Crystal Structure of Mycobacterium tuberculosis D-3-Phosphoglycerate Dehydrogenase (2005)

Phosphoglycerate dehydrogenases exist in at least three different structural motifs. The first d-3-phosphoglycerate dehydrogenase structure to be determined was from Escherichia coli and is a tetramer composed of identical subunits that contain three discernable structural domains. The crystal structure of d-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis has been determined at 2.3 Å. This enzyme represents a second structural motif of the d-3-phosphoglycerate dehydrogenase family, one that contains an extended C-terminal region. This structure is also a tetramer of identical subunits, and the extended motif of 135 amino acids exists as a fourth structural domain. This intervening domain exerts quite a surprising characteristic to the structure by introducing significant asymmetry in the tetramer. The asymmetric unit is composed of two identical subunits that exist in two different conformations characterized by rotation of ∼180° around a hinge connecting two of the four domains. This asymmetric arrangement results in the formation of two different and distinct domain interfaces between identical domains in the asymmetric unit. As a result, the surface of the intervening domain that is exposed to solvent in one subunit is turned inward in the other subunit toward the center of the structure where it makes contact with other structural elements. Significant asymmetry is also seen at the subunit level where different conformations exist at the NAD-binding site and the putative serine-binding site in the two unique subunits. Phosphoglycerate dehydrogenases exist in at least three different structural motifs. The first d-3-phosphoglycerate dehydrogenase structure to be determined was from Escherichia coli and is a tetramer composed of identical subunits that contain three discernable structural domains. The crystal structure of d-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis has been determined at 2.3 Å. This enzyme represents a second structural motif of the d-3-phosphoglycerate dehydrogenase family, one that contains an extended C-terminal region. This structure is also a tetramer of identical subunits, and the extended motif of 135 amino acids exists as a fourth structural domain. This intervening domain exerts quite a surprising characteristic to the structure by introducing significant asymmetry in the tetramer. The asymmetric unit is composed of two identical subunits that exist in two different conformations characterized by rotation of ∼180° around a hinge connecting two of the four domains. This asymmetric arrangement results in the formation of two different and distinct domain interfaces between identical domains in the asymmetric unit. As a result, the surface of the intervening domain that is exposed to solvent in one subunit is turned inward in the other subunit toward the center of the structure where it makes contact with other structural elements. Significant asymmetry is also seen at the subunit level where different conformations exist at the NAD-binding site and the putative serine-binding site in the two unique subunits. l-Serine acts as a key intermediate in a number of important metabolic pathways. It can be synthesized from the glycolytic intermediate, d-3-phosphoglycerate, in the phosphorylated pathway of serine biosynthesis. d-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) 1The abbreviations used are: PGDH, d-3-phosphoglycerate dehydrogenase; SeMet, selenomethione; MAD, multiple anomalous dispersion; MES, 2-morpholinoethanesulfonic acid; r.m.s.d., root mean square deviation. catalyzes the first committed step in this conversion of d-3-phosphoglycerate to phosphohydroxypyruvate utilizing NAD+ as a cofactor. Subsequently, phosphohydroxypyruvate is converted to phosphoserine by phosphoserine transaminase and then to l-serine by phosphoserine phosphatase (1Walsh D.A. Sallach H.J. J. Biol. Chem. 1966; 241: 4068-4076Abstract Full Text PDF PubMed Google Scholar, 2Ichihara A. Greenberg D.M. J. Biol. Chem. 1957; 224: 331-340Abstract Full Text PDF PubMed Google Scholar). PGDH also belongs to a family of proteins classified as 2-hydroxy acid dehydrogenases that are generally specific for substrates with a D-configuration (3Grant G.A. Biochem. Biophys. Res. Commun. 1989; 165: 1371-1374Crossref PubMed Scopus (77) Google Scholar). It shares sequence homology with other members of this family such as formate dehydrogenase and glycerate dehydrogenase whose structures are also known (4Lamzin V.S. Dauter Z. Popov V.O. Harutyunyan E.H. Wilson K.S. J. Mol. Biol. 1994; 236: 759-785Crossref PubMed Scopus (165) Google Scholar, 5Goldberg J.D. Yoshida T. Brick P. J. Mol. Biol. 1994; 236: 1123-1140Crossref PubMed Scopus (102) Google Scholar). PGDH appears to be ubiquitously found in all organisms. It exists in at least three different basic structural motifs that do not appear to be strictly specific for organism type (6Dey S. Hu Z. Lan Xu X. Sacchettini J.C. Grant G.A. J. Biol. Chem. 2005; 280: 14884-14891Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The PGDH of some bacteria and some lower eukaryotes, such as yeast and Neurospora are similar to the Escherichia coli enzyme. In addition to substrate and nucleotide-binding domains, they possess a homologous C-terminal domain that, in E. coli, is involved in regulation of activity through binding the effector, l-serine. Other bacteria such as Mycobacterium, Bacillus subtilis, Corynebacterium, plants such as Arabidopsis, and higher order eukaryotes, including mammals, possess a large polypeptide insertion (∼150–200 amino acids) in their C-terminal segment immediately following the substrate-binding domain and before the C-terminal domain. A third motif, which lacks the C-terminal regulatory domain altogether, is also found in some bacteria such as Clostridium and including some, such as Mycobacterium tuberculosis, that also produce PGDH with the extended C-terminal motif. Recent studies on the parasite Entamoeba histolytica (7Ali V. Hashimoto T. Shigeta Y. Nozaki T. Eur. J. Biochem. 2004; 271: 2670-2681Crossref PubMed Scopus (42) Google Scholar) found a variation of this third motif where the dimeric enzyme appears to have a lysine residue rather than a histidine at the active site. The SerA1 gene from M. tuberculosis (H37Rv lab strain) coding for PGDH has been found to be an essential gene in mycobacterium by Himar1-based transposon mutagenesis in H37Rv strain (8Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (2025) Google Scholar). Kinetic studies (6Dey S. Hu Z. Lan Xu X. Sacchettini J.C. Grant G.A. J. Biol. Chem. 2005; 280: 14884-14891Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) have revealed that M. tuberculosis PGDH has a Km value of ∼85 μm for hydroxypyruvic acid phosphate and a kcat/Km value of ∼5.6 × 106 m–1 s–1. Unlike E. coli PGDH, it displays no activity with α-ketoglutarate. In addition, M. tuberculosis PGDH exhibits substrate inhibition similar to what has been reported for rat liver PGDH (9Achouri Y. Rider M.H. Van Schaftingen E. Robbi M. Biochem. J. 1997; 323: 365-370Crossref PubMed Scopus (86) Google Scholar). Increased ionic strength (100–400 mm) reduces the degree of substrate inhibition and shifts the maximal activity to higher substrate concentrations, also seen in rat liver PGDH. In addition, the activity of the enzyme is also dependent on the ionic strength of the buffer. E. coli PGDH is strongly inhibited by l-serine (I0.5 ∼ 2–4 μm) and classified as a V-type enzyme in which the velocity of the reaction rather than the affinity of the substrates is mainly affected (10Sugimoto E. Pizer L.I. J. Biol. Chem. 1968; 243: 2081-2089Abstract Full Text PDF PubMed Google Scholar). In B. subtilis (11Saski R. Pizer L.I. Eur. J. Biochem. 1975; 51: 415-427Crossref PubMed Scopus (29) Google Scholar) and Corynebacterium glutamicum (12Peters-Wendisch P. Netzer R. Eggeling L. Sahm H. Appl. Microbiol. Biotechnol. 2002; 60: 437-441Crossref PubMed Scopus (50) Google Scholar), PGDH is less sensitive to l-serine and requires extensive preincubation of the enzyme with the inhibitor. In the pea (Pisum sativum), the sensitivity to l-serine has been reported to be cold labile (13Slaughter J.C. Davies D.D. Methods Enzymol. 1975; 41: 278-281Crossref PubMed Scopus (10) Google Scholar). l-Serine has been reported to have no effect on the activity of PGDH from rat liver and chicken liver (9Achouri Y. Rider M.H. Van Schaftingen E. Robbi M. Biochem. J. 1997; 323: 365-370Crossref PubMed Scopus (86) Google Scholar, 14Walsh D.A. Sallach H.J. Biochemistry. 1965; 4: 1076-1085Crossref PubMed Scopus (43) Google Scholar). On the contrary, M. tuberculosis PGDH is strongly inhibited by l-serine, with an I0.5 value for l-serine of ∼30 μm (6Dey S. Hu Z. Lan Xu X. Sacchettini J.C. Grant G.A. J. Biol. Chem. 2005; 280: 14884-14891Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), making it the next most sensitive enzyme after E. coli to be reported. Unlike the E. coli enzyme, M. tuberculosis PGDH displays a Hill coefficient of ∼1.0, indicating the absence of cooperativity for serine inhibition. However, kinetic studies show that cooperativity can be induced in M. tuberculosis PGDH by the presence of chloride ions (6Dey S. Hu Z. Lan Xu X. Sacchettini J.C. Grant G.A. J. Biol. Chem. 2005; 280: 14884-14891Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and to a lesser by and In this the crystal structure of PGDH from M. tuberculosis in the absence of This structure represents the second of PGDH with extended motif at the This structure this is the structure of this of PGDH and to important the regulation of this the tetramer is composed of identical subunits, significant asymmetry is seen in the structure of the subunits. and of M. tuberculosis PGDH SerA1 gene from M. tuberculosis H37Rv was and The site the and the site was immediately by a following the C-terminal residue of the enzyme. of a for multiple was by of at to a mutagenesis This site was the homologous in E. coli PGDH is a The gene was the in the as in the The was the E. coli strain The was in with at the and then induced with and at the was E. coli The in with all of the amino acids and also to the The was induced with and then at The by at for and in phosphate The a and the was by at for The was then on to a that was with phosphate and was a The PGDH and by and to a with phosphate and PGDH in MES, at by in This was found to produce of after a with different and and from The in of of PGDH and of The used for SeMet, the than a to The in as for and for M. tuberculosis PGDH was at at of of M. tuberculosis PGDH was with the E. coli PGDH nucleotide-binding domain as a and However, no was Subsequently, different such as and to produce an with PGDH was not this acid not contain the which was a residue was at that was to and for PGDH at at in for PGDH at 2.3 was also at in The and Z. Methods Enzymol. 1997; PubMed Scopus Google Scholar). The was found to be a a in the asymmetric unit. found Chem. 51: PubMed Scopus Google Scholar) and P. Biol. PubMed Scopus Google Scholar). solvent was also two in the asymmetric unit. this and was in Sacchettini J.C. Biol. 2002; PubMed Scopus (45) Google Scholar). of the of PGDH the first two be in the The was then the E.J. Biol. 1997; PubMed Scopus Google Scholar). The of the was and then at P. J. M. T. Biol. PubMed Scopus Google Scholar). of with J. Biol. PubMed Scopus Google Scholar) the P. B. Biol. 2002; PubMed Scopus Google Scholar) and the a was to the structure in as as the was and and the structure has an of and an value of with as by J. Appl. Google Scholar) and asymmetric in for the in for the in for the in for the where is the and is the of multiple of value value where and are the and structure of of asymmetric in for the where is the and is the of multiple of where and are the and structure in a of the structure The Scholar) for A and of which Methods Enzymol. 1997; PubMed Scopus Google Scholar), and the surface in and which J. The for A Scholar). The structure of M. tuberculosis PGDH has been to 2.3 C.M. Methods Enzymol. 1997; PubMed Scopus Google Scholar). this the residue at was to on sequence this is by a residue in E. coli PGDH. it is generally that be one for amino acids to C.M. Methods Enzymol. 1997; PubMed Scopus Google Scholar), a and at an of from this one in the acid no The structure was to an of and of with The structure contains all the amino acid the first two of the The structure that are two found in the asymmetric unit A and that a tetramer with the of domains can be in subunit of are to as the substrate-binding domain and the nucleotide-binding domain the regulatory domain and the intervening domain The and regulatory domains are homologous to found in E. coli PGDH and The intervening domain between the substrate-binding and regulatory domains is not in E. coli PGDH. of M. tuberculosis on A with the nucleotide-binding domain and substrate-binding domain as a rotation of the other two domains by indicating the presence of two different conformations the of the asymmetric unit nucleotide-binding domain in M. tuberculosis PGDH of to and contains with This is a variation of the that is a characteristic of of the nucleotide-binding domain in M. tuberculosis PGDH and E. coli PGDH sequence and of domains in A a root mean square of for This is to the presence of a between and in M. tuberculosis PGDH where is a in E. coli PGDH, as as in the structures of in M. tuberculosis and in E. of M. tuberculosis PGDH nucleotide-binding domain on A for that they have different conformations in the where binding In the in the where is On the other in the of the that this is different and is in such a that it appears to to the NAD-binding site The nucleotide-binding site in E. coli PGDH is different from the seen in A the seen in it is exposed This most the M. tuberculosis PGDH not to the to the not the to to the binding site the substrate-binding domain is to the nucleotide-binding domain and the intervening domain and is composed of and This domain contains by similar to E. coli PGDH for it has sequence with E. coli PGDH in and is no significant in the structure of the substrate-binding domain in A and of M. tuberculosis PGDH for to E. coli PGDH, and as seen in most of the the nucleotide-binding domain and substrate-binding domain the active site The presence of the active site in this is by the presence of the nucleotide-binding site to this NAD-binding site on the nucleotide-binding domain are and which are homologous to and in E. coli PGDH and which a to dehydrogenases and also seen in serine as a to dehydrogenases is a basic residue in the that to the which is in M. tuberculosis PGDH and in E. coli PGDH. acid sequence of PGDH with homologous proteins that three are homologous found in the are and that the residue on the which to and in E. coli PGDH. is by the nucleotide-binding domain. with a in the binding of the in M. tuberculosis PGDH is homologous to in E. coli PGDH, that is also in the nucleotide-binding domain of the subunit and whose a the active site of the subunit where the the of the This residue is to an important in subunit and tetramer formation in E. coli PGDH. The active site in this structure of M. tuberculosis PGDH to be in an exposed to dehydrogenases a their and ions are in the active site be from solvent This is similar to the seen in the crystal structure of inhibited E. coli PGDH and to a intervening domain to is as such it is a domain not in E. coli PGDH that is between the substrate-binding domain and the regulatory domain where l-serine It of of which two are by two and the four an of the intervening domains of on A in M. tuberculosis PGDH for not show significant structure However, as the of the intervening domain in is and with that in A The from to for all the domains from the and are not This results in the regulatory domain of A a to nucleotide-binding domain than in B. conformations be to as and to from other for the of of this other than the of the domains, is that are two different interfaces between the nucleotide-binding domain and the intervening domain in the two unique subunits. of the also results in the of a of in to the This appears to to of this to in the in this region. A and structural the intervening domain found with a of domain of from E. coli of for This enzyme is involved in and catalyzes the conversion of to the of this domain in this enzyme is not from the other with a of also found with in B. subtilis of for H. S. A. PubMed Scopus Google Scholar) that is a with the a domain that catalyzes the conversion of to in amino acid biosynthesis. was also some structural with from of 2.3 for J. J. Mol. Biol. 2004; PubMed Scopus Google Scholar) involved in the histidine the structural is seen in the that is of the NAD-binding domain in this enzyme, no such NAD-binding is in the intervening domain of the PGDH. are two distinct of in the where intervening domains of are by the and appear to which are as a of the The is of from one subunit and and from the is found in the regulatory domain and an with the of which is found in the connecting the intervening domain and the regulatory ionic contact with the of of to this of the of the structure and appear to an Kinetic studies (6Dey S. Hu Z. Lan Xu X. Sacchettini J.C. Grant G.A. J. Biol. Chem. 2005; 280: 14884-14891Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) have that serine inhibition of M. tuberculosis PGDH is not a as seen for the E. coli enzyme. However, cooperativity can be induced by the presence of ions such as chloride at This site a for the of cooperativity of inhibition in M. tuberculosis PGDH. is also in the intervening domain seen in which also appears to In this appears to in a with the it is not known this has results from crystal The C-terminal regulatory domain of M. tuberculosis PGDH of and contains four with two It is a of the domain family, the of which is the regulatory domain of E. coli PGDH. proteins a regulatory with the the of binding is not domains are that in amino acid and the domain has been the domain in amino acid D.M. B. Biol. PubMed Scopus Google Scholar). The domain has also been found in proteins as the for binding Y. Y. A. L. A. J. Mol. Biol. 2004; PubMed Scopus (43) Google Scholar), in the as the for binding Y. Biol. 2003; PubMed Scopus Google Scholar), and in of M. tuberculosis Y. V. Sacchettini J.C. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) where it the of the In the PGDH two regulatory domains an that a of Grant G.A. L. Biol. PubMed Scopus Google Scholar). This type of arrangement is also seen in other proteins of the domain family not involved in amino acid such as and proteins appear to an site at their interfaces Y. Y. A. L. A. J. Mol. Biol. 2004; PubMed Scopus (43) Google Scholar). of the E. coli regulatory domain on the M. tuberculosis regulatory domain for a in one of the in M. tuberculosis PGDH and of in M. tuberculosis PGDH and Kinetic studies on serine inhibition of M. tuberculosis PGDH show that is a type of inhibition with l-serine (6Dey S. Hu Z. Lan Xu X. Sacchettini J.C. Grant G.A. J. Biol. Chem. 2005; 280: 14884-14891Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), to serine binding at a site other than the active site. In the inhibited structure of E. coli PGDH, and with l-serine, an of PGDH, at the regulatory domain The sequence of M. tuberculosis PGDH with E. coli PGDH that and the of serine in the of and at this site in M. tuberculosis PGDH A and of this structure A and have also been the regulatory domains are for in the the of and in have also been In a with in A and with in B. is also in of the of in which a with This in has the of of In with whose is from the in B. the regulatory domain the serine-binding site not to be to serine to binding the regulatory domains from other to some a to the surface large for serine to This of and at the serine-binding site in one subunit a for serine a surface of the regulatory domain and an in subunit A not in subunit B. However, an to the of what is this in one subunit of an not the is not of the two unique subunits at the between the substrate-binding domain and the intervening domain results in two different and unique of between the nucleotide-binding domains and the intervening domains. are in and of the unique between the nucleotide-binding domain and the intervening domain of subunit and the nucleotide-binding domain and intervening domain of subunit A A the subunit and domain arrangement of E. coli and M. tuberculosis PGDH is in domain domain of domain of domain of A domain of A in a the E. coli PGDH are two of between nucleotide-binding domains and between regulatory domains. This structure that domain are extensive in M. tuberculosis PGDH. are in and in of between the nucleotide-binding domain of subunit with the intervening domain of subunit by the nucleotide-binding domain of and the intervening domain of between the nucleotide-binding domain of subunit A with the intervening domain of subunit and by the domain of and the intervening domain of and between the intervening domains of subunits A and and domain binding domain of domain of binding domain of A domain of domain of A domain of in a The M. tuberculosis PGDH is composed of identical subunits and contains four active and four binding The binding are at the between regulatory domains by homology to E. coli PGDH. The active appear to be in the by the and substrate-binding domains, homologous to E. coli PGDH. This is by the presence of the NAD-binding in this region. E. coli PGDH with two This not to be the in this enzyme no for is In addition, also appears to be four binding for a where the intervening and regulatory domains from subunits In this the are by be the of with chloride that has been to the cooperativity of inhibition (6Dey S. Hu Z. Lan Xu X. Sacchettini J.C. Grant G.A. J. Biol. Chem. 2005; 280: 14884-14891Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). not be the for it is and the site contains basic to a On the other chloride show effect by the ionic at this site seen with the However, the chloride effect is not strictly ionic the ionic strength of the with other is not are two of between the subunits in the tetramer. which is is between the nucleotide-binding domains of subunits with surface of and this is also in the homologous such as glycerate formate and E. coli PGDH. The other is between the regulatory domains from subunits with surface of This is for tetramer and a similar contact is found in E. coli PGDH. It is where l-serine a between the two domains. different conformations are seen at the putative serine-binding In one the appear to be a through which serine In the other a appears to this that is In E. coli PGDH, two of one at are for inhibition. the is for M. tuberculosis PGDH, this structural asymmetry two that serine from two that The polypeptide between domains is similar to that in E. coli PGDH. The substrate-binding domain is to the nucleotide-binding domain through two of polypeptide that to in domain. The substrate-binding domain is to the intervening domain by a polypeptide in E. coli PGDH. A in the connecting the substrate-binding domain to the regulatory domain acts as a hinge that is important for serine binding and inhibition. In M. tuberculosis PGDH the between the substrate-binding domain to the regulatory domain is by the presence of the intervening domain. However, E. coli PGDH, the connecting from the substrate-binding domain to the next domain contains a sequence of multiple in this three a similar type of hinge be in M. tuberculosis PGDH. It is not to what the of this be one be that the intervening and regulatory domains are as a unit in M. tuberculosis PGDH, an extended regulatory with the hinge found in homologous in enzyme. this is the between the of the regulatory domain with the substrate-binding domain in E. coli PGDH and the intervening and substrate-binding domain in M. tuberculosis PGDH to in the pathway of inhibition of the active site. The arrangement of subunits in this tetramer is different from that seen in E. coli PGDH. The presence of the 135 amino acid polypeptide intervening has to the of the regulatory domain toward the nucleotide-binding domain a structure in subunit as with the seen in E. coli PGDH. is also the presence of asymmetry in the tetramer of the of this regulatory domain from the as in an E. coli PGDH tetramer to the of a M. tuberculosis PGDH which is In addition, the asymmetry in the of the intervening and regulatory domain in the two unique subunits is quite one of the ∼180° to the other to produce a different domain in one subunit to the The of rotation is in the connecting the substrate-binding domain to the intervening domain. of the of domains is that two different of the intervening domain the nucleotide-binding domain two different subunit specific domain In the the of the intervening domain that the nucleotide-binding domain In the are exposed to and the nucleotide-binding domain. asymmetry has been in other structures composed of However, this asymmetry structural such as found the active site. The asymmetry seen large in the of domains, This asymmetry also important and the of inhibition. the to the regulatory domain of subunit in a similar to that in E. coli PGDH. In M. tuberculosis PGDH the of subunits to the active site is different in two of the subunits to the other are different for of to the active site in can the of be induced by the two different On the other M. tuberculosis PGDH exhibits activity E. coli PGDH, are two subunits that are the other two with the different domain arrangement are This for the of can be with In this mutagenesis to the of this are