Crystal Structure of Mycobacterium tuberculosis D-3-Phosphoglycerate Dehydrogenase

Abstract

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 Cterminal 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 ϳ180°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 serinebinding site in the two unique subunits. 1 catalyzes the first committed step in this conversion of D-3-phosphoglycerate to phosphohydroxypyruvate utilizing NAD ϩ as a cofactor. Subsequently, phosphohydroxypyruvate is converted to phosphoserine by phosphoserine transaminase and then to L-serine by phosphoserine phosphatase (1, 2). PGDH also belongs to a family of proteins classified as 2-hydroxy acid dehydrogenases that are generally specific for substrates with a D-configuration (3). It shares sequence homology with other members of this family such as formate dehydrogenase and glycerate dehydrogenase whose structures are also known (4, 5).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 (6). 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 (ϳ150 -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 altog...

D-3-Phosphoglycerate Dehydrogenase from Mycobacterium tuberculosis Is a Link between the Escherichia coli and Mammalian Enzymes

“…However, the M. tuberculosis enzyme does not display the affinity for 5Ј-AMP-Sepharose that the rat liver and E. coli enzyme do, although all three utilize NAD as a cofactor. A possible reason for this is addressed in the accompanying article (21), which describes the crystal structure of M. tuberculosis PGDH.…”

“…The crystal structure of M. tuberculosis PGDH does not reveal the site for chloride binding. A potential site for the binding of anionic molecules has been identified by virtue of the binding of tartrate molecules from the crystallization buffer at a site between the intervening and regulatory domains (21). However, tartrate itself does not exhibit an effect on the cooperativity of inhibition by serine.…”

High Throughput Screen Identifies Small Molecule Inhibitors Specific for Mycobacterium tuberculosis Phosphoserine Phosphatase

“…Phosphoserine aminotransferase (PSAT), a PLP (pyridoxal-5Ј-phosphate)-dependent enzyme converts 3-phosphohydroxy pyruvate to O-phospho-L-serine that is subsequently dephosphorylated by phosphoserine phosphatase (PSP) into L-serine (11). Both 3-phosphoglycerate dehydrogenase and PSAT homologs in M. tuberculosis have been extensively biochemically characterized, and their crystal structures have also been determined (12)(13)(14). In a recent study, it has been shown that intracellular cyclic AMP regulates the levels of PSAT enzyme, and extracellular addition of L-serine restores the growth defect of M. tuberculosis crp mutant in vitro (15).…”

“…1A and Table 2). M. tuberculosis PGDH and PSAT enzymes catalyzing the first two steps of L-serine biosynthesis have been biochemically well characterized (13)(14)(15). Phylogenetic analysis among PSP proteins from various organisms revealed that PSP proteins from actinobacter and helicobacter species were more similar to each other in comparison with enterobacterial and human PSP enzymes (Fig.…”

Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

“…These structurally related ferredoxin-like fold proteins mediate allosteric regulation and ligand binding and typically undergo oligomerization through different strat- egies resulting in different oligomer architectures (14), in some cases similar to that presented by the peptidase M20/M25/M40 family ( Fig. 4B) (15,22). It has also been demonstrated that the sequence divergence of SMBDs such as ACT can expand beyond the detection limits of the sequence-based algorithm of PSI-BLAST (17), which would explain why this is the first time that the ACT-like nature of the dimerization domain of the peptidase M20/M25/M40 family has been found.…”