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Proline racemases (PRAC), catalyzing the l-proline and d-proline interconversion, are essential factors in eukaryotic pathogens such as Trypanosoma cruzi, Trypanosoma vivax, and Clostridioides difficile. If the discovery of irreversible inhibitors of T. cruzi PRAC (TcPRAC) led to innovative therapy of the Chagas disease, no inhibitors of CdPRAC have been discovered to date. However, C. difficile, due to an increased incidence in recent years, is considered as a major cause of health threat. In this work, we have taken into account the similarity between TcPRAC and CdPRAC enzymes to design new inhibitors of CdPRAC. Starting from (E) 4-oxopent-2-enoic acid TcPRAC irreversible inhibitors, we synthesized 4-aryl substituted analogs and evaluated their CdPRAC enzymatic inhibition against eleven strains of C. difficile. This study resulted in promising candidates and allowed for identification of (E)-4-(3-bromothiophen-2-yl)-4-oxobut-2-enoic acid 20 that was chosen for complementary in vivo studies and did not reveal in vivo toxicity., (© 2021 John Wiley & Sons A/S.)

Enterococcus faecalis (E. faecalis) is a Gram-positive coccoid, non-sporulating, facultative anaerobic, multidrug resistance bacterium responsible for almost 65% to 80% of all enterococcal nosocomial infections. It usually causes infective endocarditis, urinary tract and surgical wound infections. The increase in E. faecalis resistance to conventionally available antibiotic has rekindled intense interest in developing useful antibacterial drugs. In E. faecalis, diaminopimelate epimerase (DapF) is involved in the lysine biosynthetic pathway. The product of this pathway is precursors of peptidoglycan synthesis, which is a component of bacterial cell wall. Also, because mammals lack this enzyme, consequently E. faecalis diaminopimelate epimerase (EfDapF) represents a potential target for developing novel class of antibiotics. In this regard, we have successfully cloned, overexpressed the gene encoding DapF in BL-21(DE3) and purified with Ni-NTA Agarose resin. In addition to this, binding studies were performed using fluorescence spectroscopy in order to confirm the bindings of the identified lead compounds (acetaminophen and dexamethasone) with EfDapF. Docking studies revealed that acetaminophen found to make hydrogen bonds with Asn72 and Asn13 while dexamethasone interacted by forming hydrogen bonds with Asn205 and Glu223. Thus, biochemical studies indicated acetaminophen and dexamethasone, as potential inhibitors of EfDapF and eventually can reduce the catalytic activity of EfDapF., (© 2021 John Wiley & Sons Ltd.)

Glutamate racemases (GR) are members of the family of bacterial enzymes known as cofactor-independent racemases and epimerases and catalyze the stereoinversion of glutamate. D-amino acids are universally important for the proper construction of viable bacterial cell walls, and thus have been repeatedly validated as attractive targets for novel antimicrobial drug design. Significant aspects of the mechanism of this challenging stereoinversion remain unknown. The current study employs a combination of MD and QM/MM computational approaches to show that the GR from H. pylori must proceed via a pre-activation step, which is dependent on the enzyme's flexibility. This mechanism is starkly different from previously proposed mechanisms. These findings have immediate pharmaceutical relevance, as the H. pylori GR enzyme is a very attractive allosteric drug target. The results presented in this study offer a distinctly novel understanding of how AstraZeneca's lead series of inhibitors cripple the H. pylori GR's native motions, via prevention of this critical chemical pre-activation step. Our experimental studies, using SPR, fluorescence and NMR WaterLOGSY, show that H. pylori GR is not inhibited by the uncompetitive mechanism originally put forward by Lundqvist et al.. The current study supports a deep connection between native enzyme motions and chemical reactivity, which has strong relevance to the field of allosteric drug discovery., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Streptococcus iniae is a pathogenic and zoonotic bacterium responsible for human diseases and mortality of many fish species. Recently, this bacterium has demonstrated an increasing trend for antibiotics resistance, which has warranted a search for new approaches to tackle its infection. Glutamate racemase (MurI) is a ubiquitous enzyme of the peptidoglycan synthesis pathway that plays an important role in the cell wall integrity maintenance; however, the significance of this enzyme differs in different species. In this study, we knocked out the MurI gene in S. iniae in order to elucidate the role of glutamate racemase in maintaining cell wall integrity in this bacterial species. We also cloned, expressed, and purified MurI and determined its biochemical characteristics. Biochemical analysis revealed that the MurI gene in S. iniae encodes a functional enzyme with a molecular weight of 30 kDa, temperature optimum at 35°C, and pH optimum at 8.5. Metal ions, such as Cu2+, Mn2+, Co2+ and Zn2+, inhibited the enzyme activity. MurI was found to be essential for the viability and cell wall integrity of S. iniae. The optimal growth of the MurI-deficient S. iniae mutant can be achieved only by adding a high concentration of D-glutamate to the medium. Membrane permeability assay of the mutant showed an increasing extent of the cell wall damage with time upon D-glutamate starvation. Moreover, the mutant lost its virulence when incubated in fish blood. Our results demonstrated that the MurI knockout leads to the generation of S. iniae auxotroph with damaged cell walls.

Covering: up to March 2019 Amino acid racemases and epimerases are key enzymes that invert the configuration of common amino acids and supply many corresponding d-isomers in living organisms. Some d-amino acids are inherently bioactive, whereas others are building blocks for important biomolecules, for example lipid II, the bacterial cell wall precursor. Peptides containing them have enhanced proteolytic stability and can act as important recognition elements in mammalian systems. Selective inhibition of certain amino acid racemases (e.g. glutamate racemase) is believed to offer a promising target for new antibacterial drugs effective against pathogens resistant to current antibiotics. Many amino acid racemases employ imine formation with pyridoxal phosphate (PLP) as a cofactor to accelerate the abstraction of the alpha proton. However, the group reviewed herein achieves racemization of free amino acids without the use of cofactors or metals, and uses a thiol/thiolate pair for deprotonation and reprotonation. All bacteria and higher plants contain such enzymes, for example diaminopimelate epimerase, which is required for lysine biosynthesis in these organisms. This process cannot be accomplished without an enzyme catalyst as the acidities of a thiol and the substrate α-hydrogen are inherently mismatched by at least 10 orders of magnitude. This review describes the structural and mechanistic studies on PLP-independent racemases and the evolving view of key enzymatic machinery that accomplishes these remarkable transformations.

Increasing drug resistance in pathogens including Mycobacterium tuberculosis (MTB) has been ascribed to mutations in the known target genes. However, many of these drugs have multiple targets; some of which have not been identified so far. Understanding the mechanism of action of these drugs holds a great promise in better management of disease especially by drug-resistant strains. In this study, we report glutamate racemase (MurI), a crucial enzyme of phase I peptidoglycan (PG) biosynthesis pathway of MTB, as an additional target of ethambutol (EMB). The effect on EMB on the MurI protein at structural and functional level was studied using different spectroscopic, biochemical, and insilico approaches. Spectroscopic analysis revealed that EMB-modified protein undergoes conformational alterations. Furthermore, in vitro racemization studies of the MurI protein suggest that EMB decreases its functional activity. Docking studies revealed that EMB interacts with most of the active residues at the binding site and blocks the binding pocket. Overall, data suggests that EMB, a primary drug used for the treatment of tuberculosis (TB), acts as a competitive inhibitor of substrate for binding to mycobacterial MurI protein. The study also points out to our lacunae in understanding the site and mechanism of action of existing drugs. Furthermore, glutamate racemase is a conserved protein of the bacterial kingdom; therefore, ethambutol could be a promising candidate as a broad-spectrum antibiotic for many other bacterial diseases.

The application of covalent inhibitors has experienced a renaissance within drug discovery programs in the last decade. To leverage the superior potency and drug target residence time of covalent inhibitors, there have been extensive efforts to develop highly specific covalent modifications to decrease off-target liabilities. Herein, we present a series of covalent inhibitors of an antimicrobial drug target, glutamate racemase, discovered through structure-based virtual screening. A combination of enzyme kinetics, mass spectrometry, and surface-plasmon resonance experiments details a highly specific 1,4-conjugate addition of a small-molecule inhibitor with a catalytic cysteine of glutamate racemase. Molecular dynamics simulations and quantum mechanics-molecular mechanics geometry optimizations reveal the chemistry of the conjugate addition. Two compounds from this series of inhibitors display antimicrobial potency similar to β-lactam antibiotics, with significant activity against methicillin-resistant S. aureus strains. This study elucidates a detailed chemical rationale for covalent inhibition and provides a platform for the development of antimicrobials with a novel mechanism of action against a target in the cell wall biosynthesis pathway., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chagas disease, caused by Trypanosoma cruzi, affects millions of people in South America and no satisfactory therapy exists, especially for its life threatening chronic phase. We targeted the Proline Racemase of T. cruzi, which is present in all stages of the parasite life cycle, to discover new inhibitors against this disease. The first published crystal structures of the enzyme revealed that the catalytic site is too small to allow any relevant drug design. In previous work, to break through the chemical space afforded to virtual screening and drug design, we generated intermediate models between the open (ligand free) and closed (ligand bound) forms of the enzyme. In the present work, we co-crystallized the enzyme with the selected inhibitors and found that they were covalently bound to the catalytic cysteine residues in the active site, thus explaining why these compounds act as irreversible inhibitors. These results led us to the design of a novel, more potent specific inhibitor, NG-P27. Co-crystallization of this new inhibitor with the enzyme allowed us to confirm the predicted protein functional motions and further characterize the chemical mechanism. Hence, the catalytic Cys300 sulfur atom of the enzyme attacks the C2 carbon of the inhibitor in a coupled, regiospecific-stereospecific Michael reaction with trans-addition of a proton on the C3 carbon. Strikingly, the six different conformations of the catalytic site in the crystal structures reported in this work had key similarities to our intermediate models previously generated by inference of the protein functional motions. These crystal structures span a conformational interval covering roughly the first quarter of the opening mechanism, demonstrating the relevance of modeling approaches to break through chemical space in drug design., Competing Interests: The authors have declared that no competing interests exist.

In the present study, we attempted to develop novel class of Mycobacterium tuberculosis (Mtb) inhibitors by exploring the pharmaceutically underexploited enzyme targets which are majorly involved in cell wall biosynthesis of mycobacteria. For this purpose glutamate racemase was selected which racemizes d-glutamate from l-glutamate, a key step in peptidoglycan synthesis. Furthermore, enzyme is neither expressed nor its product, d-glutamate is produced in mammals, and hence inhibiting this enzyme will have no vulnerable effect in host organism. A library of our in-house compounds were screened against glutamate racemase using a biophysical technique; thermal shift assay and further by enzyme inhibition assay to identify Lead 1 molecule. Lead 1 optimization and expansion resulted in twenty four compounds. Among the synthesized compounds twelve compounds shown good enzyme inhibition than Lead 1 (IC 50 20.07 ± 0.29 μM). Among all the compounds; compound 22 (IC 50 1.1 ± 0.52 μM) showed potent non-competitive mode of inhibition in enzyme assay. Further showed good susceptibility (in replicating bacteria) of MIC 8.72 μM and bactericidal time dependant kill on dormant culture. It also exhibited significant activity in Mtb nutrient starvation model (2.5) and Mtb biofilm model (2.4) and in vivo M. marinum infected Zebra fish model studies (3.6) reduction at logarithmic scale., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Mycobacterium tuberculosis glutamate racemase is an essential enzyme involved in peptidoglycan synthesis and conserved in most bacteria. Small molecule inhibitors were reported on other bacterial species whereas in M. tuberculosis it wasn't explored much. In this study we have screened in house compound library using fluorescence thermal shift assay and enzyme inhibition assay, form this (1-(3-(benzo[d]thiazol-2-yl)phenyl)-3-(p-tolyl)thiourea) was identified as lead compound with IC 50 19.47 ± 0.81 μM. Further lead optimization by synthesis resulted in twenty-three compounds, of which Compound 25 has shown more efficacy compared to lead 1 showing non-competitive mode of inhibition with IC 50 1.32 ± 0.43 μM. It also showed significant activity (represented in log reduction) in nutrient starved dormant M. tuberculosis model (2.1), M. tuberculosis biofilm assay (2.0) and in vivo M. marinum infected zebrafish model (3.5)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In 2013, the Centers for Disease Control highlighted Clostridium difficile as an urgent threat for antibiotic-resistant infections, in part due to the emergence of highly virulent fluoroquinolone-resistant strains. Limited therapeutic options currently exist, many of which result in disease relapse. We sought to identify molecules specifically targeting C. difficile in high-throughput screens of our diversity-oriented synthesis compound collection. We identified two scaffolds with apparently novel mechanisms of action that selectively target C. difficile while having little to no activity against other intestinal anaerobes; preliminary evidence suggests that compounds from one of these scaffolds target the glutamate racemase. In vivo efficacy data suggest that both compound series may provide lead optimization candidates.

The greatest obstacle for the treatment of cystic fibrosis patients infected with the Burkholderia species is their intrinsic antibiotic resistance. For this reason, there is a need to develop new effective compounds. Glutamate racemase, an essential enzyme for the biosynthesis of the bacterial cell wall, is an excellent candidate target for the design of new antibacterial drugs. To this aim, we recombinantly produced and characterized glutamate racemase from Burkholderia cenocepacia J2315. From the screening of an in-house library of compounds, two Zn (II) and Mn (III) 1,3,5-triazapentadienate complexes were found to efficiently inhibit the glutamate racemase activity with IC50 values of 35.3 and 10.0 μM, respectively. Using multiple biochemical approaches, the metal complexes have been shown to affect the enzyme activity by binding to the enzyme-substrate complex and promoting the formation of an inhibited dimeric form of the enzyme. Our results corroborate the value of glutamate racemase as a good target for the development of novel inhibitors against Burkholderia., Competing Interests: The authors have declared that no competing interests exist.

The increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except in Mycobacterium tuberculosis, where it has so far been underexploited. d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within the d-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies of M. tuberculosis During our studies on alternative inhibitors of the d-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor β-chloro-d-alanine (BCDA) is a very poor inhibitor of recombinant M. tuberculosis Alr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit the d-alanine pathway in intact cells, consistent with its poor in vitro activity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI in M. tuberculosis and thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development., (Copyright © 2016 Prosser et al.)

We describe competitive activity-based protein profiling (ABPP) to accelerate the functional prediction and assessment of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) in proteomic environments. Using a library of sulfamoyloxy-linked aminoacyl-AMP analogs, the competitive ABPP technique offers a simple and rapid assay system for adenylating enzymes and provides insight into enzyme substrate candidates and enzyme active-site architecture.

D-Aspartate (D-Asp) has important physiological functions, and recent studies have shown that substantial amounts of free D-Asp are present in a wide variety of mammalian tissues and cells. Biosynthesis of D-Asp has been observed in several cultured rat cell lines, and a murine gene (glutamate-oxaloacetate transaminase 1-like 1, Got1l1) that encodes Asp racemase, a synthetic enzyme that produces D-Asp from L-Asp, was proposed recently. The product of this gene is homologous to mammalian glutamate-oxaloacetate transaminase (GOT). Here, we tested the hypothesis that rat and human homologs of mouse GOT1L1 are involved in Asp synthesis. The following two approaches were applied, since the numbers of attempts were unsuccessful to prepare soluble GOT1L1 recombinant proteins. First, the relationship between the D-Asp content and the expression levels of the mRNAs encoding GOT1L1 and D-Asp oxidase, a primary degradative enzyme of D-Asp, was examined in several rat and human cell lines. Second, the effect of knockdown of the Got1l1 gene on D-Asp biosynthesis during culture of the cells was determined. The results presented here suggest that the rat and human homologs of mouse GOT1L1 are not involved in D-Asp biosynthesis. Therefore, D-Asp biosynthetic pathway in mammals is still an urgent issue to be resolved.

A pyridodiazepine amine inhibitor of Helicobacter pylori glutamate racemase (MurI) was characterized. The compound was selectively active against H. pylori, and growth suppression was shown to be mediated through the inhibition of MurI by several methods. In killing kinetics experiments, the compound showed concentration-independent activity, with about a 2-log loss of viability in 24 h. A demonstration of efficacy in a mouse infection model was attempted but not achieved, and this was attributed to the failure to attain extended exposure levels above the MIC for >95% of the time. This index and magnitude were derived from pharmacokinetic-pharmacodynamic (PK-PD) studies with amoxicillin, another inhibitor of peptidoglycan biosynthesis that showed slow killing kinetics similar to those of the pyridodiazepine amines. These studies indicate that MurI and other enzymes involved in peptidoglycan biosynthesis may be less desirable targets for monotherapy directed against H. pylori if once-a-day dosing is required., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

D-Glutamate is an essential biosynthetic building block of the peptidoglycans that encapsulate the bacterial cell wall. Glutamate racemase catalyzes the reversible formation of D-glutamate from L-glutamate and, hence, the enzyme is a potential therapeutic target. We show that the novel cyclic substrate-product analogue (R,S)-1-hydroxy-1-oxo-4-amino-4-carboxyphosphorinane is a modest, partial noncompetitive inhibitor of glutamate racemase from Fusobacterium nucleatum (FnGR), a pathogen responsible, in part, for periodontal disease and colorectal cancer (Ki=3.1±0.6 mM, cf. Km=1.41±0.06 mM). The cyclic substrate-product analogue (R,S)-4-amino-4-carboxy-1,1-dioxotetrahydro-thiopyran was a weak inhibitor, giving only ∼30% inhibition at a concentration of 40 mM. The related cyclic substrate-product analogue 1,1-dioxo-tetrahydrothiopyran-4-one was a cooperative mixed-type inhibitor of FnGR (Ki=18.4±1.2 mM), while linear analogues were only weak inhibitors of the enzyme. For glutamate racemase, mimicking the structure of both enantiomeric substrates (substrate-product analogues) serves as a useful design strategy for developing inhibitors. The new cyclic compounds developed in the present study may serve as potential lead compounds for further development., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

d-Amino acids can play important roles as specific biosynthetic building blocks required by organisms or act as regulatory molecules. Consequently, amino acid racemases that catalyze the formation of d-amino acids are potential therapeutic targets. Serine racemase catalyzes the reversible formation of d-serine (a modulator of neurotransmission) from l-serine, while proline racemase (an essential enzymatic and mitogenic protein in trypanosomes) catalyzes the reversible conversion of l-proline to d-proline. We show the substrate-product analogue α-(hydroxymethyl)serine is a modest, linear mixed-type inhibitor of serine racemase from Schizosaccharomyces pombe (Ki=167±21mM, Ki'=661±81mM, cf. Km=19±2mM). The bicyclic substrate-product analogue of proline, 7-azabicyclo[2.2.1]heptan-7-ium-1-carboxylate is a weak inhibitor of proline racemase from Clostridium sticklandii, giving only 29% inhibition at 142.5mM. However, the more flexible bicyclic substrate-product analogue tetrahydro-1H-pyrrolizine-7a(5H)-carboxylate is a noncompetitive inhibitor of proline racemase from C. sticklandii (Ki=111±15mM, cf. Km=5.7±0.5mM). These results suggest that substrate-product analogue inhibitors of racemases may only be effective when the active site is capacious and/or plastic, or when the inhibitor is sufficiently flexible., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A novel lead compound for inhibition of the antibacterial drug target, glutamate racemase (GR), was optimized for both ligand efficiency and lipophilic efficiency. A previously developed hybrid molecular dynamics-docking and scoring scheme, FERM-SMD, was used to predict relative potencies of potential derivatives prior to chemical synthesis. This scheme was successful in distinguishing between high- and low-affinity binders with minimal experimental structural information, saving time and resources in the process. In vitro potency was increased approximately fourfold against GR from the model organism, B. subtilis. Lead derivatives show two- to fourfold increased antimicrobial potency over the parent scaffold. In addition, specificity toward B. subtilis over E. coli and S. aureus depends on the substituent added to the parent scaffold. Finally, insight was gained into the capacity for these compounds to reach the target enzyme in vivo using a bacterial cell wall lysis assay. The outcome of this study is a novel small-molecule inhibitor of GR with the following characteristics: Ki=2.5 μM, LE=0.45 kcal mol(-1) atom(-1), LiPE=6.0, MIC50=260 μg mL(-1) against B. subtilis, EC50, lysis=520 μg mL(-1) against B. subtilis., (Copyright © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Three glutamic acid derivatives, two boron-containing and one imide-containing compound, were synthesized and tested for antimicrobial activity targeting glutamate-racemase. Antimicrobial effect was evaluated over Bacillus spp. Docking analysis shown that the test compounds bind near the active site of racemase isoforms, suggesting an allosteric effect. The boron derivatives had greater affinity than the imide derivative. In vitro assays shown good antimicrobial activity for the boron-containing compounds, and no effectiveness for the imide-containing compounds. The minimum inhibitory concentration of tetracycline, used as standard, was lower than that of the boron-containing derivatives. However, it seems that the boron-containing derivatives are more selective for bacteria. Experimental evidence suggests that the boron-containing derivatives act by inhibiting the racemase enzyme. Therefore, these test compounds probably impede the formation of the bacterial cell wall. Thus, the boron-containing glutamic acid derivatives should certainly be of interest for future studies as antimicrobial agents for Bacillus spp.

Glutamate racemase (GR) is a cofactor independent amino acid racemase that has recently garnered increasing attention as an antimicrobial drug target. There are numerous high resolution crystal structures of GR, yet these are invariably bound to either D-glutamate or very weakly bound oxygen-based salts. Recent in silico screens have identified a number of new competitive inhibitor scaffolds, which are not based on D-Glu, but exploit many of the same hydrogen bond donor positions. In silico studies on 1-H-benzimidazole-2-sulfonic acid (BISA) show that the sulfonic acid points to the back of the GR active site, in the most buried region, analogous to the C2-carboxylate binding position in the GR-d-glutamate complex. Furthermore, BISA has been shown to be the strongest nonamino acid competitive inhibitor. Previously published computational studies have suggested that a portion of this binding strength is derived from complexation with a more closed active site, relative to weaker ligands, and in which the internal water network is more isolated from the bulk solvent. In order to validate key contacts between the buried sulfonate moiety of BISA and moieties in the back of the enzyme active site, as well as to probe the energetic importance of the potentially large number of interstitial waters contacted by the BISA scaffold, we have designed several mutants of Asn75. GR-N75A removes a key hydrogen bond donor to the sulfonate of BISA, but also serves to introduce an additional interstitial water, due to the newly created space of the mutation. GR- N75L should also show the loss of a hydrogen bond donor to the sulfonate of BISA, but does not (a priori) seem to permit an additional interstitial water contact. In order to investigate the dynamics, structure, and energies of this water-mediated complexation, we have employed the extended linear response (ELR) approach for the calculation of binding free energies to GR, using the YASARA2 knowledge based force field on a set of ten GR complexes, and yielding an R-squared value of 0.85 and a RMSE of 2.0 kJ/mol. Surprisingly, the inhibitor set produces a uniformly large interstitial water contribution to the electrostatic interaction energy (), ranging from 30 to >50%, except for the natural substrate (D-glutamate), which has only a 7% contribution of from water. The broader implications for predicting and exploiting significant interstitial water contacts in ligand-enzyme complexation are discussed.

Two independent series of inhibitors of Helicobacter pylori glutamate racemase (MurI) were characterized for their kinetic mechanism, and one was used to generate resistant mutants in vitro. Mutant MurI enzymes from these strains were characterized by structural, genetic, kinetic and biophysical methods. Both inhibitor series, pyrazolopyrimidinediones and benzodiazepines, are uncompetitive with respect to the glutamate substrate, and the resistance mutations were found to act by reducing the affinity of MurI for substrate, thereby reducing the pool of enzyme-substrate complex available for binding inhibitor, while still allowing sufficient glutamate racemase activity for peptidoglycan construction. Uncompetitive inhibitors of a single-substrate, single-product enzyme are rare, and this work gives insight into an remarkable resistance mechanism. This article will discuss the projected clinical impact of H. pylori MurI resistance on these types of inhibitors.

Chagas' disease is caused by Trypanosoma cruzi, a protozoan transmitted to humans by blood-feeding insects, blood transfusion or congenitally. Previous research led us to discover a parasite proline racemase (TcPRAC) and to establish its validity as a target for the design of new chemotherapies against the disease, including its chronic form. A known inhibitor of proline racemases, 2-pyrrolecarboxylic acid (PYC), is water-insoluble. We synthesized soluble pyrazole derivatives, but they proved weak or inactive TcPRAC inhibitors. TcPRAC catalytic site is too small and constrained when bound to PYC to allow efficient search for new inhibitors by virtual screening. Forty-nine intermediate conformations between the opened enzyme structure and the closed liganded one were built by calculating a transition path with a method we developed. A wider range of chemical compounds could dock in the partially opened intermediate active site models in silico. Four models were selected for known substrates and weak inhibitors could dock in them and were used to screen chemical libraries. Two identified soluble compounds, (E)-4-oxopent-2-enoic acid (OxoPA) and its derivative (E)-5-bromo-4-oxopent-2-enoic acid (Br-OxoPA), are irreversible competitive inhibitors that presented stronger activity than PYC on TcPRAC. We show here that increasing doses of OxoPA and Br-OxoPA hamper T. cruzi intracellular differentiation and fate in mammalian host cells. Our data confirm that through to their binding mode, these molecules are interesting and promising as lead compounds for the development of chemotherapies against diseases where active proline racemases play essential roles.

Imidazole, a five-membered heterocycle having three carbon atoms, and two double bonds, having efficient antibacterial Escherichia coli, Bacillus subtili, Bacillus proteus, Staphylococcus aureus, Pseudomonas aeruginosa, and Helicobacter pyloriurease etc, shows a broad-spectrum of antibacterial activities. To Search new antibacterial drugs to overcome resistance of microorganisms to antibiotics, to date hundreds of this sort of derivatives have been synthesized and possess potent antibacterial activity. As the structure of imidazole derivatives is various, the target of antibacterial is also diverse including β-Lactamases, β-ketoacyl-acyl carrier protein synthase III (FabH), DNA gyrase and topoisomerase, glutamate racemase and urease. In this review, we will discuss the emergence of resistance to antibiotics and attempt to summarize the main developments of imidazole derivatives in the past ten years. We hope that increasing knowledge of the structure-activity relationship (SAR) will be beneficial to the rational design of new generation of small molecule antibacterial drugs.

Structure-activity relationships are presented around a series of pyrazolopyrimidinediones that inhibit the growth of Helicobacter pylori by targeting glutamate racemase, an enzyme that provides d-glutamate for the construction of N-acetylglucosamine-N-acetylmuramic acid peptidoglycan subunits assimilated into the bacterial cell wall. Substituents on the inhibitor scaffold were varied to optimize target potency, antibacterial activity and in vivo pharmacokinetic stability. By incorporating an imidazole ring at the 7-position of scaffold, high target potency was achieved due to a hydrogen bonding network that occurs between the 3-position nitrogen atom, a bridging water molecule and the side chains Ser152 and Trp244 of the enzyme. The lipophilicity of the scaffold series proved important for expression of antibacterial activity. Clearances in vitro and in vivo were monitored to identify compounds with improved plasma stability. The basicity of the imidazole may contribute to increased aqueous solubility at lower pH allowing for improved oral bioavailability., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Allostery is the process by which remote sites of a system are energetically coupled to elicit a functional response. The early models of allostery such as the Monod-Wyman-Changeux model and the Koshland-Némethy-Filmer model explain the allosteric behavior of multimeric proteins. However, these models do not explain how allostery arises from atomic level in detail. Recent developments in computational methods and experimental techniques have led the beginning of a new age in studying allostery. The combination of computational methods and experiments is a powerful research approach to help answering questions regarding allosteric mechanism at atomic resolution. In this review, three case studies are discussed to illustrate how this combined approach helps to increase our understanding of protein allostery., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Isomerases involved in the metabolism of D/L-amino acids represent promising therapeutic targets for treatment of disease. Herein, we report a tunable platform for the assessment of enzymatic kinetics involving amino acid isomerization by CE that offers improved selectivity and sensitivity over traditional methods. Enzyme activity and competition assays were evaluated for various hydroxyproline diastereoisomers, proline enantiomers and their structural analogs using 4-hydroxyproline-2-epimerase as a model system. In this work, pyrrole 2-carboxylic acid was found to be a selective inhibitor of 4-hydroxyproline-2-epimerase with a half-maximal inhibition concentration of (2.3 + or - 0.1) mM. Reliable methods for unambiguous characterization of amino acid isomerases are required for the screening of novel inhibitors with epimerase and/or racemase activity.

Proline racemase is an important enzyme of Trypanosoma cruzi and has been shown to be an effective mitogen for B cells, thus contributing to the parasite's immune evasion and persistence in the human host. Recombinant epimastigote parasites overexpressing TcPRAC genes coding for proline racemase present an augmented ability to differentiate into metacyclic infective forms and subsequently penetrate host-cells in vitro. Here we demonstrate that both anti T. cruzi proline racemase antibodies and the specific proline racemase inhibitor pyrrole-2-carboxylic acid significantly affect parasite infection of Vero cells in vitro. This inhibitor also hampers T. cruzi intracellular differentiation.

A successful scaffold-hopping approach gave a novel series of inhibitors of bacterial glutamate racemase (MurI). Early SAR studies of the 8-benzyl pteridine-6,7-diones led to compounds with micromolar enzyme potency and antibacterial activity.

The bacterial cell wall is a highly cross-linked polymeric structure consisting of repeating peptidoglycan units, each of which contains a novel pentapeptide substitution which is cross-linked through transpeptidation. The incorporation of D-glutamate as the second residue is strictly conserved across the bacterial kingdom. Glutamate racemase, a member of the cofactor-independent, two-thiol-based family of amino acid racemases, has been implicated in the production and maintenance of sufficient d-glutamate pool levels required for growth. The subject of over four decades of research, it is now evident that the enzyme is conserved and essential for growth across the bacterial kingdom and has a conserved overall topology and active site architecture; however, several different mechanisms of regulation have been observed. These traits have recently been targeted in the discovery of both narrow and broad spectrum inhibitors. This review outlines the biological history of this enzyme, the recent biochemical and structural characterization of isozymes from a wide range of species and developments in the identification of inhibitors that target the enzyme as possible therapeutic agents., (© 2008 The Author. Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd.)

High-throughput screening uncovered a pyrazolopyrimidinedione hit as a selective, low micromolar inhibitor of Helicobacter pylori glutamate racemase (MurI). Variation of the substituents around the scaffold led to low nanomolar inhibitors and improved antibacterial activity. The challenge in this program was to translate excellent enzyme inhibition into potent antibacterial activity and pharmacokinetics suitable for oral therapy. Compounds were profiled for MurI inhibition, activity against H. pylori, microsomal stability, and pharmacokinetics in mice. Iterative cycles of analog synthesis and biological testing led to compounds with substituents optimized for both low MICs (2 microg/ml) and good microsomal stability. In order to achieve high bioavailability, a novel pro-drug approach was implemented wherein a solubilizing sulfoxide moiety is oxidized in vivo to a sulfone.

An early SAR study of a screening hit series has generated a series of 9-benzyl purines as inhibitors of bacterial glutamate racemase (MurI) with micromolar enzyme potency and improved physical properties. X-ray co-crystal EI structures were obtained.

Bioassay-guided fractionation of the methanol extract of the Australian sponge Neopetrosia exigua led to the isolation of exiguaquinol (2), a new pentacyclic hydroquinone that inhibited Helicobacter pylori glutamate racemase (MurI) with an IC(50) of 4.4 microM. Its structure and relative configuration were assigned on the basis of spectroscopic data. Exiguaquinol (2), bearing a novel pentacyclic ring skeleton, is the first natural product to show inhibition of H. pylori MurI. Its protein-ligand modeling is also discussed.

Peptidoglycan, a key constituent of bacterial cell walls, is currently the target of broad spectrum antibiotics and a new research field involves both design and synthesis of inhibitors of its biosynthesis. Most bacteria require either lysine, or its biosynthetic precursor, diaminopimelate (meso-DAP), as a component of the peptidoglycan layer of the cell wall. In this paper, molecular modelling studies were undertaken in order to shed light on the molecular basis of interaction between (2S,6S)-diaminopimelic acid (l,l-DAP) (1) with its target enzyme DAP-epimerase, since this is a key step in the lysine biosynthetic path leading to (2R,6S)-diaminopimelic acid (meso-DAP) (2). In particular, the docking of the ligand-enzyme complex was studied by means of MD simulations and DFT computations in order to ascertain the optimal structural requirements for the epimerization reaction. Molecular dynamics simulations clearly showed that the configuration of the distal carbon C6 of l,l-DAP is critical for complex formation since both amino and carboxylate groups are involved in Hbonding interactions with the active site residues. Furthermore, the interactions occurring between the functional groups bonded to the C2 and some residues of the binding cavity immobilize the ligand in a position appropriate for the epimerization reaction, i.e., exactly in the middle of the two catalytic residues Cys73 and Cys217 as confirmed by DFT quantum mechanical computation of the Michaelis complex. All this mechanistic information could be useful for the rational design of new potential antibiotic drugs effective as inhibitors of peptidoglycan biosynthesis.

Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K(M) value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis.

Identification of novel drug targets in silico in Vibrio vulnificus is important as it is one of the emerging pathogenic microorganisms. Glutamate racemase, an important constituent of bacterial cell wall is chosen for structure prediction using homology modeling. With the aid of tools and software like MODELLER and Swiss-PdbViewer, the 3D structure is predicted and the final model is refined by energy minimization. The quality of the refined model is assessed using PROCHECK. The interaction between the predicted structure of glutamate racemase and its potential inhibitors namely L-serine O-sulfate, (2R,4R)-2-amino-4-(2-benzo[b]thienyl)methyl pentanedioic acid, aziridino glutamate, exiguaquinol, gamma-2 naphthylmethyl-D-glutamate and D-glutamine is analysed in silico by Autodock. The results indicate that certain residues like Aspl3, Tyr45, Gly46, Asn78, Thr79, Cys185, His187 are highly conserved across the active site stretches of different bacterial species and may possibly assume precedence over the other residues for inhibitory action. This study provides an insight into the structure of glutamate racemase in V. vulnificus and also gives an idea about potential sites responsible for inhibitory action that could further be substantiated by experimental investigations.

D-Glutamic acid is a required biosynthetic building block for peptidoglycan, and the enzyme glutamate racemase (GluR) catalyzes the inter-conversion of D and L-glutamate enantiomers. Therefore, GluR is considered as an attractive target for the design of new antibacterial drugs. Here, we report the crystal structures of GluR from Streptococcus pyogenes in both inhibitor-free and inhibitor-bound forms. The inhibitor free GluR crystallized in two different forms, which diffracted to 2.25 A and 2.5 A resolution, while the inhibitor-bound crystal diffracted to 2.5 A resolution. GluR is composed of two domains of alpha/beta protein that are related by pseudo-2-fold symmetry and the active site is located at the domain interface. The inhibitor, gamma-2-naphthylmethyl-D-glutamate, which was reported earlier as a novel potent competitive inhibitor, makes several hydrogen bonds with protein atoms, and the naphthyl moiety is located in the hydrophobic pocket. The inhibitor binding induces a disorder in one of the loops near the active site. In both crystal forms, GluR exists as a dimer and the interactions seen at the dimer interface are almost identical. This agrees well with the results from gel filtration and dynamic light-scattering studies.

Glutamate racemase is an enzyme essential to the bacterial cell wall biosynthesis pathway, and has therefore been considered as a target for antibacterial drug discovery. We characterized the glutamate racemases of several pathogenic bacteria using structural and biochemical approaches. Here we describe three distinct mechanisms of regulation for the family of glutamate racemases: allosteric activation by metabolic precursors, kinetic regulation through substrate inhibition, and D-glutamate recycling using a d-amino acid transaminase. In a search for selective inhibitors, we identified a series of uncompetitive inhibitors specifically targeting Helicobacter pylori glutamate racemase that bind to a cryptic allosteric site, and used these inhibitors to probe the mechanistic and dynamic features of the enzyme. These structural, kinetic and mutational studies provide insight into the physiological regulation of these essential enzymes and provide a basis for designing narrow-spectrum antimicrobial agents.

Platypus venom contains an isomerase that reversibly interconverts the second amino-acid residue in some peptides between the L-form and the D-form. The enzyme acts on the natriuretic peptides OvCNPa and OvCNPb, and on the defensin-like peptides DLP-2 and DLP-4, but it does not act on DLP-1. While the isomerization of DLP-2 to DLP-4 is inhibited by the amino-peptidase inhibitor amastatin, it is not affected by the leucine amino-peptidase inhibitor bestatin. The enzyme, that is only present in minute quantities in an extract of the venom gland, is thermally stable up to 55 degrees C, and it was found by anion-exchange chromatography to be acidic. Isolation of the isomerase was carried out by combined ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC).

The widespread emergence of pathogenic bacterial strains with resistance to antibiotics is becoming a serious threat to public health. Continuous development of novel antibacterials therefore remains one of the biggest challenges to science and unmet needs in the clinics. The biosynthetic pathway of bacterial peptidoglycan, an essential building block of cell walls, has been well studied and appears to be a rich source of attractive enzyme targets for new antibacterials. We have therefore reviewed the intracellular part of peptidoglycan biosynthesis, including the enzymes GlmS, GlmM, GlmU for formation of UDP-GlcNAc, subsequent pentapeptide synthesis by MurA-MurF, and its connection to lipid carrier by MraY and MurG. Naturally occurring inhibitors and the development of low-molecular weight inhibitors of the intracellular part of peptidoglycan synthesis are presented.

D-amino acids are much less common than their L-isomers but are widely distributed in most organisms. Many D-amino acids, including those necessary for bacterial cell wall formation, are synthesized from the corresponding L-isomers by alpha-amino acid racemases. The important class of pyridoxal phosphate-independent racemases function by an unusual mechanism whose details have been poorly understood. It has been proposed that the stereoinversion involves two active-site cysteine residues acting in concert as a base (thiolate) and an acid (thiol). Although crystallographic structures of several such enzymes are available, with the exception of the recent structures of glutamate racemase from Bacillus subtilis and of proline racemase from Trypanosoma cruzi, the structures either are of inactive forms (e.g., disulfide) or do not allow unambiguous modeling of the substrates in the active sites. Here, we present the crystal structures of diaminopimelate (DAP) epimerase from Haemophilus influenzae with two different isomers of the irreversible inhibitor and substrate mimic aziridino-DAP at 1.35- and 1.70-A resolution. These structures permit a detailed description of this pyridoxal 5'-phosphate-independent amino acid racemase active site and delineate the electrostatic interactions that control the exquisite substrate selectivity of DAP epimerase. Moreover, the active site shows how deprotonation of the substrates' nonacidic hydrogen at the alpha-carbon (pKa approximately 29) by a seemingly weakly basic cysteine residue (pKa approximately 8-10) is facilitated by interactions with two buried alpha-helices. Bacterial racemases, including glutamate racemase and DAP epimerase, are potential targets for the development of new agents effective against organisms resistant to conventional antibiotics.

The presence of d-amino-acid-containing polypeptides, defensin-like peptide (DLP)-2 and Ornithorhyncus venom C-type natriuretic peptide (OvCNP)b, in platypus venom suggested the existence of a mammalian d-amino-acid-residue isomerase(s) responsible for the modification of the all-l-amino acid precursors. We show here that this enzyme(s) is present in the venom gland extract and is responsible for the creation of DLP-2 from DLP-4 and OvCNPb from OvCNPa. The isomerisation reaction is freely reversible and under well defined laboratory conditions catalyses the interconversion of the DLPs to full equilibration. The isomerase is approximately 50-60 kDa and is inhibited by methanol and the peptidase inhibitor amastatin. This is the first known l-to-d-amino-acid-residue isomerase in a mammal.

Amino acid racemases catalyze the stereoinversion of the chiral C alpha to produce the d-enantiomers that participate in biological processes, such as cell wall construction in prokaryotes. Within this large protein family, bacterial proline racemases have been extensively studied as a model of enzymes acting with a pyridoxal-phosphate-independent mechanism. Here we report the crystal structure of the proline racemase from the human parasite Trypanosoma cruzi (TcPRACA), a secreted enzyme that triggers host B cell polyclonal activation, which prevents specific humoral immune responses and is crucial for parasite evasion and fate. The enzyme is a homodimer, with each monomer folded in two symmetric alpha/beta subunits separated by a deep crevice. The structure of TcPRACA in complex with a transition-state analog, pyrrole-2-carboxylic acid, reveals the presence of one reaction center per monomer, with two Cys residues optimally located to perform acid/base catalysis through a carbanion stabilization mechanism. Mutation of the catalytic Cys residues abolishes the enzymatic activity but preserves the mitogenic properties of the protein. In contrast, inhibitor binding promotes the closure of the interdomain crevice and completely abrogates B cell proliferation, suggesting that the mitogenic properties of TcPRACA depend on the exposure of transient epitopes in the ligand-free enzyme.

Aziridine analogues of diaminopimelic acid (DAP) have been prepared stereoselectively for the first time and evaluated as inhibitors of DAP epimerase. (2R,3S,3'S)-3-(3'-Aminopropane)aziridine-2,3'-dicarboxylate was synthesised and shown to be a reversible inhibitor of DAP epimerase with an IC(50) value of 2.88 mM. (2S,4S)- and (2S,4R)-2-(4-Amino-4-carboxybutyl)aziridine-2-carboxylic acid (ll-azi-DAP and dl-azi-DAP ) were made as pure diastereomers, and both were shown to be irreversible inhibitors of DAP epimerase. ll-Azi-DAP selectively binds to Cys-73 of the enzyme active site whereas dl-azi-DAP binds to Cys-217 via attack of sulfhydryl on the methylene of the inhibitor aziridine ring. These observations are consistent with the two base mechanism proposed for the epimerization of ll-DAP and meso-DAP by DAP epimerase.

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.

Glutamate and aspartate are frequently recognized as key structural elements for the biological activity of natural peptides and synthetic compounds. The acidic side-chain functionality of both the amino acids provides the basis for the ionic interaction and subsequent molecular recognition by specific receptor sites that results in the regulation of physiological or pathophysiological processes in the organism. In the development of new biologically active compounds that possess the ability to modulate these processes, compounds offering the same type of interactions are being designed. Thus, using a peptidomimetic design approach, glutamate and aspartate mimetics are incorporated into the structure of final biologically active compounds. This review covers different bioisosteric replacements of carboxylic acid alone, as well as mimetics of the whole amino acid structure. Amino acid analogs presented include those with different distances between anionic moieties, and analogs with additional functional groups that result in conformational restriction or alternative interaction sites. The article also provides an overview of different cyclic structures, including various cycloalkane, bicyclic and heterocyclic analogs, that lead to conformational restriction. Higher di- and tripeptide mimetics in which carboxylic acid functionality is incorporated into larger molecules are also reviewed. In addition to the mimetic structures presented, emphasis in this article is placed on their steric and electronic properties. These mimetics constitute a useful pool of fragments in the design of new biologically active compounds, particularly in the field of RGD mimetics and excitatory amino acid agonists and antagonists.

Bacterial biosynthesis of lysine has come under increased scrutiny as a target for novel antibacterial agents as it provides both lysine for protein synthesis and meso-diaminopimelate for construction of the bacterial peptidoglycan cell wall. Recent studies of the enzymes of the lysine biosynthetic pathway, development of inhibitors and investigations of their antibacterial properties are discussed.

Ribosomally produced peptides that contain D-amino acids have been isolated from a number of vertebrate and invertebrate sources. In each case, the D-amino acids are introduced by a posttranslational modification of a parent peptide containing only amino acids of the L-configuration. The only known enzyme to catalyze such a reaction is the peptide epimerase (also known as peptide isomerase) from the venom of the funnel web spider, Agelenopsis aperta. This enzyme interconverts two 48-amino-acid-long peptide toxins that differ only by the stereochemistry at a single serine residue. In this paper we report the synthesis and testing of two pentapeptide analogues that contain modified amino acids at the site normally occupied by the substrate serine residue. When the L-chloroalanine-containing peptide 3 was incubated with the epimerase it was converted into the dehydroalanine-containing peptide 4 via an elimination of HCl. The dehydroalanine peptide 4 was independently synthesized and found to act as a potent inhibitor of the epimerase (IC50 = 0.5 microM). These results support a direct deprotonation/reprotonation mechanism in which a carbanionic intermediate is formed. The observed inhibition by 4 can be attributed to the sp(2)-hybridization of the alpha-carbon in the dehydroalanine unit that mimics the planar geometry of the anionic intermediate.

The first potent inhibitors of glutamate racemase (MurI) enzyme that show whole cell antibacterial activity are described. Optically pure 4-substituted D-glutamic acid analogues with (2R,4S) stereochemistry and bearing aryl-, heteroaryl-, cinnamyl-, or biaryl-methyl substituents represent a novel class of glutamate racemase inhibitors. Exploration of the D-Glu core led to the identification of lead compounds (-)-8 and 10. 2-Naphthylmethyl derivative 10 was found to be a potent competitive inhibitor of glutamate racemase activity (K(i) = 16 nM, circular dichroism assay; IC(50) = 0.1 microg/mL high-performance liquid chromatography (HPLC) assay). Thorough structure-activity relationship (SAR) studies led to benzothienyl derivatives such as 69 and 74 with increased potency (IC(50) = 0.036 and 0.01 microg/mL, respectively, HPLC assay). These compounds showed potent whole cell antibacterial activity against S. pneumoniae PN-R6, and good correlation with the enzyme assay. Compounds 69, 74 and biaryl derivative 52 showed efficacy in an in vivo murine thigh infection model against Streptococcus pneumoniae. Data described herein suggest that glutamate racemase may be a viable target for developing new antibacterial agents.

Free D-amino acid content in some archaea was investigated and D-forms of several amino acids were found in them. In the acidothermophilic archaeon, Thermoplasma acidophilum, the proportion of D-aspartate (D-Asp) to total Asp was as high as 39.7%. Crude extracts of Thermoplasma acidophilum had Asp-specific racemase activity that was pyridoxal 5'-phosphate (PLP)-dependent. The relative insensitivity to a SH-modifying reagent distinguished this activity from those of the PLP-independent Asp racemases found in other hyperthermophilic archaea (Matsumoto, M., et al., J. Bacteriol. 181, 6560-6563 1999). Thus, high levels of d-Asp should be produced by a new type(s) of Asp-specific racemase in Thermoplasma acidophilum, although the function of d-Asp in this archaeon remains unknown., (Copyright 2001 Academic Press.)