Substrate-induced conformational changes in Bacillus subtilis glutamate racemase and their implications for drug discovery

D-glutamate is an essential building block of the peptidoglycan layer in bacterial cell walls and can be synthesized from L-glutamate by glutamate racemase (RacE). The structure of a complex of B. subtilis RacE with D-glutamate reveals that the glutamate is buried in a deep pocket, whose formation at the interface of the enzyme's two domains involves a large-scale conformational rearrangement. These domains are related by pseudo-2-fold symmetry, which superimposes the two catalytic cysteine residues, which are located at equivalent positions on either side of the alpha carbon of the substrate. The structural similarity of these two domains suggests that the racemase activity of RacE arose as a result of gene duplication. The structure of the complex is dramatically different from that proposed previously and provides new insights into the RacE mechanism and an explanation for the potency of a family of RacE inhibitors, which have been developed as novel antibiotics.     

Crystal structure of aspartate racemase from Pyrococcus horikoshii OT3 and its implications for molecular mechanism of PLP-independent racemization

There exists a d-enantiomer of aspartic acid in lactic acid bacteria and several hyperthermophilic archaea, which is biosynthesized from the l-enantiomer by aspartate racemase. Aspartate racemase is a representative pyridoxal 5'-phosphate (PLP)-independent amino acid racemase. The "two-base" catalytic mechanism has been proposed for this type of racemase, in which a pair of cysteine residues are utilized as the conjugated catalytic acid and base. We have determined the three-dimensional structure of aspartate racemase from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 at 1.9 A resolution by X-ray crystallography and refined it to a crystallographic R factor of 19.4% (R(free) of 22.2%). This is the first structure reported for aspartate racemase, indeed for any amino acid racemase from archaea. The crystal structure revealed that this enzyme forms a stable dimeric structure with a strong three-layered inter-subunit interaction, and that its subunit consists of two structurally homologous alpha/beta domains, each containing a four-stranded parallel beta-sheet flanked by six alpha-helices. Two strictly conserved cysteine residues (Cys82 and Cys194), which have been shown biochemically to act as catalytic acid and base, are located on both sides of a cleft between the two domains. The spatial arrangement of these two cysteine residues supports the "two-base" mechanism but disproves the previous hypothesis that the active site of aspartate racemase is located at the dimeric interface. The structure revealed a unique pseudo mirror-symmetry in the spatial arrangement of the residues around the active site, which may explain the molecular recognition mechanism of the mirror-symmetric aspartate enantiomers by the non-mirror-symmetric aspartate racemase.     

The Escherichia coli Dga (MurI) protein shares biological activity and structural domains with the Pediococcus pentosaceus glutamate racemase.

The Pediococcus pentosaceus glutamate racemase gene product complemented the D-glutamate auxotrophy of Escherichia coli WM335. Amino acid sequence analysis of the two proteins revealed 28% identity, primarily in six clusters scattered throughout the sequence. Further analyses indicated secondary structure similarities between the two proteins. These data support a recent report that the dga (murI) gene product is a glutamate racemase.     

Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus

A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts l- or d-glutamate to d- or l-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The K _m and k_cat values of the overexpressed A. pyrophilus glutamate racemase for the racemization of l-glutamate to the d-form and the conversion of d-glutamate to the l-form were measured as 1.8 ± 0.4 mM and 0.79 ± 0.06 s⁻¹ or 0.50 ± 0.07 mM and 0.25 ± 0.01 s⁻¹, respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85°C for 90 min. Received: September 11, 1998 / Accepted: January 12, 1999     

Crystal Structures of Lysine-Preferred Racemases,the Non-Antibiotic Selectable Markers for Transgenic Plants

Lysine racemase, a pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase that catalyzes the interconversion of lysine enantiomers, is valuable to serve as a novel non-antibiotic selectable marker in the generation of transgenic plants. Here, we have determined the first crystal structure of a lysine racemase (Lyr) from Proteus mirabilis BCRC10725, which shows the highest activity toward lysine and weaker activity towards arginine. In addition, we establish the first broad-specificity amino acid racemase (Bar) structure from Pseudomonas putida DSM84, which presents not only the highest activity toward lysine but also remarkably broad substrate specificity. A complex structure of Bar-lysine is also established here. These structures demonstrate the similar fold of alanine racemase, which is a head-to-tail homodimer with each protomer containing an N-terminal (α/β)₈ barrel and a C-terminal β-stranded domain. The active-site residues are located at the protomer interface that is a funnel-like cavity with two catalytic bases, one from each protomer, and the PLP binding site is at the bottom of this cavity. Structural comparisons, site-directed mutagenesis, kinetic, and modeling studies identify a conserved arginine and an adjacent conserved asparagine that fix the orientation of the PLP O3 atom in both structures and assist in the enzyme activity. Furthermore, side chains of two residues in α-helix 10 have been discovered to point toward the cavity and define the substrate specificity. Our results provide a structural foundation for the design of racemases with pre-determined substrate specificity and for the development of the non-antibiotic selection system in transgenic plants.     

Glutamate racemase is an endogenous DNA gyrase inhibitor

Almost all bacteria possess glutamate racemase to synthesize d-glutamate as an essential component of peptidoglycans in the cell walls. The enforced production of glutamate racemase, however, resulted in suppression of cell proliferation. In the Escherichia coli JM109/pGR3 clone, the overproducer of glutamate racemase, the copy number (i.e. replication efficiency) of plasmid DNA declined dramatically, whereas the E. coli WM335 mutant that is defective in the gene of glutamate racemase showed little genetic competency. The comparatively low and high activities for DNA supercoiling were contained in the E. coli JM109/pGR3 and WM335 cells, respectively. Furthermore, we found that the DNA gyrase of E. coli was modulated by the glutamate racemase of E. coli in the presence of UDP-N-acetylmuramyl-l-alanine, which is a peptidoglycan precursor and functions as an absolute activator for the racemase. This is the first finding of the enzyme protein participating in both d-amino acid metabolism and DNA processing.     

Functional comparison of the two Bacillus anthracis glutamate racemases

Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because L-glutamate stereoisomerization to D-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and racE2. To evaluate whether racE1 and racE2 encode functional glutamate racemases, we cloned and expressed racE1 and racE2 in Escherichia coli. Size exclusion chromatography of the two purified recombinant proteins suggested differences in their quaternary structures, as RacE1 eluted primarily as a monomer, while RacE2 demonstrated characteristics of a higher-order species. Analysis of purified recombinant RacE1 and RacE2 revealed that the two proteins catalyze the reversible stereoisomerization of L-glutamate and D-glutamate with similar, but not identical, steady-state kinetic properties. Analysis of the pH dependence of L-glutamate stereoisomerization suggested that RacE1 and RacE2 both possess two titratable active site residues important for catalysis. Moreover, directed mutagenesis of predicted active site residues resulted in complete attenuation of the enzymatic activities of both RacE1 and RacE2. Homology modeling of RacE1 and RacE2 revealed potential differences within the active site pocket that might affect the design of inhibitory pharmacophores. These results suggest that racE1 and racE2 encode functional glutamate racemases with similar, but not identical, active site features.     

Genetic design of conditional D-glutamate auxotrophy for Bacillus subtilis: use of a vector-borne poly-gamma-glutamate synthetic system

Bacillus subtilis possesses two glutamate racemase isozymes, RacE and YrpC. For the first time, we succeeded in constructing glutamate racemase-gene disruptants of B. subtilis. Phenotypic analysis of their D-glutamate auxotrophy indicated that the RacE-type glutamate racemase is important for ensuring maximum growth rate but dispensable. The YrpC-type glutamate racemase probably operates as an anaplerotic enzyme for RacE, especially under liquid culture conditions. We found novel applicability of RacE-less mutants inheriting only a marginal activity for endogenous D-glutamate supply, viz. the employment for the in vivo identification of D-glutamate-consuming systems. In fact, the genetic induction of a poly-gamma-glutamate synthetic system led a RacE-less mutant to severe growth suppression, which was overcome in the presence of a high concentration of exogenous D-glutamate. The results indicate that a significant amount of D-glutamate is consumed during poly-glutamate biosynthesis. To our knowledge, this is the first report of conditional D-glutamate auxotrophy for B. subtilis.     

Thermostable alanine racemase from Bacillus stearothermophilus: DNA and protein sequence determination and secondary structure prediction

The nucleotide sequence of the alanine racemase (EC 5.1.1.1) gene from a thermophile, Bacillus stearothermophilus, was determined by the dideoxy chain termination method with universal and synthetic site-specific primers. The amino acid sequence of the enzyme predicted from the nucleotide sequence was confirmed by peptide sequence information derived from the N-terminal amino acid residues and several tryptic fragments. The alanine racemase gene consists of 1158 base pairs encoding a protein of 386 amino acid residues; the molecular weight of the apoenzyme is estimated as 43,341. The racemase gene of B. stearothermophilus has a closely similar size (1158 vs 1167 base pairs) to that of the gene of a mesophile, B. subtilis, but shows a higher preference for codons ending in G or C. A comparison of the amino acid sequence with those of Bacillus subtilis and Salmonella typhimurium dadB and alr enzymes revealed overall sequence homologies of 31-54%, including an identical octapeptide bearing the pyridoxal 5'-phosphate binding site. Although the residues common in the four racemases are not continuously arrayed, these constitute distinct domains and their hydropathy profiles are very similar. The secondary structure of B. stearothermophilus alanine racemase was predicted from the results obtained by theoretical analysis and circular dichroism measurement.     

Characterization of yrpC gene product of Bacillus subtilis IFO 3336 as glutamate racemase isozyme.

Glr, the glutamate racemase of Bacillus subtilis (formerly Bacillus natto) IFO 3336 encoded by the glr gene, and YrpC, a protein encoded by the yrpC gene, which is located at a different locus from that of the glr gene in the B. subtilis genome, share a high sequence similarity. The yrpC gene complemented the D-glutamate auxotrophy of Escherichia coli WM335 cells defective in the glutamate racemase gene. Glutamate racemase activity was found in the extracts of E. coli WM335 clone cells harboring a plasmid, pYRPC1, carrying its gene. Thus, the yrpC gene encodes an isozyme of glutamate racemase of B. subtilis IFO 3336. YrpC is mostly found in an inactive inclusion body in E. coli JM109/pYRPC1 cells. YrpC was solubilized readily, but glutamate racemase activity was only slightly restored. We purified YrpC from the extracts of E. coli JM109/pYRPC2 cells using a Glutathione S-transferase Gene Fusion System to characterize it. YrpC is a monomeric protein and contains no cofactors, like Glr. Enzymological properties of YrpC, such as the substrate specificity and optimum pH, are also similar to those of Glr. The thermostability of YrpC, however, is considerably lower than that of Glr. In addition, YrpC showed higher affinity and lower catalytic efficiency for L-glutamate than Glr. This is the first example showing the occurrence and properties of a glutamate racemase isozyme.     

The 1.9 A crystal structure of alanine racemase from Mycobacterium tuberculosis contains a conserved entryway into the active site

We report the crystal structure of alanine racemase from Mycobacterium tuberculosis (Alr(Mtb)) at 1.9 A resolution. In our structure, Alr(Mtb) is found to be a dimer formed by two crystallographically different monomers, each comprising 384 residues. The domain makeup of each monomer is similar to that of Bacillus and Pseudomonas alanine racemases and includes both an alpha/beta-barrel at the N-terminus and a C-terminus primarily made of beta-strands. The hinge angle between these two domains is unique for Alr(Mtb), but the active site geometry is conserved. In Alr(Mtb), the PLP cofactor is covalently bound to the protein via an internal aldimine bond with Lys42. No guest substrate is noted in its active site, although some residual electron density is observed in the enzyme's active site pocket. Analysis of the active site pocket, in the context of other known alanine racemases, allows us to propose the inclusion of conserved residues found at the entrance to the binding pocket as additional targets in ongoing structure-aided drug design efforts. Also, as observed in other alanine racemase structures, PLP adopts a conformation that significantly distorts the planarity of the extended conjugated system between the PLP ring and the internal aldimine bond.     

Structure of aspartate racemase complexed with a dual substrate analogue, citric acid, and implications for the reaction mechanism

Pyrococcus horikoshii OT3 aspartate racemase (PhAspR) catalyzes the interconversion between L- and D-aspartate. The X-ray structure of PhAspR revealed a pseudo mirror-symmetric distribution of the residues around its active site, which is very reasonable for its chiral substrates, L-aspartate and D-aspartate. In this study, we have determined the crystal structure of an inactive mutant PhAspR complexed with a citric acid (Cit) at a resolution of 2.0 A. Cit contains the substrate analogue moieties of both L- and D-aspartate and exhibits a low competitive inhibition activity against PhAspR. In the structure, Cit binds to the catalytic site of PhAspR, which induced the conformational change to close the active site. The distance between the thiolates was estimated to be 7.4 A, representing a catalytic state and the substrate binding modes of PhAspR. Two conserved basic residues, Arg48 and Lys164, seem to be indispensable for PhAspR activity. Arg48 is thought to be responsible for recognizing carboxyl groups of the substrates L-/D-aspartates and stabilizing a reaction intermediate, and Lys164 is responsible for stabilizing a closed state structure. In this structure, the L-aspartate moiety of Cit is likely to take the substrate position of the PhAspR-substrate complex, which is very similar to that of Glutamate racemase. There is also another possibility that the two substrate analogue moieties of the bound Cit reflect the binding modes of both L- and D-aspartates. Based on the PhAspR-Cit complex structure, the reaction mechanism of aspartate racemase was elucidated.     

Human serine racemase: moleular cloning, genomic organization and functional analysis

High levels of D-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl D-aspartate type of glutamate receptors. D-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from L-serine. We now report cloning and expression of human serine racemase, an enzyme that catalyses the synthesis of D-serine from L-serine. The enzyme displays a high homology to the murine serine racemase. It contains a pyridoxal 5'-phosphate attachment sequence similar to bacterial biosynthetic threonine dehydratase. Northern-blot analysis show high levels of human serine racemase in areas known to contain large amounts of endogenous D-serine, such as hippocampus and corpus callosum. Robust synthesis of D-serine was detected in cells transfected with human serine racemase, demonstrating the conservation of D-amino acid metabolism in humans. Serine racemase may be a therapeutic target in psychiatric diseases as supplementation of D-serine greatly improves schizophrenia symptoms. We identify the human serine racemase genomic structure and show that the gene encompasses seven exons and localizes to chromosome 17q13.3. Identification of the intron-exon boundaries of the human serine racemase gene will be useful to search for mutations in neuropsychiatric disorders.     

Staphylococcus haemolyticus contains two D-glutamic acid biosynthetic activities, a glutamate racemase and a D-amino acid transaminase.

Two D-glutamic acid biosynthetic activities, glutamate racemase and D-amino acid transaminase, have been described previously for bacteria. To date, no bacterial species has been reported to possess both activities. Genetic complementation studies using Escherichia coli WM335, a D-glutamic acid auxotroph, and cloned chromosomal DNA fragments from Staphylococcus haemolyticus revealed two distinct DNA fragments containing open reading frames which, when present, allowed growth on medium without exogenous D-glutamic acid. Amino acid sequences of the two open reading frames derived from the DNA nucleotide sequences indicated extensive identity with the amino acid sequence of Pediococcus pentosaceous glutamate racemase in one case and with that of the D-amino acid transaminase of Bacillus spp. in the second case. Enzymatic assays of lysates of E. coli WM335 strains containing either the cloned staphylococcal racemase or transminase verified the identities of these activities. Subsequent DNA hybridization experiments indicated that Staphylococcus aureus, in addition to S. haemolyticus, contained homologous chromosomal DNA for each of these genes. These data suggest that S. haemolyticus, and probably S. aureus, contains genes for two D-glutamic acid biosynthetic activities, a glutamate racemase (dga gene) and a D-amino acid transaminase (dat gene).     

Structural and functional analysis of two glutamate racemase isozymes from Bacillus anthracis and implications for inhibitor design

Glutamate racemase (RacE) is responsible for converting l-glutamate to d-glutamate, which is an essential component of peptidoglycan biosynthesis, and the primary constituent of the poly-gamma-d-glutamate capsule of the pathogen Bacillus anthracis. RacE enzymes are essential for bacterial growth and lack a human homolog, making them attractive targets for the design and development of antibacterial therapeutics. We have cloned, expressed and purified the two glutamate racemase isozymes, RacE1 and RacE2, from the B. anthracis genome. Through a series of steady-state kinetic studies, and based upon the ability of both RacE1 and RacE2 to catalyze the rapid formation of d-glutamate, we have determined that RacE1 and RacE2 are bona fide isozymes. The X-ray structures of B. anthracis RacE1 and RacE2, in complex with d-glutamate, were determined to resolutions of 1.75 A and 2.0 A. Both enzymes are dimers with monomers arranged in a "tail-to-tail" orientation, similar to the B. subtilis RacE structure, but differing substantially from the Aquifex pyrophilus RacE structure. The differences in quaternary structures produce differences in the active sites of racemases among the various species, which has important implications for structure-based, inhibitor design efforts within this class of enzymes. We found a Val to Ala variance at the entrance of the active site between RacE1 and RacE2, which results in the active site entrance being less sterically hindered for RacE1. Using a series of inhibitors, we show that this variance results in differences in the inhibitory activity against the two isozymes and suggest a strategy for structure-based inhibitor design to obtain broad-spectrum inhibitors for glutamate racemases.     

Isolation of peptide ligands that inhibit glutamate racemase activity from a random phage display library

Several new antibacterial agents are currently being developed in response to the emergence of bacterial resistance to existing antibiotic substances. The new agents include compounds that interfere with bacterial membrane function. The peptidoglycan component of the bacterial cell wall is synthesized by glutamate racemase, and this enzyme is responsible for the biosynthesis of d-glutamate, which is an essential component of cell wall peptidoglycan. In this study, we screened a phage display library expressing random dodecapeptides on the surface of bacteriophage against an Escherichia coli glutamate racemase, and isolated specific peptide sequences that bind to the enzyme. Twenty-seven positive phage clones were analyzed, and seven different peptide sequences were obtained. Among them, the peptide sequence His-Pro-Trp-His-Lys-Lys-His-Pro-Asp-Arg-Lys-Thr was found most frequently, suggesting that this peptide might have the highest affinity to glutamate racemase. The positive phage clones and HPWHKKHPDRKT synthetic peptide were able to inhibit glutamate racemase activity in vitro, implying that our peptide inhibitors may be utilized for the molecular design of new potential antibacterial agents targeting cell wall synthesis.     

Crystal structure at 1.45 A resolution of alanine racemase from a pathogenic bacterium, Pseudomonas aeruginosa, contains both internal and external aldimine forms

The structure of the catabolic alanine racemase, DadX, from the pathogenic bacterium Pseudomonas aeruginosa, reported here at 1.45 A resolution, is a dimer in which each monomer is comprised of two domains, an eight-stranded alpha/beta barrel containing the PLP cofactor and a second domain primarily composed of beta-strands. The geometry of each domain is very similar to that of Bacillus stearothermophilus alanine racemase, but the rotation between domains differs by about 15 degrees. This change does not alter the structure of the active site in which almost all residues superimpose well with a low rms difference of 0.86 A. Unexpectedly, the active site of DadX contains a guest substrate that is located where acetate and propionate have been observed in the Bacillus structures. It is modeled as d-lysine and oriented such that its terminal NZ atom makes a covalent bond with C4' of PLP. Since the internal aldimine bond between the protein lysine, Lys33, and C4' of PLP is also unambiguously observed, there appears to be an equilibrium between both internally and externally reacted forms. The PLP cofactor adopts two partially occupied conformational states that resemble previously reported internal and external aldimine complexes.     

Enzymatic production of d-glutamate from l-glutamate by a glutamate racemase

d-Glutamate was produced from l-glutamate by two successive cellular reactions with a glutamate racemase produced by Escherichia coli TM93 harboring a plasmid containing a glutamate racemase gene from Lactobacillus brevis ATCC 8287 and a glutamate decarboxylase produced by E. coli ATCC 11246. l-Glutamate was first racemized to dl-glutamate at pH 8.5 and l-glutamate was then decarboxylated at pH 4.2. Starting from 100 g/l of l-glutamate, 50 g/l of d-glutamate remained after 15 h reaction.     

Active site residues of glutamate racemase

Glutamate racemase, MurI, catalyzes the interconversion of glutamate enantiomers in a cofactor-independent fashion and provides bacteria with a source of D-Glu for use in peptidoglycan biosynthesis. The enzyme uses a "two-base" mechanism involving a deprotonation of the substrate at the alpha-position to form an anionic intermediate, followed by a reprotonation in the opposite stereochemical sense. In the Lactobacillus fermenti enzyme, Cys73 is responsible for the deprotonation of D-glutamate, and Cys184 is responsible for the deprotonation of L-glutamate; however, very little is known about the roles of other active site residues. This work describes the preparation of four mutants in which strictly conserved residues containing ionizable side chains were modified (D10N, D36N, E152Q, and H186N). During the course of this research, the structural analysis of a crystallized glutamate racemase indicated that three of these residues (D10, E152, and H186) are in the active site of the enzyme [Hwang, K. Y., Cho, C.-S., Kim, S. S., Sung, H.-C., Yu, Y. G., and Cho, Y. (1999) Nat. Struct. Biol. 6, 422-426]. Two of the mutants, D10N and H186N, displayed a marked decrease in the values of k(cat), but not K(M), and are therefore implicated as important catalytic residues. Further analysis of the primary kinetic isotope effects observed with alpha-deuterated substrates showed that a significant asymmetry was introduced into the free energy profile by these two mutations. This is interpreted as evidence that the mutated residues normally assist the catalytic thiols in acting as bases (D10 with C73 and H186 with C184). An alternate possibility is that the residues may serve to stabilize the carbanionic intermediate in the racemization reaction.     

Enantioselective synthesis of various D-amino acids by a multi-enzyme system

We have developed an effective method for the synthesis of various D-amino acids from the corresponding α-keto acids and ammonia by coupling four enzyme reactions catalyzed by D-amino acid aminotransferase, glutamate racemase, glutamate dehydrogenase, and formate dehydrogenase. In this system, D-glutamate is continuously regenerated from α-ketoglutarate, ammonia and NADH by the coupled reaction of glutamate dehydrogenase and glutamate racemase, and used as an amino donor for the enantioselective D-amino acid synthesis by the D-amino acid aminotransferase reaction. The unidirectional formate dehydrogenase reaction is also coupled to regenerate NADH consumed. Under the optimum conditions, D-enantiomers of valine, alanine, α-keto analogues with a molar yield higher than 80%.