (10 μM) or excess amounts (1 mM) of FeCl2, FeCl3, ZnCl2, CoCl2, NiCl2, CuCl2, and MnCl2 to monitor metal ion specificity of the ectoine synthase. Prior to initiation of the enzyme reaction (by addition of the substrate), the (Sa)EctC protein solution was incubated with the different indicated metal ions for 10 minutes. METHODS title_2 19242 Determination of the oligomeric state of (Sa)EctC protein METHODS paragraph 19300 To determine the oligomeric state of the (Sa)EctC protein in solution, we used high-performance liquid chromatography coupled to multi-angle light scattering detection (HPLC-MALS). A Bio SEC-5 HPLC column (Agilent Technologies Deutschland GmbH, Böblingen, Germany) with a pore size of 300 Å was equilibrated with 20 mM Tris-HCl (pH 7.5), 200 mM NaCl for high-performance liquid chromatography analysis. For these experiments, an Agilent Technologies system connected to a triple-angle light scattering detector (miniDAWN TREOS, Wyatt Technology Europe GmbH, Dernbach, Germany) followed by a differential refractive index detection system (Optilab t-rEX, Wyatt Technology) was used. Typically, 100 μl of purified (Sa)EctC protein (2 mg ml-1) was loaded onto the Bio SEC-5 HPLC column and the obtained data were analyzed with the ASTRA software package (Wyatt Technology). METHODS title_2 20174 Determination of metal content of recombinant (Sa)EctC protein by ICP-MS METHODS paragraph 20247 The elemental contents of P, Fe, Ni, Cu and Zn of the (Sa)EctC-Strep-Tag-II protein sample were determined by inductive-coupled plasma mass spectrometry (ICP-MS) using an Agilent 7900 ICP-MS system equipped with a HEN nebulizer and cooled scott spray chamber under standard operating conditions. The isotopes 31P, 56Fe, 57Fe, 58Ni, 60Ni, 62Ni, 63Cu, 65Cu, 64Zn, 66Zn, and 67Zn were measured under NoGas, He collision and H2 reaction mode conditions. Some isotopes are strongly interfered from the matrix (mainly 56Fe, 63Cu) in the NoGas mode and are therefore rejected. The (Sa)EctC protein samples and buffer blanks were diluted 100-fold with ultra pure water and spiked with 10 μg kg-1 Y as the internal standard. The calibration of the ICP-MS was performed in the concentration range between 0.1 to 100 μg kg-1 using a homemade P standard solution prepared from titrimetrically analyzed H3PO4 solution and from dilutions of a Merck ICP multi-element standard solution IV (Merck No. 111355, Darmstadt, Germany). METHODS title_2 21263 Photometric determination of non-heme-iron in (Sa)EctC and its mutant derivatives METHODS paragraph 21345 To determine the iron content in our (Sa)EctC-Strep-Tag-II preparations photometrically, 10 nmol of the purified proteins were heated at 80°C for 10 min in 250 μl of a 1% HCl solution. The reaction assay was cooled down on ice and then centrifuged (13000 rpm, 10 min at room temperature). The supernatant was transferred to a new reaction tube, and 750 μl H2O, 50 μl of 10% hydroxylamine/HCl, and 250 μl of 0.1% phenanthroline were added to the reaction vessel. After 30 min of incubation at room temperature, the absorbance of the solution was measured at 512 nm. 5 to 40 nmol of ammonium iron(II) sulfate were used for calibration of the assay. METHODS title_2 21997 Crystallization of the (Sa)EctC protein METHODS paragraph 22037 Several conditions under which the (Sa)EctC protein formed crystals were found by using commercial screens (Nextal, Qiagen, Hilden, Germany; Molecular Dimensions, Suffolk, UK) in 96-well sitting drop plates (Corning 3553) at 12°C. Homogeneous (Sa)EctC protein (0.1 μl from a solution of 11 mg protein ml-1) was mixed with 0.1 μl reservoir solution and equilibrated against 50 μl reservoir solution. The most promising condition was found with a solution containing 0.05 M calcium acetate, 0.1 M sodium acetate (pH 4.5), and 40% (v/v) 1,2-propanediol from the Nextal Core IV suite (Qiagen, Hilden, Germany). A second condition under which (Sa)EctC crystallized was identified in microbatch setups (1 μl + 1 μl drops) using 20% (w/v) PEG 6000, 0.9 M lithium chloride, and 0.1 M citric acid (pH 5) from the Nextal Core II suite (Qiagen, Hilden, Germany). These conditions were optimized by grid screens around the initial condition and/or after the addition of tert-butanol as an additive. Large crystals were obtained either without any additive or after the addition of tert-butanol to the (Sa)EctC protein solution 30 minutes before the drops were spotted. Crystals reached their maximum dimensions of about 50 × 50 × 70 μm3 after 3–10 weeks. The crystals were fished after overlaying the drop with 2 μl mineral oil and flash frozen in liquid nitrogen. To obtain heavy atom derivatized crystals, methylmercury(II) chloride was added (final concentration: 0.5 mM) to the crystals in their drop for 30 minutes before they were fished and flash frozen in liquid nitrogen. METHODS title_2 23618 Data processing and structure determination METHODS paragraph 23662 Native data sets were collected from a single crystal of (Sa)EctC obtained from the various crystallization trials at the ERSF beamline ID23eh2 (Grenoble, France) at 100 K. These data sets were processed using the XDS package and scaled with XSCALE showing a maximum resolution of 1.2 Å. To obtain initial phases of (Sa)EctC, a mercury-derivatized crystal was used to collect a conservative dataset at 2.8 Å resolution. The data were processed and scaled as described above, before the program AUTORICKSHAW using single isomorphous replacement (SIRAS), was used to localize the Hg atom, phase and built an initial model of the (Sa)EctC protein. This initial model was used as a template for molecular replacement on the 2.0 Å dataset revealing four monomers in the asymmetric unit. Once the 2.0 Å structure was refined, a single monomer of this structure was used as a template for molecular replacement to phase the 1.2 Å resolution dataset using the PHENIX software. Model building and refinement were performed using COOT, Refmac5 and Phenix_refine. Data refinement statistics and model content are summarized in Table 1. METHODS title_2 24792 PDB accession numbers METHODS paragraph 24814 The atomic coordinates and structural factors have been deposited into the Protein Data Bank (PDB) (Brookhaven, USA) under the following accession codes: 5BXX (for the “semi-closed” (Sa)EctC structure) and 5BY5 (for the “open” (Sa)EctC structure). METHODS title_2 25070 Figure preparation of crystal structures METHODS paragraph 25111 Figures of the crystal structures of SaEctC were prepared using the PyMol software suite (www.pymol.org). RESULTS title_1 25217 Results RESULTS title_2 25225 Overproduction, purification and oligomeric state of the ectoine synthase in solution 0.9935509 experimental_method cleaner0 2023-07-20T14:28:59Z MESH: Overproduction 0.8892884 experimental_method cleaner0 2023-07-20T14:29:04Z MESH: purification 0.99961185 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase RESULTS paragraph 25311 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. 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. The (Sa)EctC protein was overproduced and isolated with good yields (30–40 mg L-1 of culture) and purity (S2a Fig). 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. 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). 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). Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus. 0.9994874 experimental_method cleaner0 2023-07-20T14:29:08Z MESH: biochemical and structural studies 0.9996302 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.99917936 species cleaner0 2023-07-20T10:21:23Z MESH: S. alaskensis species MESH: cleaner0 2023-07-20T10:22:10Z Sa protein PR: cleaner0 2023-07-20T10:09:40Z EctC taxonomy_domain DUMMY: cleaner0 2023-07-20T14:21:30Z marine ultra-microbacterium 0.99960726 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure 0.99947584 protein_type cleaner0 2023-07-20T10:22:20Z MESH: ectoine hydroxylase 0.99979144 protein_type cleaner0 2023-07-20T10:22:39Z MESH: EctD 0.9992048 protein_state cleaner0 2023-07-20T13:57:28Z DUMMY: in complex with 0.99922276 species cleaner0 2023-07-20T10:21:24Z MESH: S. alaskensis 0.99774545 gene cleaner0 2023-07-20T10:22:58Z GENE: ectC 0.99888724 species cleaner0 2023-07-20T10:23:06Z MESH: E. coli 0.90680575 experimental_method cleaner0 2023-07-20T14:19:30Z MESH: Strep-tag II affinity peptide species MESH: cleaner0 2023-07-20T10:06:57Z Sa protein PR: cleaner0 2023-07-20T10:09:40Z EctC experimental_method MESH: cleaner0 2023-07-20T10:23:30Z Strep-Tag-II 0.9994743 experimental_method cleaner0 2023-07-20T14:29:28Z MESH: affinity chromatography 0.3394467 species cleaner0 2023-07-20T10:06:57Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:40Z EctC 0.9861797 experimental_method cleaner0 2023-07-20T14:29:37Z MESH: size-exclusion chromatography 0.99960774 experimental_method cleaner0 2023-07-20T13:22:12Z MESH: SEC 0.4025306 species cleaner0 2023-07-20T10:06:57Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.99941814 oligomeric_state cleaner0 2023-07-20T14:22:55Z DUMMY: dimers 0.9995423 experimental_method cleaner0 2023-07-20T14:29:41Z MESH: High performance liquid chromatography 0.99823344 experimental_method cleaner0 2023-07-20T14:29:45Z MESH: multi-angle light-scattering detection 0.99953747 experimental_method cleaner0 2023-07-20T13:22:18Z MESH: HPLC-MALS 0.44877085 species cleaner0 2023-07-20T10:06:57Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.34720233 species cleaner0 2023-07-20T10:06:57Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.9993968 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer 0.99939466 oligomeric_state cleaner0 2023-07-20T10:12:09Z DUMMY: monomer 0.85244745 experimental_method cleaner0 2023-07-20T14:19:43Z MESH: Strep-tag II affinity peptide 0.9994011 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer 0.9995308 protein_type cleaner0 2023-07-20T10:24:20Z MESH: EctC proteins 0.9994009 species cleaner0 2023-07-20T10:21:29Z MESH: H. elongata 0.99942994 species cleaner0 2023-07-20T10:21:34Z MESH: N. maritimus RESULTS title_2 26746 Biochemical properties of the ectoine synthase 0.9996504 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase RESULTS paragraph 26793 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. 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). This procedure also yielded the isomer of N-γ-ADABA, N-α-acetyl-L-2,4-diaminobutyric acid (N-α-ADABA) (S1b Fig). 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. 0.5488887 protein cleaner0 2023-07-20T10:13:49Z PR: EctA 0.999634 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.9997744 chemical cleaner0 2023-07-20T10:24:44Z CHEBI: N-γ-acetyl-L-2,4-diaminobutyric acid 0.99975413 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.99936503 chemical cleaner0 2023-07-20T10:07:53Z CHEBI: ectoine 0.9997587 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.99974823 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.99976957 chemical cleaner0 2023-07-20T10:24:47Z CHEBI: N-α-acetyl-L-2,4-diaminobutyric acid 0.9997544 chemical cleaner0 2023-07-20T10:24:51Z CHEBI: N-α-ADABA 0.9997612 chemical cleaner0 2023-07-20T10:24:52Z CHEBI: N-α-ADABA 0.9994758 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.9994418 taxonomy_domain cleaner0 2023-07-20T11:32:08Z DUMMY: microorganisms 0.9997521 chemical cleaner0 2023-07-20T10:07:53Z CHEBI: ectoine RESULTS paragraph 27398 Using N-γ-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein. 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. 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). 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). 0.9997508 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.5292546 species cleaner0 2023-07-20T10:06:57Z MESH: Sa 0.6270969 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.99938875 species cleaner0 2023-07-20T10:21:24Z MESH: S. alaskensis 0.9996713 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.9974341 taxonomy_domain cleaner0 2023-07-20T14:21:36Z DUMMY: microorganism species MESH: cleaner0 2023-07-20T10:06:57Z Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.9985224 protein_state cleaner0 2023-07-20T14:46:40Z DUMMY: enzymatically active 0.6991387 protein_state cleaner0 2023-07-20T14:47:02Z DUMMY: alkaline 0.99969196 protein_type cleaner0 2023-07-20T14:17:02Z MESH: ectoine synthases 0.9490307 protein_state cleaner0 2023-07-20T14:47:07Z DUMMY: halo-tolerant 0.999429 species cleaner0 2023-07-20T10:21:30Z MESH: H. elongata 0.98819995 taxonomy_domain cleaner0 2023-07-20T14:21:42Z DUMMY: alkaliphile 0.99946517 species cleaner0 2023-07-20T10:25:07Z MESH: M. alcaliphilum 0.9690805 taxonomy_domain cleaner0 2023-07-20T14:21:45Z DUMMY: acidophile 0.99937564 species cleaner0 2023-07-20T10:17:43Z MESH: Acidiphilium cryptum 0.9995845 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.9994826 species cleaner0 2023-07-20T10:21:35Z MESH: N. maritimus 0.9332136 protein_state cleaner0 2023-07-20T14:47:11Z DUMMY: neutral pH RESULTS paragraph 28279 The salinity of the assay buffer had a significant influence on the maximal enzyme activity of the (Sa)EctC protein. An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity. The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively. (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). The stimulation of EctC enzyme activity by salts has previously also been observed for other ectoine synthases. species MESH: cleaner0 2023-07-20T10:06:57Z Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.99961096 chemical cleaner0 2023-07-20T10:26:08Z CHEBI: NaCl 0.9996014 chemical cleaner0 2023-07-20T10:26:14Z CHEBI: KCl 0.9996583 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase species MESH: cleaner0 2023-07-20T10:06:57Z Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.9995086 chemical cleaner0 2023-07-20T10:26:10Z CHEBI: NaCl 0.99956685 chemical cleaner0 2023-07-20T10:26:15Z CHEBI: KCl species MESH: cleaner0 2023-07-20T10:06:57Z Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.9996302 chemical cleaner0 2023-07-20T10:26:10Z CHEBI: NaCl 0.9996433 chemical cleaner0 2023-07-20T10:26:15Z CHEBI: KCl 0.9996387 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.99969184 protein_type cleaner0 2023-07-20T10:26:36Z MESH: ectoine synthases RESULTS title_2 28902 The ectoine synthase is a metal-containing protein 0.99967754 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.985259 protein_type cleaner0 2023-07-20T14:17:21Z MESH: metal-containing protein RESULTS paragraph 28953 Considerations based on bioinformatics suggests that EctC belongs to the cupin superfamily. Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel. 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). 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. An abbreviated alignment of the amino acid sequence of EctC-type proteins is shown in Fig 2. 0.99895215 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.9994916 protein_type cleaner0 2023-07-20T10:07:22Z MESH: cupin superfamily 0.99914396 chemical cleaner0 2023-07-20T11:21:59Z CHEBI: iron 0.9990225 chemical cleaner0 2023-07-20T11:22:06Z CHEBI: copper 0.9989623 chemical cleaner0 2023-07-20T11:22:12Z CHEBI: zinc 0.9989072 chemical cleaner0 2023-07-20T11:22:20Z CHEBI: manganese 0.9986871 chemical cleaner0 2023-07-20T11:22:25Z CHEBI: cobalt 0.99882394 chemical cleaner0 2023-07-20T11:22:32Z CHEBI: nickel 0.99960715 protein_type cleaner0 2023-07-20T11:22:38Z MESH: Cupins protein_state DUMMY: cleaner0 2023-07-20T11:24:19Z conserved structure_element SO: cleaner0 2023-07-20T11:23:06Z G(X)5HXH(X)3,4E(X)6G structure_element SO: cleaner0 2023-07-20T11:23:28Z G(X)5PXG(X)2H(X)3N chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.9817178 experimental_method cleaner0 2023-07-20T14:30:15Z MESH: alignment of the amino acid sequences 0.9996746 protein_type cleaner0 2023-07-20T11:23:47Z MESH: EctC-type proteins 0.70793587 structure_element cleaner0 2023-07-20T11:23:52Z SO: metal-binding motif 0.9996796 protein_type cleaner0 2023-07-20T11:23:56Z MESH: cupin-type proteins 0.9992851 protein_state cleaner0 2023-07-20T11:24:00Z DUMMY: not conserved protein_type MESH: cleaner0 2023-07-20T11:24:39Z ectoine synthase protein family experimental_method MESH: cleaner0 2023-07-20T14:30:36Z alignment of the amino acid sequence 0.9996861 protein_type cleaner0 2023-07-20T11:24:52Z MESH: EctC-type proteins pone.0151285.g002.jpg pone.0151285.g002 FIG fig_title_caption 29679 Abbreviated alignment of EctC-type proteins. 0.97866905 experimental_method cleaner0 2023-07-20T14:30:40Z MESH: alignment 0.99968976 protein_type cleaner0 2023-07-20T11:24:53Z MESH: EctC-type proteins pone.0151285.g002.jpg pone.0151285.g002 FIG fig_caption 29724 The amino acid sequences of 20 selected EctC-type proteins are compared. Strictly conserved amino acid residues are shown in yellow. 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). The conserved residue Tyr-52 with so-far undefined functions is indicated by a green dot circled in red. 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. 0.99966794 protein_type cleaner0 2023-07-20T11:24:53Z MESH: EctC-type proteins 0.9993727 protein_state cleaner0 2023-07-20T11:25:19Z DUMMY: Strictly conserved 0.43636063 species cleaner0 2023-07-20T10:06:57Z MESH: Sa 0.32576406 protein cleaner0 2023-07-20T10:09:41Z PR: EctC chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.99935764 protein_state cleaner0 2023-07-20T11:25:40Z DUMMY: conserved 0.99919754 residue_name_number cleaner0 2023-07-20T13:38:45Z DUMMY: Tyr-52 0.99957323 structure_element cleaner0 2023-07-20T14:40:15Z SO: α-helices 0.999482 structure_element cleaner0 2023-07-20T14:40:19Z SO: β-sheets 0.6731305 species cleaner0 2023-07-20T10:06:57Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.9995988 evidence cleaner0 2023-07-20T11:25:35Z DUMMY: crystal structure 0.99965817 protein_type cleaner0 2023-07-20T11:24:53Z MESH: EctC-type proteins RESULTS paragraph 30315 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). For this analysis we used recombinant (Sa)EctC preparations from three independent protein overproduction and purification experiments. 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). All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively). 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. Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing 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. 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. 0.9835426 structure_element cleaner0 2023-07-20T11:25:52Z SO: metal-binding motif chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.40864503 species cleaner0 2023-07-20T10:06:57Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.9995365 experimental_method cleaner0 2023-07-20T14:30:45Z MESH: inductive-coupled plasma mass spectrometry 0.9995499 experimental_method cleaner0 2023-07-20T13:55:27Z MESH: ICP-MS 0.2987374 species cleaner0 2023-07-20T10:06:57Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.99955463 experimental_method cleaner0 2023-07-20T13:55:27Z MESH: ICP-MS 0.9986418 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.99890316 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.41933072 species cleaner0 2023-07-20T10:06:57Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.99888176 chemical cleaner0 2023-07-20T11:22:14Z CHEBI: zinc 0.9990158 chemical cleaner0 2023-07-20T11:22:14Z CHEBI: zinc 0.99899393 chemical cleaner0 2023-07-20T11:22:07Z CHEBI: copper 0.9987733 chemical cleaner0 2023-07-20T11:22:33Z CHEBI: nickel chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.9990601 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.37607437 species cleaner0 2023-07-20T10:06:57Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC experimental_method MESH: cleaner0 2023-07-20T11:27:02Z colorimetric method chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.9954098 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.40473375 species cleaner0 2023-07-20T10:06:57Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.99956316 experimental_method cleaner0 2023-07-20T13:55:27Z MESH: ICP-MS 0.8751861 experimental_method cleaner0 2023-07-20T11:27:02Z MESH: colorimetric method 0.9994973 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.9993145 species cleaner0 2023-07-20T10:21:24Z MESH: S. alaskensis 0.65454566 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.98509324 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.5177903 species cleaner0 2023-07-20T10:06:57Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.8369187 experimental_method cleaner0 2023-07-20T11:27:07Z MESH: colorimetric assay 0.9820753 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.45383352 species cleaner0 2023-07-20T10:06:57Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC RESULTS title_2 31796 A metal cofactor is important for the catalytic activity of EctC chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.9996711 protein cleaner0 2023-07-20T10:09:41Z PR: EctC RESULTS paragraph 31861 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. 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. 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). 0.9994646 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.99937034 protein_type cleaner0 2023-07-20T10:07:22Z MESH: cupin superfamily 0.9995086 experimental_method cleaner0 2023-07-20T14:31:05Z MESH: incubated species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC experimental_method MESH: cleaner0 2023-07-20T14:31:27Z with increasing concentrations chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.9996955 chemical cleaner0 2023-07-20T11:27:57Z CHEBI: ethylene-diamine-tetraacetic-acid 0.9996581 chemical cleaner0 2023-07-20T11:28:02Z CHEBI: EDTA 0.9993819 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.9996971 chemical cleaner0 2023-07-20T11:28:04Z CHEBI: EDTA 0.99951756 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.99938935 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.99970055 chemical cleaner0 2023-07-20T11:28:04Z CHEBI: EDTA pone.0151285.g003.jpg pone.0151285.g003 FIG fig_title_caption 32488 Dependency of the ectoine synthase activity on metals. 0.9996023 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase pone.0151285.g003.jpg pone.0151285.g003 FIG fig_caption 32543 (a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein. Metal depletion and reconstitution experiments with (b) stoichiometric and (c) excess amounts of metals. 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. chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.99977547 chemical cleaner0 2023-07-20T11:28:04Z CHEBI: EDTA species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.81504804 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.99951935 experimental_method cleaner0 2023-07-20T11:28:35Z MESH: Metal depletion and reconstitution experiments species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.3710249 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.99964386 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.99955547 experimental_method cleaner0 2023-07-20T14:31:34Z MESH: enzyme assays 0.9997687 chemical cleaner0 2023-07-20T11:29:01Z CHEBI: FeCl2 RESULTS paragraph 32944 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. 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. All other tested metals, including Fe3+, were unable to restore activity (Fig 3b). 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). 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). 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. 0.9988016 protein_state cleaner0 2023-07-20T14:47:17Z DUMMY: inactivated 0.99844754 chemical cleaner0 2023-07-20T11:28:04Z CHEBI: EDTA 0.9991221 experimental_method cleaner0 2023-07-20T14:31:38Z MESH: dialysis species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.77772117 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.99972755 chemical cleaner0 2023-07-20T11:29:00Z CHEBI: FeCl2 0.99939704 experimental_method cleaner0 2023-07-20T11:29:12Z MESH: enzyme assay 0.99973184 chemical cleaner0 2023-07-20T11:28:55Z CHEBI: ZnCl2 0.99973637 chemical cleaner0 2023-07-20T11:29:05Z CHEBI: CoCl2 species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.7600365 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.99975914 chemical cleaner0 2023-07-20T11:29:26Z CHEBI: Fe3+ 0.99914694 experimental_method cleaner0 2023-07-20T11:29:13Z MESH: enzyme assay 0.99975884 chemical cleaner0 2023-07-20T11:29:23Z CHEBI: Fe2+ species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.8068256 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.99578047 chemical cleaner0 2023-07-20T11:28:04Z CHEBI: EDTA 0.9994541 chemical cleaner0 2023-07-20T11:22:14Z CHEBI: zinc 0.99934477 chemical cleaner0 2023-07-20T11:22:27Z CHEBI: cobalt 0.999358 chemical cleaner0 2023-07-20T11:22:33Z CHEBI: nickel 0.9994154 chemical cleaner0 2023-07-20T11:22:08Z CHEBI: copper 0.9994241 chemical cleaner0 2023-07-20T11:22:21Z CHEBI: manganese 0.99947345 protein_type cleaner0 2023-07-20T10:07:22Z MESH: cupin superfamily 0.99968255 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.999539 protein_type cleaner0 2023-07-20T14:17:27Z MESH: cupin-type enzymes chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal RESULTS title_2 34093 Kinetic parameters of EctC for N-γ-ADABA and N-α-ADABA 0.99944764 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.9997571 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.99975604 chemical cleaner0 2023-07-20T10:24:52Z CHEBI: N-α-ADABA RESULTS paragraph 34156 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. 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). 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. This was indeed the case. (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). Hence, N-α-ADABA is a newly recognized substrate for ectoine synthase. 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. 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. 0.9988838 experimental_method cleaner0 2023-07-20T11:30:39Z MESH: activity assay 0.99960184 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.9992654 species cleaner0 2023-07-20T10:21:24Z MESH: S. alaskensis 0.33515686 species cleaner0 2023-07-20T10:06:58Z MESH: Sa 0.6267819 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.9997531 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.9997481 chemical cleaner0 2023-07-20T10:24:53Z CHEBI: N-α-ADABA 0.9973105 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.9997524 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.9990388 chemical cleaner0 2023-07-20T10:07:53Z CHEBI: ectoine 0.9958072 experimental_method cleaner0 2023-07-20T11:30:32Z MESH: Michaelis-Menten-kinetics 0.99950933 evidence cleaner0 2023-07-20T11:30:04Z DUMMY: Km 0.99886847 evidence cleaner0 2023-07-20T11:31:42Z DUMMY: vmax 0.9994772 evidence cleaner0 2023-07-20T11:30:13Z DUMMY: kcat 0.9997486 chemical cleaner0 2023-07-20T10:24:53Z CHEBI: N-α-ADABA 0.9997503 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.99965304 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.77422124 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.9997444 chemical cleaner0 2023-07-20T10:24:53Z CHEBI: N-α-ADABA 0.9989826 chemical cleaner0 2023-07-20T10:07:53Z CHEBI: ectoine species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.82548434 protein cleaner0 2023-07-20T10:09:41Z PR: EctC 0.9978008 experimental_method cleaner0 2023-07-20T11:30:34Z MESH: Michaelis-Menten-kinetics 0.9995671 evidence cleaner0 2023-07-20T11:30:04Z DUMMY: Km 0.99924266 evidence cleaner0 2023-07-20T11:31:43Z DUMMY: vmax 0.9994406 evidence cleaner0 2023-07-20T11:30:12Z DUMMY: kcat 0.99975175 chemical cleaner0 2023-07-20T10:24:53Z CHEBI: N-α-ADABA 0.9996559 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.99953246 evidence cleaner0 2023-07-20T14:10:42Z DUMMY: affinity 0.99958366 evidence cleaner0 2023-07-20T11:30:03Z DUMMY: Km species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.736817 protein cleaner0 2023-07-20T10:09:41Z PR: EctC evidence DUMMY: cleaner0 2023-07-20T11:31:31Z catalytic efficiency 0.99560183 evidence cleaner0 2023-07-20T11:31:19Z DUMMY: kcat/Km 0.9997519 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.99958986 evidence cleaner0 2023-07-20T11:30:04Z DUMMY: Km evidence DUMMY: cleaner0 2023-07-20T11:31:33Z catalytic efficiency RESULTS paragraph 35415 Both N-γ-ADABA and N-α-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism. 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. 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). 0.99971217 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.9997239 chemical cleaner0 2023-07-20T10:24:53Z CHEBI: N-α-ADABA 0.99963677 chemical cleaner0 2023-07-20T10:07:53Z CHEBI: ectoine 0.99970275 chemical cleaner0 2023-07-20T10:24:53Z CHEBI: N-α-ADABA 0.99965507 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.99938774 taxonomy_domain cleaner0 2023-07-20T11:32:08Z DUMMY: microorganisms 0.999724 chemical cleaner0 2023-07-20T10:07:54Z CHEBI: ectoine 0.9992518 taxonomy_domain cleaner0 2023-07-20T11:32:07Z DUMMY: microorganisms 0.9993874 evidence cleaner0 2023-07-20T11:31:59Z DUMMY: affinity 0.9996618 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.99972785 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.99971116 chemical cleaner0 2023-07-20T10:24:53Z CHEBI: N-α-ADABA evidence DUMMY: cleaner0 2023-07-20T11:31:33Z catalytic efficiency RESULTS title_2 36001 Crystallization of the (Sa)EctC protein 0.9996445 experimental_method cleaner0 2023-07-20T14:31:43Z MESH: Crystallization 0.18065484 species cleaner0 2023-07-20T10:06:58Z MESH: Sa 0.49151045 protein cleaner0 2023-07-20T10:09:41Z PR: EctC RESULTS paragraph 36041 Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)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. However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained. Crystal form A diffracted to 1.2 Å and had a unit cell of a = 72.71 b = 72.71 c = 52.33 Å and α = 90 β = 90 γ = 120° displaying a P3221 symmetry (S1 Table). Crystal form B diffracted to 2.0 Å and had a unit cell of a = 97.52 b = 43.96 c = 138.54 Å and α = 90 β = 101.5 γ = 120° and displayed a C2 symmetry (S1 Table). Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed. 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). 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). 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). 0.99962616 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure 0.99959636 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.99956125 experimental_method cleaner0 2023-07-20T14:31:46Z MESH: crystallize species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:41Z EctC 0.9958228 evidence cleaner0 2023-07-20T14:10:52Z DUMMY: crystals species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.99944985 protein_state cleaner0 2023-07-20T14:47:22Z DUMMY: in complex 0.99971485 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.999548 chemical cleaner0 2023-07-20T10:07:54Z CHEBI: ectoine 0.9986279 evidence cleaner0 2023-07-20T14:11:01Z DUMMY: crystal forms species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9994385 protein_state cleaner0 2023-07-20T13:34:29Z DUMMY: absence of 0.9996357 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.99954855 experimental_method cleaner0 2023-07-20T14:31:50Z MESH: molecular replacement 0.9473007 evidence cleaner0 2023-07-20T14:11:06Z DUMMY: crystals 0.9993457 chemical cleaner0 2023-07-20T11:33:32Z CHEBI: mercury 0.9893433 evidence cleaner0 2023-07-20T14:11:10Z DUMMY: structural model species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9995866 experimental_method cleaner0 2023-07-20T14:31:53Z MESH: molecular replacement 0.9993136 oligomeric_state cleaner0 2023-07-20T13:33:48Z DUMMY: monomers species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.99962366 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure 0.99959403 evidence cleaner0 2023-07-20T11:33:19Z DUMMY: Rcryst 0.99952066 evidence cleaner0 2023-07-20T11:33:24Z DUMMY: Rfree 0.9993229 oligomeric_state cleaner0 2023-07-20T10:12:09Z DUMMY: monomer 0.99949193 evidence cleaner0 2023-07-20T14:11:15Z DUMMY: structure 0.9995883 experimental_method cleaner0 2023-07-20T14:31:56Z MESH: molecular replacement 0.9995921 evidence cleaner0 2023-07-20T11:33:20Z DUMMY: Rcryst 0.99957305 evidence cleaner0 2023-07-20T11:33:24Z DUMMY: Rfree RESULTS title_2 37632 Overall fold of the (Sa)EctC protein species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC RESULTS paragraph 37669 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). However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2). 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. 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). In this structure no metal co-factor was identified. 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. Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2). 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. 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. 0.997881 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.9990163 evidence cleaner0 2023-07-20T14:11:19Z DUMMY: structures 0.99481285 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.99909353 protein_type cleaner0 2023-07-20T10:07:22Z MESH: cupin superfamily 0.9970457 residue_range cleaner0 2023-07-20T14:37:05Z DUMMY: 137 amino acids species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.30935073 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.99948114 evidence cleaner0 2023-07-20T14:11:23Z DUMMY: structure species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.421482 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.94030523 residue_range cleaner0 2023-07-20T14:11:35Z DUMMY: Met-1 to Glu-115 0.99928635 protein_state cleaner0 2023-07-20T14:47:26Z DUMMY: lacks 0.96660024 residue_range cleaner0 2023-07-20T14:37:19Z DUMMY: 22 amino acids structure_element SO: cleaner0 2023-07-20T14:44:23Z carboxy-terminus species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.37216732 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.99869365 evidence cleaner0 2023-07-20T14:11:48Z DUMMY: structure 0.9996549 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.9987495 structure_element cleaner0 2023-07-20T14:40:25Z SO: cupin barrels 0.9996737 protein_state cleaner0 2023-07-20T11:35:51Z DUMMY: open species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.45329663 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.99948573 evidence cleaner0 2023-07-20T14:11:53Z DUMMY: structure 0.9987312 evidence cleaner0 2023-07-20T14:11:57Z DUMMY: structure chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.99953264 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.81937975 experimental_method cleaner0 2023-07-20T14:32:01Z MESH: solved oligomeric_state DUMMY: cleaner0 2023-07-20T14:24:15Z protomer structure_element SO: cleaner0 2023-07-20T14:23:59Z A 0.9478492 residue_range cleaner0 2023-07-20T14:11:44Z DUMMY: Met-1 to Gly-121 0.99966216 protein_state cleaner0 2023-07-20T14:47:40Z DUMMY: closed 0.8067061 protein_state cleaner0 2023-07-20T14:47:43Z DUMMY: lacks 0.9836785 residue_range cleaner0 2023-07-20T14:37:24Z DUMMY: 16 amino acid structure_element SO: cleaner0 2023-07-20T14:44:23Z carboxy-terminus 0.9969917 residue_range cleaner0 2023-07-20T14:37:29Z DUMMY: 137 amino acids species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.99961877 evidence cleaner0 2023-07-20T11:35:41Z DUMMY: crystal structure 0.99947864 protein_state cleaner0 2023-07-20T11:35:58Z DUMMY: fully closed 0.99901295 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.9992604 protein_state cleaner0 2023-07-20T11:36:03Z DUMMY: semi-closed species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.5132443 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.9994236 evidence cleaner0 2023-07-20T14:12:00Z DUMMY: structure 0.9989819 oligomeric_state cleaner0 2023-07-20T13:33:48Z DUMMY: monomers 0.8134865 residue_range cleaner0 2023-07-20T14:12:12Z DUMMY: Met-1 to Glu-115 0.9996649 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.91006696 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.9993042 evidence cleaner0 2023-07-20T14:12:04Z DUMMY: structure pone.0151285.g004.jpg pone.0151285.g004 FIG fig_title_caption 39144 Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC. 0.99957496 evidence cleaner0 2023-07-20T14:12:18Z DUMMY: structure 0.999647 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.9995422 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.99960786 evidence cleaner0 2023-07-20T13:57:50Z DUMMY: crystal structures species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC pone.0151285.g004.jpg pone.0151285.g004 FIG fig_caption 39234 (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. The β-strands are numbered β1-β11 and the helices α-I to α-II. (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. The entrance to the active site of the ectoine synthase is marked. (c) Overlay of the “semi-closed” and “open” (Sa)EctC structures. 0.9980724 evidence cleaner0 2023-07-20T14:12:21Z DUMMY: structure 0.99951744 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.54517436 species cleaner0 2023-07-20T10:06:58Z MESH: Sa 0.99927276 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.99968773 structure_element cleaner0 2023-07-20T14:40:31Z SO: β-strands 0.9995842 structure_element cleaner0 2023-07-20T13:54:20Z SO: β1-β11 0.99951017 structure_element cleaner0 2023-07-20T14:40:34Z SO: helices 0.9993981 structure_element cleaner0 2023-07-20T14:40:37Z SO: α-I to α-II 0.99891806 evidence cleaner0 2023-07-20T14:12:24Z DUMMY: structure 0.99967027 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.5190078 species cleaner0 2023-07-20T10:06:58Z MESH: Sa 0.9992449 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.9996062 site cleaner0 2023-07-20T13:43:15Z SO: active site 0.9995431 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.9994759 experimental_method cleaner0 2023-07-20T14:32:08Z MESH: Overlay 0.9995149 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.9996666 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.5526954 species cleaner0 2023-07-20T10:06:58Z MESH: Sa 0.9992661 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.99936575 evidence cleaner0 2023-07-20T14:12:26Z DUMMY: structures RESULTS paragraph 39782 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. 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. 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. 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. 0.9994179 evidence cleaner0 2023-07-20T14:12:41Z DUMMY: structure species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9992894 evidence cleaner0 2023-07-20T14:12:44Z DUMMY: crystals structure_element SO: cleaner0 2023-07-20T14:44:23Z carboxy-terminus 0.99950683 structure_element cleaner0 2023-07-20T13:33:43Z SO: cupin barrel oligomeric_state DUMMY: cleaner0 2023-07-20T13:23:23Z monomer structure_element SO: cleaner0 2023-07-20T14:23:26Z A 0.9995088 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.99846053 evidence cleaner0 2023-07-20T14:12:49Z DUMMY: structure 0.99302316 evidence cleaner0 2023-07-20T14:12:52Z DUMMY: root mean square deviation 0.9995968 evidence cleaner0 2023-07-20T13:17:42Z DUMMY: RMSD 0.9996617 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.9990269 oligomeric_state cleaner0 2023-07-20T13:33:48Z DUMMY: monomers 0.9995272 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.99902415 oligomeric_state cleaner0 2023-07-20T10:12:10Z DUMMY: monomer 0.99965227 evidence cleaner0 2023-07-20T13:17:42Z DUMMY: RMSD 0.9996704 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.9994696 evidence cleaner0 2023-07-20T14:12:57Z DUMMY: structure 0.9996313 evidence cleaner0 2023-07-20T14:13:00Z DUMMY: structure 0.9995301 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9996687 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.9995306 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed RESULTS paragraph 40595 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). 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. These two β-sheets pack against each other, forming a cup-shaped β-sandwich with a topology characteristic for the cupin-fold. Hence, (Sa)EctC adopts an overall bowl shape in which one side is opened towards the solvent (Fig 4a to 4c). 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). 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). 0.9994899 evidence cleaner0 2023-07-20T14:13:04Z DUMMY: structure 0.99953014 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.32368964 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9989476 structure_element cleaner0 2023-07-20T14:40:40Z SO: β-strands 0.9996641 structure_element cleaner0 2023-07-20T13:54:20Z SO: β1-β11 0.9994936 structure_element cleaner0 2023-07-20T14:40:43Z SO: α-helices 0.9996903 structure_element cleaner0 2023-07-20T14:40:47Z SO: α-I 0.9996945 structure_element cleaner0 2023-07-20T14:40:50Z SO: α-II 0.9995985 structure_element cleaner0 2023-07-20T14:40:53Z SO: β-strands 0.9996136 structure_element cleaner0 2023-07-20T14:40:57Z SO: anti-parallel β-sheets 0.9997389 structure_element cleaner0 2023-07-20T14:41:04Z SO: β2 0.97667503 structure_element cleaner0 2023-07-20T14:41:07Z SO: β3 0.99975175 structure_element cleaner0 2023-07-20T14:41:10Z SO: β4 0.99974126 structure_element cleaner0 2023-07-20T13:26:56Z SO: β11 0.9997428 structure_element cleaner0 2023-07-20T13:24:59Z SO: β6 0.99973994 structure_element cleaner0 2023-07-20T14:41:14Z SO: β9 0.9830336 structure_element cleaner0 2023-07-20T14:41:17Z SO: three-stranded β-sheet 0.99974567 structure_element cleaner0 2023-07-20T14:41:20Z SO: β7 0.9997398 structure_element cleaner0 2023-07-20T14:41:23Z SO: β8 0.99974865 structure_element cleaner0 2023-07-20T14:41:26Z SO: β10 0.99933386 structure_element cleaner0 2023-07-20T14:41:29Z SO: β-sheets 0.9995074 structure_element cleaner0 2023-07-20T14:41:31Z SO: cup-shaped β-sandwich 0.9976778 structure_element cleaner0 2023-07-20T13:28:25Z SO: cupin-fold 0.4580153 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.999531 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.99948585 evidence cleaner0 2023-07-20T14:13:08Z DUMMY: structure 0.9988877 structure_element cleaner0 2023-07-20T14:41:37Z SO: carboxy-terminal tail 0.9995363 evidence cleaner0 2023-07-20T13:31:03Z DUMMY: electron density structure_element SO: cleaner0 2023-07-20T14:42:03Z small helix 0.99971133 structure_element cleaner0 2023-07-20T14:42:06Z SO: α-II 0.9995864 site cleaner0 2023-07-20T13:43:15Z SO: active site 0.4139034 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9987648 structure_element cleaner0 2023-07-20T14:42:11Z SO: α-II helix 0.45860088 protein_state cleaner0 2023-07-20T14:42:26Z DUMMY: unstructured 0.99947053 structure_element cleaner0 2023-07-20T14:42:30Z SO: loop 0.99964976 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.99924123 evidence cleaner0 2023-07-20T14:13:29Z DUMMY: structure 0.9997776 structure_element cleaner0 2023-07-20T14:42:33Z SO: β4 0.9997366 structure_element cleaner0 2023-07-20T13:24:59Z SO: β6 0.99904567 protein_state cleaner0 2023-07-20T14:48:01Z DUMMY: stable 0.9996109 structure_element cleaner0 2023-07-20T13:27:53Z SO: β-strand 0.99972516 structure_element cleaner0 2023-07-20T13:27:47Z SO: β5 0.99952763 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.5325583 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC RESULTS paragraph 41663 Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily. 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). Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction. Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily. 0.9995205 experimental_method cleaner0 2023-07-20T14:32:14Z MESH: Structural comparison analyses 0.99941206 experimental_method cleaner0 2023-07-20T14:32:17Z MESH: DALI server 0.41508162 species cleaner0 2023-07-20T10:06:58Z MESH: Sa 0.8436447 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.99930793 protein_type cleaner0 2023-07-20T10:07:22Z MESH: cupin superfamily protein PR: cleaner0 2023-07-20T13:19:00Z Cupin 2 conserved barrel domain protein protein PR: cleaner0 2023-07-20T13:18:40Z YP_751781.1 0.999388 species cleaner0 2023-07-20T13:19:06Z MESH: Shewanella frigidimarina 0.96734786 evidence cleaner0 2023-07-20T13:17:34Z DUMMY: Z-score 0.9995598 evidence cleaner0 2023-07-20T13:17:41Z DUMMY: RMSD 0.968627 evidence cleaner0 2023-07-20T14:13:35Z DUMMY: structure 0.99936956 protein cleaner0 2023-07-20T13:19:27Z PR: manganese-containing cupin 0.9408579 protein cleaner0 2023-07-20T13:19:41Z PR: TM1459 0.99944925 species cleaner0 2023-07-20T13:19:59Z MESH: Thermotoga maritima 0.8786765 evidence cleaner0 2023-07-20T13:17:35Z DUMMY: Z-score 0.99958724 evidence cleaner0 2023-07-20T13:17:43Z DUMMY: RMSD 0.99950814 protein_type cleaner0 2023-07-20T13:20:07Z MESH: cyclase 0.9994784 protein cleaner0 2023-07-20T13:20:12Z PR: RemF 0.99944437 species cleaner0 2023-07-20T13:20:17Z MESH: Streptomyces resistomycificus evidence DUMMY: cleaner0 2023-07-20T13:17:35Z Z-score 0.99956745 evidence cleaner0 2023-07-20T13:17:43Z DUMMY: RMSD protein PR: cleaner0 2023-07-20T13:20:48Z auxin-binding protein 1 0.99945617 species cleaner0 2023-07-20T13:20:53Z MESH: Zea mays evidence DUMMY: cleaner0 2023-07-20T13:17:35Z Z-score 0.9995503 evidence cleaner0 2023-07-20T13:17:43Z DUMMY: RMSD 0.9280039 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.9996645 protein_type cleaner0 2023-07-20T13:21:02Z MESH: polyketide cyclase 0.9996055 protein cleaner0 2023-07-20T13:20:13Z PR: RemF 0.99893826 protein_type cleaner0 2023-07-20T14:17:35Z MESH: cupin-related 0.999348 protein cleaner0 2023-07-20T13:20:13Z PR: RemF 0.9995912 protein_type cleaner0 2023-07-20T13:21:25Z MESH: aldos-2-ulose dehydratase 0.906959 protein_type cleaner0 2023-07-20T13:21:27Z MESH: isomerase 0.9988444 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.9995653 protein_type cleaner0 2023-07-20T13:21:31Z MESH: dehydratase 0.999416 protein_type cleaner0 2023-07-20T10:07:22Z MESH: cupin superfamily RESULTS title_2 42925 Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure 0.9020333 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.99927044 site cleaner0 2023-07-20T13:21:55Z SO: dimer interface species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9996129 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure RESULTS paragraph 43008 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. The crystal structure of this protein reflects this quaternary arrangement. In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit. 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). The strong interactions between these β-strands rely primarily on backbone contacts. In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands. 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. Both values fall within the range for known functional dimers. 0.9996774 experimental_method cleaner0 2023-07-20T13:22:11Z MESH: SEC 0.99950904 experimental_method cleaner0 2023-07-20T13:22:16Z MESH: HPLC-MALS 0.99953663 protein_type cleaner0 2023-07-20T10:07:29Z MESH: ectoine synthase 0.9993809 species cleaner0 2023-07-20T10:21:24Z MESH: S. alaskensis 0.9993979 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer 0.99958277 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure 0.99948746 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.99957633 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure 0.29558715 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.999613 experimental_method cleaner0 2023-07-20T14:32:22Z MESH: crystallized 0.99940705 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer 0.99937505 oligomeric_state cleaner0 2023-07-20T14:23:00Z DUMMY: dimers 0.99941826 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer 0.999331 oligomeric_state cleaner0 2023-07-20T13:33:48Z DUMMY: monomers 0.99941456 protein_state cleaner0 2023-07-20T10:12:24Z DUMMY: head-to-tail 0.9994602 structure_element cleaner0 2023-07-20T14:42:40Z SO: antiparallel β-strands 0.9996492 structure_element cleaner0 2023-07-20T13:27:53Z SO: β-strand 0.9997371 structure_element cleaner0 2023-07-20T13:24:53Z SO: β1 0.9195826 structure_element cleaner0 2023-07-20T13:23:50Z SO: 1MIVRN5 oligomeric_state DUMMY: cleaner0 2023-07-20T13:23:23Z monomer structure_element SO: cleaner0 2023-07-20T13:23:41Z A 0.9996632 structure_element cleaner0 2023-07-20T13:27:53Z SO: β-strand 0.9997193 structure_element cleaner0 2023-07-20T14:42:44Z SO: β8 0.99917704 oligomeric_state cleaner0 2023-07-20T10:12:10Z DUMMY: monomer 0.99017453 structure_element cleaner0 2023-07-20T13:23:46Z SO: B 0.9666013 structure_element cleaner0 2023-07-20T13:23:53Z SO: 82GVMYAL87 0.99965686 structure_element cleaner0 2023-07-20T14:42:51Z SO: β-strands bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:19:19Z hydrophobic interactions 0.9992143 oligomeric_state cleaner0 2023-07-20T13:33:48Z DUMMY: monomers 0.99970716 structure_element cleaner0 2023-07-20T14:42:54Z SO: loops 0.99967855 structure_element cleaner0 2023-07-20T14:42:49Z SO: β-strands 0.56406224 experimental_method cleaner0 2023-07-20T14:32:26Z MESH: PDBePISA 0.99939084 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer 0.9993728 oligomeric_state cleaner0 2023-07-20T10:12:10Z DUMMY: monomer 0.9993892 oligomeric_state cleaner0 2023-07-20T14:23:04Z DUMMY: dimers pone.0151285.g005.jpg pone.0151285.g005 FIG fig_title_caption 44113 Crystal structure of (Sa)EctC. 0.99959344 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: Crystal structure species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC pone.0151285.g005.jpg pone.0151285.g005 FIG fig_caption 44144 (a) Top-view of the dimer of the (Sa)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. (c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6. 0.9994184 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9997701 chemical cleaner0 2023-07-20T14:19:48Z CHEBI: water 0.9994006 oligomeric_state cleaner0 2023-07-20T13:33:48Z DUMMY: monomers species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9993993 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer 0.9994203 site cleaner0 2023-07-20T13:21:56Z SO: dimer interface 0.99967796 structure_element cleaner0 2023-07-20T14:42:58Z SO: β-strands 0.9993344 oligomeric_state cleaner0 2023-07-20T10:12:10Z DUMMY: monomer 0.9994918 site cleaner0 2023-07-20T13:21:56Z SO: dimer interface 0.99967194 structure_element cleaner0 2023-07-20T14:43:01Z SO: beta-strand 0.9997563 structure_element cleaner0 2023-07-20T13:24:52Z SO: β1 0.9997464 structure_element cleaner0 2023-07-20T13:24:58Z SO: β6 RESULTS paragraph 44533 In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit. 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. 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. 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. Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure. 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). 0.99965227 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.8590964 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.99935895 evidence cleaner0 2023-07-20T14:13:42Z DUMMY: structure 0.9992888 oligomeric_state cleaner0 2023-07-20T10:12:10Z DUMMY: monomer 0.54075 experimental_method cleaner0 2023-07-20T14:32:31Z MESH: packing oligomeric_state DUMMY: cleaner0 2023-07-20T10:12:10Z monomer oligomeric_state DUMMY: cleaner0 2023-07-20T10:12:10Z monomer 0.9993844 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer 0.7603926 evidence cleaner0 2023-07-20T14:13:46Z DUMMY: crystal form 0.99921775 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer 0.99952525 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.8735654 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9993697 evidence cleaner0 2023-07-20T14:13:49Z DUMMY: structure oligomeric_state DUMMY: cleaner0 2023-07-20T10:12:10Z monomer oligomeric_state DUMMY: cleaner0 2023-07-20T10:12:10Z monomer 0.9995718 structure_element cleaner0 2023-07-20T14:43:07Z SO: β-sheets 0.9992028 oligomeric_state cleaner0 2023-07-20T13:33:48Z DUMMY: monomers 0.9992398 oligomeric_state cleaner0 2023-07-20T10:12:10Z DUMMY: monomer 0.9993611 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer 0.9995227 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.99945027 evidence cleaner0 2023-07-20T14:13:52Z DUMMY: structure 0.6170136 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9996438 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.99921894 evidence cleaner0 2023-07-20T14:13:57Z DUMMY: structure 0.9993315 experimental_method cleaner0 2023-07-20T14:32:34Z MESH: DALI search 0.9978916 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.9994137 oligomeric_state cleaner0 2023-07-20T14:23:09Z DUMMY: dimers oligomeric_state DUMMY: cleaner0 2023-07-20T10:12:10Z monomer oligomeric_state DUMMY: cleaner0 2023-07-20T10:12:10Z monomer 0.9993254 oligomeric_state cleaner0 2023-07-20T10:12:03Z DUMMY: dimer RESULTS title_2 45634 Structural rearrangements of the flexible (Sa)EctC carboxy-terminus 0.99960583 protein_state cleaner0 2023-07-20T14:48:07Z DUMMY: flexible species MESH: cleaner0 2023-07-20T10:06:58Z Sa 0.9870107 protein cleaner0 2023-07-20T10:09:42Z PR: EctC structure_element SO: cleaner0 2023-07-20T14:44:23Z carboxy-terminus RESULTS paragraph 45702 The cupin core represents the structural framework of ectoine synthase (Figs 4 and 5). The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus. 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. 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. 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. 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). Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement. 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. 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). It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below). The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5. 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. 0.99961126 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.9996259 evidence cleaner0 2023-07-20T13:57:50Z DUMMY: crystal structures 0.61970377 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC structure_element SO: cleaner0 2023-07-20T14:44:23Z carboxy-terminus structure_element SO: cleaner0 2023-07-20T13:50:49Z carboxy-terminal region 0.99818975 residue_range cleaner0 2023-07-20T14:37:36Z DUMMY: 137 amino acids 0.5200333 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9994724 protein_state cleaner0 2023-07-20T14:48:11Z DUMMY: highly conserved 0.94543046 protein_state cleaner0 2023-07-20T14:48:15Z DUMMY: extended 0.98046684 protein_type cleaner0 2023-07-20T13:26:47Z MESH: EctC protein 0.999656 structure_element cleaner0 2023-07-20T13:27:53Z SO: β-strand 0.99964786 structure_element cleaner0 2023-07-20T13:26:55Z SO: β11 0.9928986 protein_state cleaner0 2023-07-20T14:48:18Z DUMMY: conserved 0.9993019 residue_name cleaner0 2023-07-20T13:28:56Z SO: proline 0.9992849 residue_name_number cleaner0 2023-07-20T13:28:43Z DUMMY: Pro-109 0.9992657 residue_name_number cleaner0 2023-07-20T13:28:49Z DUMMY: Pro-110 0.39014503 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9995079 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.4662466 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9996371 evidence cleaner0 2023-07-20T14:14:01Z DUMMY: structure 0.999557 evidence cleaner0 2023-07-20T13:31:03Z DUMMY: electron density structure_element SO: cleaner0 2023-07-20T14:44:23Z carboxy-terminus 0.89069974 residue_range cleaner0 2023-07-20T14:37:56Z DUMMY: 7 amino acid residues 0.9993172 residue_name_number cleaner0 2023-07-20T13:29:00Z DUMMY: Gly-121 0.9988104 structure_element cleaner0 2023-07-20T14:43:19Z SO: small helix 0.9342446 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.73618835 site cleaner0 2023-07-20T14:39:05Z SO: cavity 0.9996348 structure_element cleaner0 2023-07-20T13:28:24Z SO: cupin-fold 0.66435605 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.90157557 structure_element cleaner0 2023-07-20T14:43:25Z SO: helix 0.99969316 structure_element cleaner0 2023-07-20T14:43:28Z SO: loop region 0.9996677 structure_element cleaner0 2023-07-20T14:43:32Z SO: β-strands 0.99966717 structure_element cleaner0 2023-07-20T14:43:35Z SO: β4 0.99880993 structure_element cleaner0 2023-07-20T13:24:59Z SO: β6 0.99965495 structure_element cleaner0 2023-07-20T13:27:51Z SO: β-strand 0.9994319 structure_element cleaner0 2023-07-20T13:27:45Z SO: β5 0.9923357 structure_element cleaner0 2023-07-20T14:43:38Z SO: small C-terminal helix 0.9948067 protein_state cleaner0 2023-07-20T14:48:22Z DUMMY: absent 0.99961144 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.42274258 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.99960667 evidence cleaner0 2023-07-20T14:14:06Z DUMMY: structure 0.9996436 structure_element cleaner0 2023-07-20T13:27:53Z SO: β-strand 0.99935025 structure_element cleaner0 2023-07-20T13:27:47Z SO: β5 0.9994707 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.80144036 species cleaner0 2023-07-20T10:06:58Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9996332 evidence cleaner0 2023-07-20T14:14:09Z DUMMY: structure 0.9996664 structure_element cleaner0 2023-07-20T13:27:53Z SO: β-strand 0.99895096 structure_element cleaner0 2023-07-20T13:27:47Z SO: β5 0.99931455 residue_name_number cleaner0 2023-07-20T13:31:18Z DUMMY: His-93 chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.9986792 residue_name cleaner0 2023-07-20T13:31:09Z SO: His species MESH: cleaner0 2023-07-20T10:06:58Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.99929583 evidence cleaner0 2023-07-20T14:14:13Z DUMMY: structures 0.999655 structure_element cleaner0 2023-07-20T13:27:53Z SO: β-strand 0.9993672 structure_element cleaner0 2023-07-20T13:27:47Z SO: β5 0.99949586 structure_element cleaner0 2023-07-20T14:43:43Z SO: cupin fold 0.9996346 site cleaner0 2023-07-20T13:28:15Z SO: iron-binding site RESULTS paragraph 47466 The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2). 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. In the “semi-closed” structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c). 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. 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. The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11. 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. 0.99903864 residue_name_number cleaner0 2023-07-20T13:28:44Z DUMMY: Pro-109 0.99906427 residue_name_number cleaner0 2023-07-20T13:28:50Z DUMMY: Pro-110 0.9996564 structure_element cleaner0 2023-07-20T13:27:53Z SO: β-strand structure_element SO: cleaner0 2023-07-20T13:29:38Z β11 0.9995168 protein_state cleaner0 2023-07-20T14:48:26Z DUMMY: highly conserved 0.999655 protein_type cleaner0 2023-07-20T11:24:54Z MESH: EctC-type proteins structure_element SO: cleaner0 2023-07-20T14:44:23Z carboxy-terminus 0.99866015 residue_range cleaner0 2023-07-20T14:38:07Z DUMMY: 27 amino acid residues 0.7135056 species cleaner0 2023-07-20T10:06:58Z MESH: Sa 0.41343936 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.9989593 structure_element cleaner0 2023-07-20T14:43:47Z SO: cupin fold 0.999533 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.9993531 evidence cleaner0 2023-07-20T14:14:17Z DUMMY: structure 0.9986342 structure_element cleaner0 2023-07-20T13:33:43Z SO: cupin barrel 0.9996474 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.45168442 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.99943846 evidence cleaner0 2023-07-20T14:14:20Z DUMMY: structure 0.9989772 residue_name cleaner0 2023-07-20T13:29:05Z SO: proline 0.99952924 evidence cleaner0 2023-07-20T13:31:02Z DUMMY: electron density 0.9990189 residue_name_number cleaner0 2023-07-20T13:28:50Z DUMMY: Pro-110 0.99954295 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density structure_element SO: cleaner0 2023-07-20T14:44:23Z carboxy-terminus 0.9990298 residue_name_number cleaner0 2023-07-20T13:28:44Z DUMMY: Pro-109 0.99891084 residue_name_number cleaner0 2023-07-20T13:30:39Z DUMMY: His-55 0.9996402 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.99952775 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.4830879 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.99958783 evidence cleaner0 2023-07-20T14:14:24Z DUMMY: structures 0.9988208 residue_name_number cleaner0 2023-07-20T13:28:44Z DUMMY: Pro-109 0.9977756 residue_name_number cleaner0 2023-07-20T13:30:41Z DUMMY: His-55 0.997267 protein_state cleaner0 2023-07-20T14:48:31Z DUMMY: stable 0.99815995 residue_name cleaner0 2023-07-20T13:29:08Z SO: proline 0.9996521 structure_element cleaner0 2023-07-20T13:27:53Z SO: β-strand 0.9996872 structure_element cleaner0 2023-07-20T13:26:56Z SO: β11 0.99806434 residue_name cleaner0 2023-07-20T13:29:10Z SO: proline 0.99933326 structure_element cleaner0 2023-07-20T13:50:49Z SO: carboxy-terminal region species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.99878997 residue_name_number cleaner0 2023-07-20T13:30:41Z DUMMY: His-55 0.99891657 residue_name_number cleaner0 2023-07-20T13:28:44Z DUMMY: Pro-109 RESULTS paragraph 48657 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. 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. In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b). 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. 0.9991196 residue_name_number cleaner0 2023-07-20T13:28:44Z DUMMY: Pro-109 0.99898905 residue_name_number cleaner0 2023-07-20T13:30:41Z DUMMY: His-55 0.9983686 structure_element cleaner0 2023-07-20T13:50:49Z SO: carboxy-terminal region species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9990506 residue_name_number cleaner0 2023-07-20T13:31:31Z DUMMY: Glu-115 0.998964 residue_name_number cleaner0 2023-07-20T13:30:41Z DUMMY: His-55 0.9996947 structure_element cleaner0 2023-07-20T14:43:53Z SO: small helix structure_element SO: cleaner0 2023-07-20T14:44:22Z carboxy-terminus 0.9988809 residue_name_number cleaner0 2023-07-20T13:31:32Z DUMMY: Glu-115 0.9987626 residue_name_number cleaner0 2023-07-20T13:30:41Z DUMMY: His-55 0.9995065 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.99943584 evidence cleaner0 2023-07-20T14:14:29Z DUMMY: structure 0.99632454 protein_state cleaner0 2023-07-20T14:48:36Z DUMMY: partially extended 0.9766502 structure_element cleaner0 2023-07-20T14:44:24Z SO: carboxy-terminus 0.9995966 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density 0.99962103 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.99952877 evidence cleaner0 2023-07-20T14:14:32Z DUMMY: structure species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9989541 residue_name_number cleaner0 2023-07-20T13:31:32Z DUMMY: Glu-115 0.9691086 structure_element cleaner0 2023-07-20T14:44:24Z SO: carboxy-terminus 0.99963653 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9996598 evidence cleaner0 2023-07-20T14:14:36Z DUMMY: structure 0.99958694 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density pone.0151285.g006.jpg pone.0151285.g006 FIG fig_title_caption 49457 Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus. 0.9996562 site cleaner0 2023-07-20T14:14:46Z SO: metal-binding site species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9995962 protein_state cleaner0 2023-07-20T14:48:43Z DUMMY: flexible structure_element SO: cleaner0 2023-07-20T14:44:24Z carboxy-terminus pone.0151285.g006.jpg pone.0151285.g006 FIG fig_caption 49564 (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. The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase. His-55 interacts with the double proline motif (Pro-109 and Pro-110). 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. (b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein. 0.9997719 chemical cleaner0 2023-07-20T14:19:52Z CHEBI: water 0.99921626 residue_name_number cleaner0 2023-07-20T13:32:56Z DUMMY: Glu-57 0.9992302 residue_name_number cleaner0 2023-07-20T13:33:02Z DUMMY: Tyr-85 0.99922436 residue_name_number cleaner0 2023-07-20T13:31:19Z DUMMY: His-93 0.9997919 chemical cleaner0 2023-07-20T14:19:55Z CHEBI: water 0.99941945 chemical cleaner0 2023-07-20T14:20:01Z CHEBI: Fe2+ 0.88510096 site cleaner0 2023-07-20T14:39:11Z SO: active side 0.99900013 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.9992514 residue_name_number cleaner0 2023-07-20T13:30:41Z DUMMY: His-55 0.9992842 structure_element cleaner0 2023-07-20T13:32:18Z SO: double proline motif 0.99929523 residue_name_number cleaner0 2023-07-20T13:28:44Z DUMMY: Pro-109 0.9992961 residue_name_number cleaner0 2023-07-20T13:28:50Z DUMMY: Pro-110 0.99925417 residue_name_number cleaner0 2023-07-20T13:31:32Z DUMMY: Glu-115 0.99933594 protein_state cleaner0 2023-07-20T14:48:45Z DUMMY: flexible structure_element SO: cleaner0 2023-07-20T14:44:24Z carboxy-terminus 0.4762111 species cleaner0 2023-07-20T10:06:59Z MESH: Sa 0.5403825 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.9995139 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.46799707 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9995493 evidence cleaner0 2023-07-20T14:14:51Z DUMMY: structure 0.999553 experimental_method cleaner0 2023-07-20T14:32:39Z MESH: overlay 0.99967325 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.9995157 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.9994874 evidence cleaner0 2023-07-20T14:14:54Z DUMMY: structure 0.37436584 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC RESULTS title_2 50273 The putative iron binding site of (Sa)EctC 0.99960923 site cleaner0 2023-07-20T13:33:21Z SO: iron binding site species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC RESULTS paragraph 50316 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. 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. Only the refinement of Fe2+ resulted in a visibly improved electron density, however with a low degree of occupancy. This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b). 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. 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. 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. 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. 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). 0.9995599 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.9994135 evidence cleaner0 2023-07-20T14:14:59Z DUMMY: structure species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9990086 oligomeric_state cleaner0 2023-07-20T13:33:47Z DUMMY: monomers 0.99944574 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density 0.9995765 structure_element cleaner0 2023-07-20T13:33:41Z SO: cupin barrel 0.5870309 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.99962735 chemical cleaner0 2023-07-20T13:34:02Z CHEBI: Fe2+ 0.99964345 chemical cleaner0 2023-07-20T13:34:10Z CHEBI: Zn2+ 0.9991924 evidence cleaner0 2023-07-20T14:15:03Z DUMMY: density experimental_method MESH: cleaner0 2023-07-20T14:33:11Z refined occupancy 0.999634 chemical cleaner0 2023-07-20T13:34:07Z CHEBI: Fe2+ 0.9995745 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density 0.9994524 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.99923414 residue_name_number cleaner0 2023-07-20T13:32:58Z DUMMY: Glu-57 0.9992427 residue_name_number cleaner0 2023-07-20T13:33:03Z DUMMY: Tyr-85 0.9992547 residue_name_number cleaner0 2023-07-20T13:31:19Z DUMMY: His-93 0.9990397 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.9992247 residue_name_number cleaner0 2023-07-20T13:32:58Z DUMMY: Glu-57 0.9992345 residue_name_number cleaner0 2023-07-20T13:33:03Z DUMMY: Tyr-85 0.9992223 residue_name_number cleaner0 2023-07-20T13:31:19Z DUMMY: His-93 0.99948376 site cleaner0 2023-07-20T13:34:21Z SO: iron binding sites 0.9988392 protein_state cleaner0 2023-07-20T13:34:28Z DUMMY: absence of species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9996319 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure 0.99956316 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.99976 chemical cleaner0 2023-07-20T14:20:05Z CHEBI: water 0.9997688 chemical cleaner0 2023-07-20T14:20:10Z CHEBI: water 0.99977607 chemical cleaner0 2023-07-20T14:20:13Z CHEBI: water 0.99950516 protein_state cleaner0 2023-07-20T14:48:50Z DUMMY: strictly conserved 0.978961 experimental_method cleaner0 2023-07-20T14:33:16Z MESH: alignment 0.9155193 protein_type cleaner0 2023-07-20T13:34:49Z MESH: EctC protein 0.9996103 protein_type cleaner0 2023-07-20T11:24:54Z MESH: EctC-type proteins RESULTS paragraph 51703 In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure. 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. 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). Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above. 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. 0.9996517 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.9994394 evidence cleaner0 2023-07-20T14:15:12Z DUMMY: structure 0.33843443 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.99958664 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density 0.9984459 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.9995256 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.9993711 evidence cleaner0 2023-07-20T14:15:16Z DUMMY: structure 0.9995761 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density 0.99966013 chemical cleaner0 2023-07-20T14:20:17Z CHEBI: water 0.9975165 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.99964285 chemical cleaner0 2023-07-20T14:20:19Z CHEBI: water 0.99965155 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.47516298 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9995999 evidence cleaner0 2023-07-20T14:15:19Z DUMMY: structure 0.99967086 experimental_method cleaner0 2023-07-20T14:33:22Z MESH: superimposition 0.55314845 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC 0.9995959 evidence cleaner0 2023-07-20T13:57:50Z DUMMY: crystal structures 0.9993043 residue_name_number cleaner0 2023-07-20T13:32:58Z DUMMY: Glu-57 0.99930924 residue_name_number cleaner0 2023-07-20T13:33:03Z DUMMY: Tyr-85 0.9993059 residue_name_number cleaner0 2023-07-20T13:31:20Z DUMMY: His-93 0.99863094 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.99952936 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.9990138 evidence cleaner0 2023-07-20T14:15:23Z DUMMY: structure 0.9994984 protein_state cleaner0 2023-07-20T13:38:06Z DUMMY: iron-free 0.99965525 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.99928147 evidence cleaner0 2023-07-20T14:15:26Z DUMMY: structure 0.9992802 residue_name_number cleaner0 2023-07-20T13:31:20Z DUMMY: His-93 0.9995225 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.9994363 evidence cleaner0 2023-07-20T14:15:28Z DUMMY: structure 0.99964875 structure_element cleaner0 2023-07-20T13:27:53Z SO: β-strand 0.9996643 structure_element cleaner0 2023-07-20T13:27:47Z SO: β5 0.9996266 site cleaner0 2023-07-20T13:28:17Z SO: iron-binding site chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.99957365 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase RESULTS paragraph 52742 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. 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. Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1). 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). 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. 0.9991734 residue_name_number cleaner0 2023-07-20T13:38:45Z DUMMY: Tyr-52 0.9997297 structure_element cleaner0 2023-07-20T14:44:33Z SO: loop 0.9996465 structure_element cleaner0 2023-07-20T13:27:53Z SO: β-strand 0.9996942 structure_element cleaner0 2023-07-20T13:27:47Z SO: β5 0.9996644 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.99950296 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.41048887 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.9991352 evidence cleaner0 2023-07-20T14:15:49Z DUMMY: structures 0.99953073 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.9989262 evidence cleaner0 2023-07-20T14:15:52Z DUMMY: structure 0.9991581 residue_name_number cleaner0 2023-07-20T13:38:45Z DUMMY: Tyr-52 0.9957058 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.9991247 residue_name_number cleaner0 2023-07-20T13:38:43Z DUMMY: Tyr-52 chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.99670815 experimental_method cleaner0 2023-07-20T14:33:29Z MESH: substitution 0.9991492 residue_name cleaner0 2023-07-20T13:38:55Z SO: Ala chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.9722253 protein_state cleaner0 2023-07-20T13:40:44Z DUMMY: mutant 0.99968576 experimental_method cleaner0 2023-07-20T14:33:33Z MESH: overlay 0.9996631 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.9995298 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.42811933 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.9995607 evidence cleaner0 2023-07-20T13:57:50Z DUMMY: crystal structures 0.99912995 residue_name_number cleaner0 2023-07-20T13:38:45Z DUMMY: Tyr-52 0.99530953 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.9992278 residue_name_number cleaner0 2023-07-20T13:32:58Z DUMMY: Glu-57 0.99924177 residue_name_number cleaner0 2023-07-20T13:33:03Z DUMMY: Tyr-85 0.9992725 residue_name_number cleaner0 2023-07-20T13:31:20Z DUMMY: His-93 0.9991281 residue_name_number cleaner0 2023-07-20T13:38:45Z DUMMY: Tyr-52 0.999544 protein_state cleaner0 2023-07-20T14:49:00Z DUMMY: strictly conserved 0.99956137 experimental_method cleaner0 2023-07-20T14:33:36Z MESH: alignment 0.99964917 protein_type cleaner0 2023-07-20T11:24:54Z MESH: EctC-type proteins 0.9996603 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.999398 protein_state cleaner0 2023-07-20T13:34:29Z DUMMY: absence of 0.9994852 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.7439358 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.99959594 evidence cleaner0 2023-07-20T13:57:50Z DUMMY: crystal structures 0.99913955 residue_name_number cleaner0 2023-07-20T13:38:45Z DUMMY: Tyr-52 RESULTS paragraph 53965 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. 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). For some of the presumptive iron-coordinating residues, additional site-directed mutagenesis experiments were carried out. To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant. This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1). 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. Collectively, these data suggest that the hydroxyl group of the Tyr-85 side chain is needed for the binding of the iron (Fig 6a). 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). 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. 0.9995678 site cleaner0 2023-07-20T13:33:22Z SO: iron binding site 0.99942875 experimental_method cleaner0 2023-07-20T13:39:48Z MESH: structure-guided site-directed mutagenesis species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:42Z EctC chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.99915075 residue_name_number cleaner0 2023-07-20T13:32:58Z DUMMY: Glu-57 0.9991446 residue_name_number cleaner0 2023-07-20T13:33:03Z DUMMY: Tyr-85 0.99909955 residue_name_number cleaner0 2023-07-20T13:31:20Z DUMMY: His-93 0.9344692 site cleaner0 2023-07-20T13:40:01Z SO: mono-nuclear iron center species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.67269844 protein cleaner0 2023-07-20T10:09:42Z PR: EctC 0.9995788 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure 0.98794144 experimental_method cleaner0 2023-07-20T14:33:41Z MESH: replaced 0.99890435 residue_name cleaner0 2023-07-20T13:40:27Z SO: Ala chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.5676748 protein_state cleaner0 2023-07-20T13:40:44Z DUMMY: mutant 0.99950314 site cleaner0 2023-07-20T13:40:16Z SO: iron-coordinating residues 0.99941367 experimental_method cleaner0 2023-07-20T13:40:21Z MESH: site-directed mutagenesis 0.99917907 residue_name_number cleaner0 2023-07-20T13:32:58Z DUMMY: Glu-57 0.9856354 residue_name cleaner0 2023-07-20T13:40:30Z SO: Asp 0.8235472 protein_state cleaner0 2023-07-20T14:38:42Z DUMMY: variant 0.9981299 protein_state cleaner0 2023-07-20T13:40:44Z DUMMY: mutant chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.9215003 residue_name cleaner0 2023-07-20T13:40:33Z SO: Ala 0.7259734 experimental_method cleaner0 2023-07-20T14:33:45Z MESH: substitution 0.6129925 experimental_method cleaner0 2023-07-20T14:33:48Z MESH: replaced 0.9991696 residue_name_number cleaner0 2023-07-20T13:33:03Z DUMMY: Tyr-85 0.99924386 residue_name cleaner0 2023-07-20T13:40:36Z SO: Phe 0.99925524 residue_name cleaner0 2023-07-20T13:40:40Z SO: Trp 0.99584913 protein_state cleaner0 2023-07-20T13:40:43Z DUMMY: mutant chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.99915314 residue_name_number cleaner0 2023-07-20T13:33:03Z DUMMY: Tyr-85 0.99948275 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.9980252 experimental_method cleaner0 2023-07-20T14:33:51Z MESH: replaced 0.9995284 site cleaner0 2023-07-20T14:39:16Z SO: iron-binding residue 0.99909085 residue_name_number cleaner0 2023-07-20T13:31:20Z DUMMY: His-93 0.99927825 residue_name cleaner0 2023-07-20T13:40:51Z SO: Asn species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.58080834 protein cleaner0 2023-07-20T10:09:42Z PR: EctC chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.9995037 protein_state cleaner0 2023-07-20T13:41:16Z DUMMY: wild-type 0.99956393 site cleaner0 2023-07-20T13:40:18Z SO: iron-coordinating residues 0.9991215 residue_name_number cleaner0 2023-07-20T13:32:58Z DUMMY: Glu-57 0.99911183 residue_name_number cleaner0 2023-07-20T13:33:03Z DUMMY: Tyr-85 0.9990993 residue_name_number cleaner0 2023-07-20T13:31:20Z DUMMY: His-93 0.99946946 chemical cleaner0 2023-07-20T13:41:21Z CHEBI: Fe2+ species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.68746006 protein cleaner0 2023-07-20T10:09:43Z PR: EctC RESULTS title_2 55597 A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate 0.50598353 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.99949026 evidence cleaner0 2023-07-20T14:15:57Z DUMMY: structure 0.99971735 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA RESULTS paragraph 55715 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). 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. This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement. 0.9995761 experimental_method cleaner0 2023-07-20T14:33:56Z MESH: co-crystallization 0.99930274 experimental_method cleaner0 2023-07-20T14:34:00Z MESH: soaking experiments species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.99963343 evidence cleaner0 2023-07-20T13:57:50Z DUMMY: crystal structures 0.99975777 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.9989285 chemical cleaner0 2023-07-20T10:07:54Z CHEBI: ectoine 0.99956965 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.9995155 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.52894163 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.99962676 evidence cleaner0 2023-07-20T14:16:02Z DUMMY: structure 0.9972017 structure_element cleaner0 2023-07-20T13:43:34Z SO: carboxy-terminal loop 0.99951136 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density 0.99958414 site cleaner0 2023-07-20T13:43:15Z SO: active site 0.9973748 experimental_method cleaner0 2023-07-20T14:34:04Z MESH: crystallographic refinement 0.9996444 protein_state cleaner0 2023-07-20T11:35:52Z DUMMY: open 0.99958867 evidence cleaner0 2023-07-20T14:16:06Z DUMMY: structure 0.41580853 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.99953204 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density RESULTS paragraph 56388 We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched. 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. 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. We therefore stress that we cannot identify neither the true chemically nature of this compound, nor its precise origin. experimental_method MESH: cleaner0 2023-07-20T14:34:37Z purification 0.99943024 experimental_method cleaner0 2023-07-20T14:34:41Z MESH: crystallization 0.999591 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.9980836 species cleaner0 2023-07-20T10:23:07Z MESH: E. coli 0.99773324 experimental_method cleaner0 2023-07-20T14:34:44Z MESH: crystallization 0.9997731 chemical cleaner0 2023-07-20T13:42:28Z CHEBI: PEG 0.99964523 evidence cleaner0 2023-07-20T14:16:14Z DUMMY: density 0.9997856 chemical cleaner0 2023-07-20T13:42:30Z CHEBI: PEG 0.99977535 chemical cleaner0 2023-07-20T13:42:30Z CHEBI: PEG 0.9997731 chemical cleaner0 2023-07-20T13:42:30Z CHEBI: PEG RESULTS paragraph 57081 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. However, to the best of our knowledge, hexane-1,6-diol is not part of the E. coli metabolome. 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. We note that both N-γ-ADABA and hexane-1,6-diol are both C6-compounds and display similar length (Fig 7a). 0.9991968 evidence cleaner0 2023-07-20T13:42:46Z DUMMY: electron density feature 0.26774096 species cleaner0 2023-07-20T10:06:59Z MESH: Sa 0.70911974 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.99976677 chemical cleaner0 2023-07-20T13:42:36Z CHEBI: hexane-1,6-diol 0.99958235 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density 0.99976915 chemical cleaner0 2023-07-20T13:42:37Z CHEBI: hexane-1,6-diol 0.99940926 species cleaner0 2023-07-20T10:23:07Z MESH: E. coli 0.99940443 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.9996128 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.9996047 site cleaner0 2023-07-20T13:43:15Z SO: active site 0.9997796 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.9997713 chemical cleaner0 2023-07-20T13:42:37Z CHEBI: hexane-1,6-diol pone.0151285.g007.jpg pone.0151285.g007 FIG fig_title_caption 57795 A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure. 0.99954915 site cleaner0 2023-07-20T13:43:13Z SO: active site 0.9995071 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.9989201 species cleaner0 2023-07-20T10:06:59Z MESH: Sa 0.9980453 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.9996127 evidence cleaner0 2023-07-20T11:35:43Z DUMMY: crystal structure pone.0151285.g007.jpg pone.0151285.g007 FIG fig_caption 57909 (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. (b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted. 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. The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange. The electron density was calculated as an omit map and contoured at 1.0 σ. 0.9995905 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density 0.99960107 site cleaner0 2023-07-20T13:43:15Z SO: active site 0.9995239 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed 0.99727964 evidence cleaner0 2023-07-20T14:16:21Z DUMMY: structure 0.5305522 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.9997797 chemical cleaner0 2023-07-20T13:42:37Z CHEBI: hexane-1,6-diol 0.9995757 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density 0.9997587 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.9996003 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.999565 site cleaner0 2023-07-20T14:39:22Z SO: binding site 0.9704798 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.9997806 chemical cleaner0 2023-07-20T13:42:37Z CHEBI: hexane-1,6-diol chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.9990362 protein_state cleaner0 2023-07-20T14:49:06Z DUMMY: flexible 0.9996913 structure_element cleaner0 2023-07-20T13:43:33Z SO: carboxy-terminal loop 0.4624071 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.9996306 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density 0.99962413 evidence cleaner0 2023-07-20T13:44:12Z DUMMY: omit map RESULTS paragraph 58667 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%. We also calculated an omit map and the electron density reappeared (Fig 7b). 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. Remarkably, all of these residues are highly conserved throughout the extended EctC protein family (Fig 2). 0.99945074 experimental_method cleaner0 2023-07-20T14:34:50Z MESH: refined 0.56919813 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.99954236 evidence cleaner0 2023-07-20T14:16:25Z DUMMY: structure 0.9994499 evidence cleaner0 2023-07-20T14:16:28Z DUMMY: R- and Rfree-factor 0.99959517 evidence cleaner0 2023-07-20T13:44:11Z DUMMY: omit map 0.9995711 evidence cleaner0 2023-07-20T13:31:04Z DUMMY: electron density 0.6303048 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.9996358 protein_state cleaner0 2023-07-20T14:49:10Z DUMMY: bound 0.99931145 residue_name_number cleaner0 2023-07-20T14:22:14Z DUMMY: Trp-21 0.99931717 residue_name_number cleaner0 2023-07-20T14:22:17Z DUMMY: Ser-23 0.9996448 structure_element cleaner0 2023-07-20T14:44:38Z SO: β-sheet 0.9996158 structure_element cleaner0 2023-07-20T14:44:42Z SO: β3 0.9993132 residue_name_number cleaner0 2023-07-20T13:45:00Z DUMMY: Thr-40 0.99966 structure_element cleaner0 2023-07-20T14:44:47Z SO: β-sheet 0.99950767 structure_element cleaner0 2023-07-20T14:44:50Z SO: β4 0.9993027 residue_name_number cleaner0 2023-07-20T14:22:21Z DUMMY: Cys-105 0.99930197 residue_name_number cleaner0 2023-07-20T13:45:34Z DUMMY: Phe-107 0.99964255 structure_element cleaner0 2023-07-20T14:44:53Z SO: β-sheet 0.99967504 structure_element cleaner0 2023-07-20T13:26:56Z SO: β11 0.99954045 protein_state cleaner0 2023-07-20T14:49:14Z DUMMY: highly conserved 0.8928305 protein_type cleaner0 2023-07-20T13:44:25Z MESH: EctC protein RESULTS title_2 59274 Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase 0.9994589 experimental_method cleaner0 2023-07-20T14:35:01Z MESH: Structure-guided site-directed mutagenesis 0.98101175 site cleaner0 2023-07-20T14:39:26Z SO: catalytic core 0.99961734 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase RESULTS paragraph 59363 In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues. 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). 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. 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. 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). 0.9936033 experimental_method cleaner0 2023-07-20T14:35:05Z MESH: alignment of the amino acid sequences 0.99969447 protein_type cleaner0 2023-07-20T11:24:54Z MESH: EctC-type proteins 0.9992889 protein_state cleaner0 2023-07-20T14:49:26Z DUMMY: strictly conserved 0.99925977 residue_name_number cleaner0 2023-07-20T13:44:59Z DUMMY: Thr-40 0.999253 residue_name_number cleaner0 2023-07-20T13:38:45Z DUMMY: Tyr-52 0.99923754 residue_name_number cleaner0 2023-07-20T13:30:41Z DUMMY: His-55 0.9992304 residue_name_number cleaner0 2023-07-20T13:32:58Z DUMMY: Glu-57 0.9992234 residue_name_number cleaner0 2023-07-20T13:45:14Z DUMMY: Gly-64 residue_name_number DUMMY: cleaner0 2023-07-20T13:33:03Z Tyr-85 0.9920979 residue_name_number cleaner0 2023-07-20T13:45:24Z DUMMY: Leu-87 0.9992099 residue_name_number cleaner0 2023-07-20T13:31:20Z DUMMY: His-93 0.9992581 residue_name_number cleaner0 2023-07-20T13:45:32Z DUMMY: Phe-107 0.9992681 residue_name_number cleaner0 2023-07-20T13:28:44Z DUMMY: Pro-109 0.9992566 residue_name_number cleaner0 2023-07-20T13:45:41Z DUMMY: Gly-113 0.99928117 residue_name_number cleaner0 2023-07-20T13:31:32Z DUMMY: Glu-115 0.999279 residue_name_number cleaner0 2023-07-20T13:45:50Z DUMMY: His-117 0.7303695 species cleaner0 2023-07-20T10:06:59Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.9992512 residue_name_number cleaner0 2023-07-20T13:45:15Z DUMMY: Gly-64 0.99925476 residue_name_number cleaner0 2023-07-20T13:28:44Z DUMMY: Pro-109 0.9992551 residue_name_number cleaner0 2023-07-20T13:45:42Z DUMMY: Gly-113 0.9996702 structure_element cleaner0 2023-07-20T14:45:09Z SO: β-strands 0.99966365 structure_element cleaner0 2023-07-20T14:45:13Z SO: α-helices 0.99937916 protein_state cleaner0 2023-07-20T14:49:30Z DUMMY: flexible structure_element SO: cleaner0 2023-07-20T14:44:24Z carboxy-terminus 0.3778841 species cleaner0 2023-07-20T10:06:59Z MESH: Sa 0.6953704 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.999218 residue_name_number cleaner0 2023-07-20T13:32:58Z DUMMY: Glu-57 0.9992469 residue_name_number cleaner0 2023-07-20T13:33:03Z DUMMY: Tyr-85 0.99925154 residue_name_number cleaner0 2023-07-20T13:31:20Z DUMMY: His-93 0.99963474 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron RESULTS paragraph 60186 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). 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. 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. In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1). species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.54778117 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.9992084 evidence cleaner0 2023-07-20T14:16:35Z DUMMY: structure 0.99942106 experimental_method cleaner0 2023-07-20T13:46:32Z MESH: structure-guided site-directed mutagenesis 0.9990582 protein_state cleaner0 2023-07-20T13:40:44Z DUMMY: mutant species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.43671474 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.9973287 species cleaner0 2023-07-20T10:23:07Z MESH: E. coli 0.99090666 experimental_method cleaner0 2023-07-20T14:35:14Z MESH: affinity chromatography species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.9626147 experimental_method cleaner0 2023-07-20T13:46:48Z MESH: single time-point enzyme assay 0.99954396 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type 0.44324213 species cleaner0 2023-07-20T10:06:59Z MESH: Sa 0.34864613 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.9997834 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.9997422 chemical cleaner0 2023-07-20T10:07:54Z CHEBI: ectoine 0.99838936 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.9993874 protein_state cleaner0 2023-07-20T13:40:44Z DUMMY: mutant species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.25436148 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.9979036 experimental_method cleaner0 2023-07-20T11:27:08Z MESH: colorimetric assay RESULTS paragraph 60967 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). 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. However, their catalytic activity was substantially impaired (Table 1). 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). 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. 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. 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. However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1). 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. 0.9990735 protein_state cleaner0 2023-07-20T14:49:36Z DUMMY: evolutionarily conserved 0.9991827 residue_name_number cleaner0 2023-07-20T14:22:28Z DUMMY: Trp-21 0.999167 residue_name_number cleaner0 2023-07-20T14:22:33Z DUMMY: Ser-23 0.99915034 residue_name_number cleaner0 2023-07-20T13:45:00Z DUMMY: Thr-40 0.9991319 residue_name_number cleaner0 2023-07-20T14:22:36Z DUMMY: Cys-105 0.99915427 residue_name_number cleaner0 2023-07-20T13:45:34Z DUMMY: Phe-107 0.9995274 protein_state cleaner0 2023-07-20T11:36:05Z DUMMY: semi-closed species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.6939467 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.99955386 evidence cleaner0 2023-07-20T14:16:38Z DUMMY: structure 0.9988753 experimental_method cleaner0 2023-07-20T14:35:21Z MESH: replaced 0.99910176 residue_name cleaner0 2023-07-20T13:48:04Z SO: Ala chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.4964614 protein_state cleaner0 2023-07-20T13:40:44Z DUMMY: mutant 0.9991894 residue_name_number cleaner0 2023-07-20T13:45:00Z DUMMY: Thr-40 0.99962234 structure_element cleaner0 2023-07-20T13:27:53Z SO: β-strand 0.9996573 structure_element cleaner0 2023-07-20T13:27:47Z SO: β5 0.99946547 structure_element cleaner0 2023-07-20T13:33:43Z SO: cupin barrel species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.62484354 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.99685633 experimental_method cleaner0 2023-07-20T14:35:25Z MESH: replaced 0.99922866 residue_name_number cleaner0 2023-07-20T13:45:34Z DUMMY: Phe-107 0.99920565 residue_name cleaner0 2023-07-20T13:47:54Z SO: Tyr 0.99922764 residue_name cleaner0 2023-07-20T13:47:56Z SO: Trp mutant MESH: cleaner0 2023-07-20T13:47:41Z Phe-107/Tyr 0.8910407 experimental_method cleaner0 2023-07-20T14:35:29Z MESH: substitution 0.8363883 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.9449046 mutant cleaner0 2023-07-20T13:48:37Z MESH: Phe-107/Trp 0.7973649 experimental_method cleaner0 2023-07-20T14:35:31Z MESH: substitution chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.99957496 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type protein_state DUMMY: cleaner0 2023-07-20T13:40:44Z mutant 0.9886099 residue_number cleaner0 2023-07-20T14:36:59Z DUMMY: 107 species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.785677 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.96788937 experimental_method cleaner0 2023-07-20T14:35:34Z MESH: substitution chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.9993773 experimental_method cleaner0 2023-07-20T14:35:37Z MESH: Replacement 0.9991917 residue_name cleaner0 2023-07-20T13:47:59Z SO: Cys species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.8644455 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.9991381 residue_name_number cleaner0 2023-07-20T14:22:40Z DUMMY: Cys-105 0.99918383 residue_name cleaner0 2023-07-20T13:48:01Z SO: Ser 0.9991957 protein_type cleaner0 2023-07-20T13:49:12Z MESH: EctC proteins species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.49414182 protein_state cleaner0 2023-07-20T14:49:45Z DUMMY: variant 0.9959459 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.9995808 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type 0.9852306 mutant cleaner0 2023-07-20T13:48:20Z MESH: Cys-105/Ala 0.60997516 protein_state cleaner0 2023-07-20T14:49:49Z DUMMY: variant 0.9971433 protein_state cleaner0 2023-07-20T14:49:53Z DUMMY: catalytically inactive chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.9989643 residue_name cleaner0 2023-07-20T13:48:06Z SO: Cys 0.9991562 residue_name_number cleaner0 2023-07-20T14:22:43Z DUMMY: Cys-105 0.9991806 residue_name_number cleaner0 2023-07-20T14:22:46Z DUMMY: Ser-105 0.9995801 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase RESULTS paragraph 62729 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). 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). This is different for the His-55/Ala substitution. 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). Each of these residues is evolutionarily highly conserved. 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. 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). 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). 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. 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. 0.8126707 experimental_method cleaner0 2023-07-20T14:35:43Z MESH: amino acid substitutions chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron mutant MESH: cleaner0 2023-07-20T13:49:54Z Tyr-52/Ala 0.99514467 mutant cleaner0 2023-07-20T13:50:07Z MESH: His-55/Ala species MESH: cleaner0 2023-07-20T10:06:59Z Sa 0.4194266 protein cleaner0 2023-07-20T10:09:43Z PR: EctC species MESH: cleaner0 2023-07-20T10:06:59Z Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.99959433 evidence cleaner0 2023-07-20T13:57:50Z DUMMY: crystal structures 0.9994112 experimental_method cleaner0 2023-07-20T14:35:47Z MESH: replacement 0.99900836 residue_name_number cleaner0 2023-07-20T13:38:45Z DUMMY: Tyr-52 0.9989157 residue_name cleaner0 2023-07-20T13:50:25Z SO: Ala chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.9857507 mutant cleaner0 2023-07-20T13:50:08Z MESH: His-55/Ala 0.97617674 structure_element cleaner0 2023-07-20T13:50:48Z SO: carboxy-terminal region species MESH: cleaner0 2023-07-20T10:07:00Z Sa 0.45931357 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.9991133 residue_name_number cleaner0 2023-07-20T13:31:32Z DUMMY: Glu-115 0.9989636 residue_name_number cleaner0 2023-07-20T13:30:41Z DUMMY: His-55 0.999028 residue_name_number cleaner0 2023-07-20T13:30:41Z DUMMY: His-55 0.99918747 residue_name_number cleaner0 2023-07-20T13:28:50Z DUMMY: Pro-110 0.9993026 protein_state cleaner0 2023-07-20T14:49:58Z DUMMY: evolutionarily highly conserved 0.9575634 experimental_method cleaner0 2023-07-20T14:35:49Z MESH: substitution 0.99907035 residue_name_number cleaner0 2023-07-20T13:31:32Z DUMMY: Glu-115 0.9990751 residue_name_number cleaner0 2023-07-20T13:30:41Z DUMMY: His-55 0.99904245 residue_name cleaner0 2023-07-20T13:51:41Z SO: Ala 0.9991319 site cleaner0 2023-07-20T14:39:33Z SO: interactive network 0.96925527 mutant cleaner0 2023-07-20T13:51:08Z MESH: Glu-115/Ala 0.98248357 mutant cleaner0 2023-07-20T13:50:08Z MESH: His-55/Ala 0.9995592 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type 0.9973091 mutant cleaner0 2023-07-20T13:51:09Z MESH: Glu-115/Ala 0.9991985 protein_state cleaner0 2023-07-20T13:40:45Z DUMMY: mutant 0.9979682 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type 0.9442199 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron chemical CHEBI: cleaner0 2023-07-20T11:22:01Z iron 0.9923347 mutant cleaner0 2023-07-20T13:50:08Z MESH: His-55/Ala 0.99822956 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type 0.9986185 experimental_method cleaner0 2023-07-20T14:35:54Z MESH: replaced 0.99916977 residue_name_number cleaner0 2023-07-20T13:31:32Z DUMMY: Glu-115 0.99923563 residue_name cleaner0 2023-07-20T13:51:15Z SO: Asp species MESH: cleaner0 2023-07-20T10:07:00Z Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.99856275 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type 0.9908215 chemical cleaner0 2023-07-20T11:22:01Z CHEBI: iron 0.9920697 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type structure_element SO: cleaner0 2023-07-20T14:44:24Z carboxy-terminus 0.476489 species cleaner0 2023-07-20T10:07:00Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.99950236 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase RESULTS paragraph 64268 Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family. The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1). 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). We currently cannot comment on possible functional role Asp-91. 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. 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. 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). 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. 0.999248 residue_name_number cleaner0 2023-07-20T13:45:25Z DUMMY: Leu-87 0.9992301 residue_name_number cleaner0 2023-07-20T13:51:53Z DUMMY: Asp-91 0.9995029 protein_state cleaner0 2023-07-20T14:50:02Z DUMMY: highly conserved 0.99949646 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.9994337 experimental_method cleaner0 2023-07-20T14:35:57Z MESH: replacement 0.9992523 residue_name_number cleaner0 2023-07-20T13:45:25Z DUMMY: Leu-87 0.9991021 residue_name cleaner0 2023-07-20T13:52:17Z SO: Ala 0.9994435 experimental_method cleaner0 2023-07-20T14:36:02Z MESH: replacement 0.9992148 residue_name_number cleaner0 2023-07-20T13:51:54Z DUMMY: Asp-91 0.99909496 residue_name cleaner0 2023-07-20T13:52:12Z SO: Ala 0.999158 residue_name cleaner0 2023-07-20T13:52:14Z SO: Glu species MESH: cleaner0 2023-07-20T10:07:00Z Sa 0.5594292 protein cleaner0 2023-07-20T10:09:43Z PR: EctC 0.9992165 residue_name_number cleaner0 2023-07-20T13:51:54Z DUMMY: Asp-91 0.9992547 residue_name_number cleaner0 2023-07-20T13:45:25Z DUMMY: Leu-87 0.9996314 structure_element cleaner0 2023-07-20T14:45:18Z SO: β-sheets 0.9995923 site cleaner0 2023-07-20T13:21:56Z SO: dimer interface 0.9992736 residue_name_number cleaner0 2023-07-20T13:33:03Z DUMMY: Tyr-85 0.9993905 chemical cleaner0 2023-07-20T11:22:02Z CHEBI: iron 0.63578343 species cleaner0 2023-07-20T10:07:00Z MESH: Sa protein PR: cleaner0 2023-07-20T10:09:43Z EctC 0.99964094 site cleaner0 2023-07-20T13:43:15Z SO: active site 0.9992296 residue_name_number cleaner0 2023-07-20T13:45:51Z DUMMY: His-117 0.9994899 protein_state cleaner0 2023-07-20T14:50:05Z DUMMY: strictly conserved 0.9991002 experimental_method cleaner0 2023-07-20T14:36:05Z MESH: substitution 0.9991954 residue_name cleaner0 2023-07-20T14:22:07Z SO: Ala chemical CHEBI: cleaner0 2023-07-20T11:22:02Z iron 0.9992239 residue_name_number cleaner0 2023-07-20T13:45:51Z DUMMY: His-117 species MESH: cleaner0 2023-07-20T10:07:00Z Sa 0.632539 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.9993117 evidence cleaner0 2023-07-20T13:52:53Z DUMMY: structure 0.9996658 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase RESULTS paragraph 65360 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. Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1). This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content. experimental_method MESH: cleaner0 2023-07-20T14:36:39Z mutagenesis experiments 0.93242997 experimental_method cleaner0 2023-07-20T14:36:13Z MESH: substituted 0.99923414 residue_name_number cleaner0 2023-07-20T13:53:06Z DUMMY: Thr-41 0.9992416 residue_name_number cleaner0 2023-07-20T13:53:12Z DUMMY: His-51 0.99938995 protein_state cleaner0 2023-07-20T14:50:08Z DUMMY: not evolutionarily conserved 0.9996739 protein_type cleaner0 2023-07-20T11:24:54Z MESH: EctC-type proteins 0.99900913 residue_name cleaner0 2023-07-20T13:53:02Z SO: Ala species MESH: cleaner0 2023-07-20T10:07:00Z Sa 0.40038544 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.999278 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type chemical CHEBI: cleaner0 2023-07-20T11:22:02Z iron 0.9995473 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type species MESH: cleaner0 2023-07-20T10:07:00Z Sa protein PR: cleaner0 2023-07-20T10:09:44Z EctC chemical CHEBI: cleaner0 2023-07-20T11:22:02Z iron DISCUSS title_1 65848 Discussion DISCUSS paragraph 65859 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. 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. 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. 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. 0.99896866 evidence cleaner0 2023-07-20T13:53:40Z DUMMY: crystallographic data 0.9955268 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.7606321 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.9992632 taxonomy_domain cleaner0 2023-07-20T13:54:44Z DUMMY: microbial 0.86524945 chemical cleaner0 2023-07-20T10:07:54Z CHEBI: ectoine 0.99958295 protein_type cleaner0 2023-07-20T10:07:22Z MESH: cupin superfamily 0.58500177 species cleaner0 2023-07-20T10:07:00Z MESH: Sa 0.8823065 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.9165128 structure_element cleaner0 2023-07-20T14:45:23Z SO: β-strands 0.9996851 structure_element cleaner0 2023-07-20T13:54:19Z SO: β1-β11 0.9996598 structure_element cleaner0 2023-07-20T14:45:27Z SO: α-helices 0.99969345 structure_element cleaner0 2023-07-20T14:45:29Z SO: α-I 0.99968463 structure_element cleaner0 2023-07-20T14:45:32Z SO: α-II 0.99958915 evidence cleaner0 2023-07-20T13:57:50Z DUMMY: crystal structures 0.9995442 protein_type cleaner0 2023-07-20T11:22:39Z MESH: cupins 0.9996916 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.9996798 protein_type cleaner0 2023-07-20T14:17:41Z MESH: polyketide cyclase 0.99957806 protein cleaner0 2023-07-20T13:20:13Z PR: RemF 0.99837685 protein_type cleaner0 2023-07-20T14:17:54Z MESH: cupin-related 0.9934255 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.99956805 protein cleaner0 2023-07-20T13:20:13Z PR: RemF 0.99773055 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.9995147 protein cleaner0 2023-07-20T13:20:13Z PR: RemF 0.9996712 protein_type cleaner0 2023-07-20T11:24:54Z MESH: EctC-type proteins 0.9993017 taxonomy_domain cleaner0 2023-07-20T13:54:43Z DUMMY: microbial 0.999644 protein_type cleaner0 2023-07-20T13:54:39Z MESH: RemF-like DISCUSS paragraph 66824 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. Most cupins contain transition state metals that can promote different types of chemical reactions. 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. We report here for the first time that the ectoine synthase is a metal-dependent enzyme. ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction. 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). 0.9402873 taxonomy_domain cleaner0 2023-07-20T13:54:54Z DUMMY: pro- 0.6676487 taxonomy_domain cleaner0 2023-07-20T13:54:57Z DUMMY: eukaryotic 0.9990274 protein_type cleaner0 2023-07-20T10:07:22Z MESH: cupin superfamily 0.99962854 protein_type cleaner0 2023-07-20T11:22:39Z MESH: cupins 0.9995135 protein_type cleaner0 2023-07-20T14:18:01Z MESH: cupin-related proteins 0.99931794 protein_type cleaner0 2023-07-20T14:18:10Z MESH: metallo-chaperones 0.97990215 protein_state cleaner0 2023-07-20T14:50:18Z DUMMY: bound 0.98385483 chemical cleaner0 2023-07-20T13:55:09Z CHEBI: metal 0.99956536 site cleaner0 2023-07-20T14:39:38Z SO: active sites 0.9996103 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.9995976 experimental_method cleaner0 2023-07-20T13:55:26Z MESH: ICP-MS 0.99949604 experimental_method cleaner0 2023-07-20T13:55:31Z MESH: metal-depletion and reconstitution experiments 0.99902046 chemical cleaner0 2023-07-20T11:22:02Z CHEBI: iron chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal 0.99965394 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.9996189 protein_type cleaner0 2023-07-20T11:22:39Z MESH: cupins 0.9996886 protein cleaner0 2023-07-20T10:09:44Z PR: EctC chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal DISCUSS paragraph 67707 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). The architecture of the metal center of ectoine synthase seems to be subjected to considerable evolutionary constraints. 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). chemical CHEBI: cleaner0 2023-07-20T13:55:10Z metal species MESH: cleaner0 2023-07-20T10:07:00Z Sa protein PR: cleaner0 2023-07-20T10:09:44Z EctC 0.99939793 experimental_method cleaner0 2023-07-20T13:55:45Z MESH: structure-guided site-directed mutagenesis 0.9995793 site cleaner0 2023-07-20T13:55:55Z SO: iron-binding residues 0.99940354 site cleaner0 2023-07-20T13:56:05Z SO: metal center 0.9993664 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.99930215 residue_name_number cleaner0 2023-07-20T13:32:58Z DUMMY: Glu-57 0.9993043 residue_name_number cleaner0 2023-07-20T13:33:03Z DUMMY: Tyr-85 0.99931043 residue_name_number cleaner0 2023-07-20T13:31:20Z DUMMY: His-93 0.9994848 protein_state cleaner0 2023-07-20T13:56:17Z DUMMY: strictly conserved 0.9996911 protein_type cleaner0 2023-07-20T11:24:54Z MESH: EctC-type proteins 0.99945575 taxonomy_domain cleaner0 2023-07-20T14:21:50Z DUMMY: bacterial 0.99945897 taxonomy_domain cleaner0 2023-07-20T14:21:55Z DUMMY: archaeal DISCUSS paragraph 68362 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). 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. 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. 0.9997269 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.95461667 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.99970835 chemical cleaner0 2023-07-20T10:24:53Z CHEBI: N-α-ADABA 0.99932873 chemical cleaner0 2023-07-20T10:07:54Z CHEBI: ectoine evidence DUMMY: cleaner0 2023-07-20T11:31:33Z catalytic efficiency 0.9995589 site cleaner0 2023-07-20T13:43:15Z SO: active site 0.99962765 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.99893576 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.99979264 chemical cleaner0 2023-07-20T10:18:49Z CHEBI: ADPC 0.9997565 chemical cleaner0 2023-07-20T13:56:30Z CHEBI: glutamine 0.9997123 chemical cleaner0 2023-07-20T10:24:53Z CHEBI: N-α-ADABA 0.99957216 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.99946934 taxonomy_domain cleaner0 2023-07-20T11:32:08Z DUMMY: microorganisms 0.99968433 chemical cleaner0 2023-07-20T10:07:54Z CHEBI: ectoine 0.9997078 chemical cleaner0 2023-07-20T10:24:53Z CHEBI: N-α-ADABA DISCUSS paragraph 69193 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). 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. 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). 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. 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). 0.94073266 chemical cleaner0 2023-07-20T14:20:24Z CHEBI: C6 species MESH: cleaner0 2023-07-20T10:07:00Z Sa protein PR: cleaner0 2023-07-20T10:09:44Z EctC 0.9991386 evidence cleaner0 2023-07-20T13:56:52Z DUMMY: structure protein_type MESH: cleaner0 2023-07-20T10:07:30Z ectoine synthase 0.999745 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.9959065 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.99957526 site cleaner0 2023-07-20T13:43:15Z SO: active site 0.99945617 experimental_method cleaner0 2023-07-20T14:36:44Z MESH: site-directed mutagenesis species MESH: cleaner0 2023-07-20T10:07:00Z Sa protein PR: cleaner0 2023-07-20T10:09:44Z EctC 0.98933226 protein_state cleaner0 2023-07-20T13:41:17Z DUMMY: wild-type 0.9982116 chemical cleaner0 2023-07-20T11:22:02Z CHEBI: iron 0.9994867 protein_state cleaner0 2023-07-20T13:57:10Z DUMMY: strongly conserved 0.9996275 protein_type cleaner0 2023-07-20T11:24:54Z MESH: EctC-type proteins 0.9997193 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.999475 evidence cleaner0 2023-07-20T13:53:41Z DUMMY: crystallographic data 0.9749029 experimental_method cleaner0 2023-07-20T14:36:48Z MESH: site-directed mutagenesis study 0.99662185 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.99958104 site cleaner0 2023-07-20T13:43:15Z SO: active site DISCUSS paragraph 70236 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. 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. 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. It is hoped that these can be further employed to obtain EctC crystal structures with either the substrate or the reaction product. 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. 0.99967456 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase taxonomy_domain DUMMY: cleaner0 2023-07-20T10:19:51Z marine bacterium 0.9993682 species cleaner0 2023-07-20T10:21:25Z MESH: S. alaskensis 0.9996017 evidence cleaner0 2023-07-20T13:57:50Z DUMMY: crystal structures 0.99951786 protein_state cleaner0 2023-07-20T14:50:23Z DUMMY: full-length 0.9908317 species cleaner0 2023-07-20T10:07:00Z MESH: Sa 0.9994854 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.99932766 protein_state cleaner0 2023-07-20T13:57:27Z DUMMY: in complex with 0.9996761 chemical cleaner0 2023-07-20T10:18:35Z CHEBI: N-γ-ADABA 0.85791844 chemical cleaner0 2023-07-20T10:07:54Z CHEBI: ectoine 0.99936575 taxonomy_domain cleaner0 2023-07-20T13:54:44Z DUMMY: microbial 0.9690008 chemical cleaner0 2023-07-20T10:07:54Z CHEBI: ectoine 0.99842644 protein_type cleaner0 2023-07-20T14:18:15Z MESH: EctC proteins 0.9996333 protein cleaner0 2023-07-20T10:09:44Z PR: EctC 0.99962413 evidence cleaner0 2023-07-20T13:57:49Z DUMMY: crystal structures 0.99965763 protein_type cleaner0 2023-07-20T10:07:30Z MESH: ectoine synthase 0.99684787 protein_state cleaner0 2023-07-20T14:50:28Z DUMMY: metal dependent 0.9996215 evidence cleaner0 2023-07-20T13:57:50Z DUMMY: crystal structures 0.99962616 protein cleaner0 2023-07-20T10:09:44Z PR: EctC SUPPL title_1 71175 Supporting Information REF title 71198 References 1 24 surname:Yancey;given-names:PH 15651637 REF Sci Prog ref 87 2004 71209 Compatible and counteracting solutes: protecting cells from the Dead Sea to the deep sea 319 330 surname:Kempf;given-names:B surname:Bremer;given-names:E 9818351 REF Arch Microbiol ref 170 1998 71298 Uptake and synthesis of compatible solutes as microbial stress responses to high osmolality environments 121 147 surname:Csonka;given-names:LN 2651863 REF Microbiol Rev ref 53 1989 71403 Physiological and genetic responses of bacteria to osmotic stress 7309 7313 surname:Burg;given-names:MB surname:Ferraris;given-names:JD 10.1074/jbc.R700042200 18256030 REF J Biol Chem ref 283 2008 71469 Intracellular organic osmolytes: function and regulation 743 754 surname:Roeßler;given-names:M surname:Müller;given-names:V REF Env Microbiol Rep ref 3 2001 71526 Osmoadaptation in bacteria and archaea: common principles and differences 13997 14002 surname:Street;given-names:TO surname:Bolen;given-names:DW surname:Rose;given-names:GD 16968772 REF Proc Natl Acad Sci U S A ref 103 2006 71600 A molecular mechanism for osmolyte-induced protein stability 143 148 surname:Record;given-names:MT;suffix:Jr surname:Courtenay;given-names:ES surname:Cayley;given-names:DS surname:Guttman;given-names:HJ 9584618 REF Trends Biochem Sci ref 23 1998 71661 Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water 215 238 surname:Wood;given-names:JM 10.1146/annurev-micro-090110-102815 21663439 REF Annu Rev Microbiol ref 65 2011 71759 Bacterial osmoregulation: a paradigm for the study of cellular homeostasis 12596 12609 surname:Cayley;given-names:S surname:Record;given-names:MT;suffix:Jr 14580206 REF Biochemistry ref 42 2003 71834 Roles of cytoplasmic osmolytes, water, and crowding in the response of Escherichia coli to osmotic stress: biophysical basis of osmoprotection by glycine betaine 135 139 surname:Galinski;given-names:EA surname:Pfeiffer;given-names:HP surname:Trüper;given-names:HG 3838936 REF Eur J Biochem ref 149 1985 71996 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. 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