Glutamate Racemase Research Articles

Genetic design of conditional d-glutamate auxotrophy for Bacillus subtilis: Use of a vector-borne poly-γ-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-γ-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.

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-γ- 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 Å and 2.0 Å. 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.

Exploitation of structural and regulatory diversity in glutamate racemases

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.

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.

Structural Basis for Glutamate Racemase Inhibition

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 Å and 2.5 Å resolution, while the inhibitor-bound crystal diffracted to 2.5 Å resolution. GluR is composed of two domains of α/β protein that are related by pseudo-2-fold symmetry and the active site is located at the domain interface. The inhibitor, γ-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.

Bacillus Anthracis Race1 and RACE2 Encode Functional Glutamate Racemases with Distinct Properties

Restricted accessAbstractFirst published March 2007Bacillus Anthracis Race1 and RACE2 Encode Functional Glutamate Racemases with Distinct PropertiesD. Dodd, J.G. Reese, […], C.R. Louer, J.D. Ballard, M.A. Spies, and S.R. Blanke+3-3View all authors and affiliationsVolume 55, Issue 2https://doi.org/10.1177/108155890705500235

New Insights into the Reaction Mechanism Catalyzed by the Glutamate Racemase Enzyme: pH Titration Curves and Classical Molecular Dynamics Simulations

The mechanism of the reactions catalyzed by the pyridoxal-phosphate-independent amino acid racemases and epimerases faces the difficult task of deprotonating a relatively low acidicity proton, the amino acid's alpha-hydrogen, with a relatively poor base, a cysteine. In this work, we propose a mechanism for one of these enzymes, glutamate racemase (MurI), about which many controversies exist, and the roles that its active site residues may play. The titration curves and the pK1/2 values of all of the ionizable residues for different structures leading from reactants to products have been analyzed. From these results a concerted mechanism has been proposed in which the Cys70 residue would deprotonate the alpha-hydrogen of the substrate while, at the same time, being deprotonated by the Asp7 residue. To study the consistency of this mechanism classical molecular dynamics (MD) simulations have been carried out along with pK1/2 calculations on the MD-generated structures.

Inversion of enantioselectivity of arylmalonate decarboxylase via site-directed mutation based on the proposed reaction mechanism

Arylmalonate decarboxylase (AMDase, EC. 4.1.1.76) catalyzes asymmetric decarboxylation of α-arylmalonic acid to give optically pure ( R)-α-arylpropionic acid. This enzyme has four cysteine residues, one of which, Cys188, is estimated to be located in the active site. It is believed that it delivers a proton from the si-face of the intermediate enolate to give ( R)-product. Based on database searches, it has been revealed that AMDase has some homology with glutamate racemase and other isomerases. Glutamate racemase has a pair of Cys residues in the active site, at positions 73 and 184, which are believed to abstract and deliver a proton from both sides of the substrate. On the other hand, AMDase has only one Cys at 188, and this is considered to be the reason why this enzyme gives optically pure products. The estimated 3D-structure of AMDase suggests that Gly74 is located in the opposite side of Cys188. Then, it was expected that introduction of one Cys around the region of Gly74, i.e., from 68 to 77 in addition to the replacement of Cys188 with less acidic Ser would invert the enantioselectivity of the enzyme. Thus, 10 mutants were prepared and their activities as well as their enantioselectivities were examined. As expected, two of them, S71C/C188S and G74C/C188S, exhibited decarboxylation activity and gave the opposite enantiomer to that formed by the wild type enzyme.

Glutamate racemase from Mycobacterium tuberculosis inhibits DNA gyrase by affecting its DNA-binding

Glutamate racemase (MurI) catalyses the conversion of l-glutamate to d-glutamate, an important component of the bacterial cell wall. MurI from Escherichia coli inhibits DNA gyrase in presence of the peptidoglycan precursor. Amongst the two-glutamate racemases found in Bacillus subtilis, only one inhibits gyrase, in absence of the precursor. Mycobacterium tuberculosis has a single gene encoding glutamate racemase. Action of M.tuberculosis MurI on DNA gyrase activity has been examined and its mode of action elucidated. We demonstrate that mycobacterial MurI inhibits DNA gyrase activity, in addition to its precursor independent racemization function. The inhibition is not species-specific as E.coli gyrase is also inhibited but is enzyme-specific as topoisomerase I activity remains unaltered. The mechanism of inhibition is different from other well-known gyrase inhibitors. MurI binds to GyrA subunit of the enzyme leading to a decrease in DNA-binding of the holoenzyme. The sequestration of the gyrase by MurI results in inhibition of all reactions catalysed by DNA gyrase. MurI is thus not a typical potent inhibitor of DNA gyrase and instead its role could be in modulation of the gyrase activity.

Structural insights into stereochemical inversion by diaminopimelate epimerase: an antibacterial drug target.

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.

Biosynthesis of poly(γ-glutamic acid) in Bacillus subtilis NX-2: Regulation of stereochemical composition of poly(γ-glutamic acid)

The stereochemical composition of poly(γ-glutamic acid), γ-PGA, produced by Bacillus subtilis NX-2 could be effected by Mn 2+ in the culture medium. When the concentration of Mn 2+ varied from 0 to 0.09 g/l, the proportion of d-glutamate increased from 18 to 77%. The d-amino acid aminotransferase and the glutamate racemase activity were also analyzed with Mn 2+ concentration changing in this range, the former was stabled at 0.12 ± 0.02 U/mg, while the latter increased from 0.20 to 0.44 U/mg. Moreover, the glutamate racemase activities also increased while adding 0.03 g/l Mn 2+ to cell extracts. These results suggested that different Mn 2+ concentration could change γ-PGA stereochemical composition through regulating glutamate racemase activity, which was different from other γ-PGA producers in species of B. subtilis ever reported. On the other hand, when Mn 2+ concentration was 0.03 g/l, the proportion of d-glutamate of γ-PGA remained approximately 75% during γ-PGA production, this also made it different from other γ-PGA producers. To understand why it was different in γ-PGA stereochemical modulation, the glutamate racemase gene in B. subtilis NX-2 was cloned, and the amino acid sequence was compared with those in B. subtilis 168 and γ-PGA producer B. subtilis IFO3336. There are four different amino acids (Leu2, Ala230, Asn231 and Arg271) in B. subtilis NX-2 glutamate racemase.

On the Ionization State of the Substrate in the Active Site of Glutamate Racemase. A QM/MM Study about the Importance of Being Zwitterionic

Computer simulations on a QM/MM potential energy surface have been carried out to gain insights into the catalytic mechanism of glutamate racemase (MurI). Understanding such a mechanism is a challenging task from the chemical point of view because it involves the deprotonation of a low acidic proton by a relatively weak base to give a carbanionic intermediate. First, we have examined the dependency of the kinetics and thermodynamics of the racemization process catalyzed by MurI on the ionization state of the substrate (glutamate) main chain. Second, we have employed an energy decomposition procedure to study the medium effect on the enzyme-substrate electrostatic and polarization interactions along the reaction. Importantly, the present theoretical results quantitatively support the mechanistic proposal by Rios et al. [J. Am. Chem. Soc. 2000, 122, 9373-9385] for the PLP-independent amino acid racemases.

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.

All four Mycobacterium tuberculosis glnA genes encode glutamine synthetase activities but only GlnA1 is abundantly expressed and essential for bacterial homeostasis

Glutamine synthetases (GS) are ubiquitous enzymes that play a central role in every cell's nitrogen metabolism. We have investigated the expression and activity of all four genomic Mycobacterium tuberculosis GS - GlnA1, GlnA2, GlnA3 and GlnA4 - and four enzymes regulating GS activity and/or nitrogen and glutamate metabolism - adenylyl transferase (GlnE), gamma-glutamylcysteine synthase (GshA), UDP-N-acetylmuramoylalanine-D-glutamate ligase (MurD) and glutamate racemase (MurI). All eight genes are located in multigene operons except for glnA1, and all are transcribed in M. tuberculosis; however, some are not translated or translated at such low levels that the enzymes escape detection. Of the four GS, only GlnA1 can be detected. Each of the eight genes, as well as the glnA1-glnE-glnA2 cluster, was expressed separately in Mycobacterium smegmatis, and its gene product was characterized and assayed for enzymatic activity by analysing the reaction products. In M. smegmatis, all four recombinant-overexpressed GS are multimeric enzymes exhibiting GS activity. Whereas GlnA1, GlnA3 and GlnA4 catalyse the synthesis of L-glutamine, GlnA2 catalyses the synthesis of D-glutamine and D-isoglutamine. The generation of mutants in M. tuberculosis of the four glnA genes, murD and murI demonstrated that all of these genes except glnA1 are nonessential for in vitro growth. L-methionine-S,R-sulphoximine (MSO), previously demonstrated to inhibit M. tuberculosis growth in vitro and in vivo, strongly inhibited all four GS enzymes; hence, the design of MSO analogues with an improved therapeutic to toxic ratio remains a promising strategy for the development of novel anti-M. tuberculosis drugs.

Some special features of glyoxyl supports to immobilize proteins

This paper described that glyoxyl activated supports are only able to quantitatively immobilize proteins at alkaline pH values and using highly activated supports (otherwise, immobilization is negligible). Furthermore, the immobilization rate depends on the surface density of glyoxyl groups and not on the total concentration of groups in the suspension. Moreover, the temperature presented a dramatic effect on enzyme immobilization rate and different enzymes become immobilized at very different rates. Finally, it was not possible to detect immobilization of mono aminated compounds at neither alkaline nor neutral pH values. In fact, these supports may be used to purify enzymes due to these great differences in immobilization rate (e.g., glutamate racemase). These special features are not shared with other supports usually utilized for covalent immobilization of enzymes (e.g., glutaraldehyde, cyanogen bromide supports) that are able to immobilize proteins by just one very stable bond and suggesting an unique mechanism for glyoxyl agarose supports. In fact, all these results suggested that each individual amino-glyoxyl bond is very weak. Therefore, the first immobilization of proteins on supports activated with glyoxyl groups, in absence of reducing reagents, needs the simultaneous establishment of, at least, two attachments between the protein and the support. This mechanism promotes that proteins become immobilized by the area/s where the highest lysine residues density is located and explain the high enzyme stabilization usually achieved by using this immobilization technique.

Efficient gene inactivation in Bacillus anthracis

A procedure for high-efficiency gene inactivation in Bacillus anthracis has been developed. It is based on a highly temperature-sensitive plasmid vector carrying kanamycin resistance cassette surrounded by DNA fragments flanking the desired insertion site. The approach was tested by constructing glutamate racemase E1 ( racE1), glutamate racemase E2 ( racE2) and comEC knock-out mutants of B. anthracis strain ΔANR. Allelic replacements were observed at high frequencies, ranging from ∼0.5% for racE2 up to 50% for racE1 and comEC. The system can be used for genetic validation of potential drug targets.

Statistical criteria for the identification of protein active sites using theoretical microscopic titration curves

Theoretical Microscopic Titration Curves (THEMATICS) may be used to identify chemically important residues in active sites of enzymes by characteristic deviations from the normal, sigmoidal Henderson-Hasselbalch titration behavior. Clusters of such deviant residues in physical proximity constitute reliable predictors of the location of the active site. Originally the residues with deviant predicted behavior were identified by human observation of the computed titration curves. However, it is preferable to select the unusual residues by mathematically well-defined criteria, in order to reduce the chance of error, eliminate any possible biases, and substantially speed up the selection process. Here we present some simple statistical tests that constitute such selection criteria. The first derivatives of the predicted titration curves resemble distribution functions and are normalized. The moments of these first derivative functions are computed. It is shown that the third and fourth moments, measures of asymmetry and kurtosis, respectively, are good measures of the deviations from normal behavior. Results are presented for 44 different enzymes. Detailed results are given for 4 enzymes with 4 different types of chemistry: arginine kinase from Limulus polyphemus (horseshoe crab); beta-lactamase from Escherichia coli; glutamate racemase from Aquifex pyrophilus; and 3-isopropylmalate dehydrogenase from Thiobacillus ferrooxidans. The relationship between the statistical measures of nonsigmoidal behavior in the predicted titration curves and the catalytic activity of the residue is discussed.

Microbial production of a poly(γ-glutamic acid) derivative by Bacillus subtilis

Bacillus subtilis C1, a bacterium capable of producing a glycerol and γ-PGA bioconjugate, was studied. C1 is a glutamate independent bacterium, which produces the γ-PGA derivative in the absence of l-glutamate. A large amount of the bioconjugate, 21.4 g/l, was produced when the bacteria were cultured in medium T at 37 °C, 150 rpm for 6 days. The number-average molecular weight ( M n ) of the bioconjugate was over 1 × 10 7 as determined by gel permeation chromatography, amino acid analysis showed only glutamic acid peak and the 1H NMR spectrum showed chemical shifts of both γ-PGA and glycerol. The d-glutamate content was over 97% in every bioconjugate produced under the conditions used and the d-glutamate content was indifferent to the Mn 2+ concentrations under study. During bioconjugate production, an intracellular glutamate racemase activity was detected, suggesting the enzyme is involved in the d-glutamate supply. The molecular weight of the bioconjugate varied with salt concentration in the medium; the molecular weight decreased by a factor of approximately 10 from 7.94 × 10 6 to 0.73 × 10 6 Da at 4-day cultivation time for cultures, which contained 0.05 and 5% NaCl, respectively. The viscosity of the bioconjugate produced by C1 is significantly higher than that of γ-PGA itself. This bioconjugate is the first example of γ-PGA derivative directly produced by microbes; thus, it is a unique biomaterial.

Crystallization and preliminary X-ray crystallographic studies of glutamate racemase fromLactobacillus fermenti

Glutamate racemase catalyzes the conversion of L-glutamic acid to D-glutamic acid and vice versa. Since D-glutamic acid is one of the essential amino acids present in peptidoglycan, glutamate racemase has been considered to be an attractive target for the design of new antibacterial drugs. Glutamate racemase from Lactobacillus fermenti has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 8000 as a precipitant. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 98.32, b = 184.09, c = 45.99 A. The asymmetric unit contains one molecule, corresponding to a VM value of 1.84 A3 Da(-1). A complete data set has been collected from the native enzyme at 2.28 A resolution using a synchrotron-radiation source.

Expression, purification and preliminary X-ray analysis of crystals ofBacillus subtilisglutamate racemase

Glutamate racemase (MurI, RacE; E.C.5.1.1.3) catalyses the cofactor-independent conversion of L-glutamate to D-glutamate, an essential step in the synthesis of components of the bacterial cell wall. The gene for RacE from Bacillus subtilis has been cloned and the protein expressed in Escherichia coli, purified and crystallized in the presence of L-glutamate using the hanging-drop method of vapour diffusion with diammonium tartrate as the precipitant. The crystals belong to the monoclinic space group C2, with approximate unit-cell parameters a = 133.6, b = 60.1, c = 126.2 A, beta = 117.6 degrees . Consideration of the possible values of V(M) suggests that the asymmetric unit contains either two (V(M) = 3.75 A(3) Da(-1)) or three (V(M) = 2.5 A(3) Da(-1)) subunits. The crystals diffract X-rays to at least 2.1 A resolution on a synchrotron-radiation source and are suitable for structural studies. Determination of the structure may provide insight into the molecular basis of substrate recognition and catalysis by this enzyme.