Substrate-Induced Conformational Changes in Bacillus subtilis Glutamate Racemase and Their Implications for Drug Discovery

Ruzheinikov, S.N.; Taal, Makie A.; Sedelnikova, Svetlana E.; Baker, Patrick J.; Rice, David W.

doi:10.1016/j.str.2005.07.024

Cited by 52 publications

(110 citation statements)

References 31 publications

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“…Therefore, at neutral pH, the cysteines exist as a rapidly equilibrating thiolate-thiol pair in the presence of the substrate. Indeed, no basic residue available for deprotonation of the thiol is present in the active site, in contrast to the recently reported structures of glutamate racemase from B. subtilis with D-glutamate (19) and of proline racemase from T. cruzi with pyrrole-2-carboxylic acid (20).…”

Section: Concomitant Orientation Of the Carboxylate To Maximize Orbitmentioning

confidence: 57%

“…However, the ␣͞␤ domains of DAP epimerase (composed of mainly ␤-strands) are very different from those of aspartate racemase and glutamate racemase. A recent report on the crystal structure of glutamate racemase from B. subtilis in complex with D-glutamate reveals a similar conformational rearrangement in the enzyme upon substrate binding (19). Interestingly, despite the enzyme being cocrystallized in the presence of L-glutamate, the product D-glutamate was the species trapped in the crystal structure.…”

Section: Concomitant Orientation Of the Carboxylate To Maximize Orbitmentioning

confidence: 90%

“…However, the observation of a highly ordered D-glutamate in the crystal structure is surprising, because the enzyme forms a racemic mixture of L-and D-glutamate under the conditions of crystallization. Also, on the basis of the proximity of the catalytic cysteines (Cys-74 and Cys-185) to residues of the enzyme that could function as base catalysts and to groups on the substrate, Rice and coworkers (19) suggest a mechanism using a neutral cysteine thiol. However, a clear role for these groups in the activation of the catalytic cysteines could not be identified from their structure.…”

Section: Concomitant Orientation Of the Carboxylate To Maximize Orbitmentioning

confidence: 99%

“…The x-ray crystal structure of glutamate racemase has been reported with the substrate analogue D-glutamine (3), but the lack of any strong interactions with the enzyme allows for a variety of possible binding modes in molecular modeling studies and precludes convincing mechanistic deductions (18). Recently, Rice and coworkers (19) have reported that the crystal structure of D-glutamate bound to glutamate racemase from Bacillus subtilis; this structure revealed a ''closed conformation'' for this enzyme upon ligand binding. The authors suggested that, in glutamate racemase, an aspartate (Asp-10) and͞or histidine (His-187) could act as a base catalyst but that the epimerization mechanism utilizes a neutral thiol.…”

mentioning

confidence: 99%

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

Pillai

Cherney

Diaper

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

113

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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 ␣-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-Å 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 ␣-carbon (pKa Ϸ 29) by a seemingly weakly basic cysteine residue (pK a Ϸ 8 -10) is facilitated by interactions with two buried ␣-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.racemase ͉ stereochemical mechanism T he increase in microbial resistance to conventional antibiotics has rekindled intense interest in new methods of inhibiting bacterial cell wall biosynthesis (1), including blocking formation or utilization of D-amino acids such as D-alanine (2), D-glutamate (3), and meso-diaminopimelic acid (4) present in peptidoglycan (5). The inclusion of D-amino acids in the peptidoglycan layer of the cell wall is thought to provide bacteria with protection from the action of host proteases (2). Glutamate racemase (6, 7) and diaminopimelate (DAP) epimerase (EC 5.1.1.7) (8), as well as aspartate racemase (9) and proline racemase (10, 11), are examples of pyridoxal 5Ј-phosphate (PLP)-independent amino acid racemases that invert the configuration at the ␣-carbon of an amino acid without the use of cofactors, metals, or reducible keto or imino functionalities. The proposed ''two-base'' mechanism (11) for these enzymes involves one active-site cysteine thiolate acting as a base to deprotonate the ␣-carbon, while a second cysteine thiol functions as an acid to reprotonate the resulting planar carbanionic intermediate from the opposite face (Scheme 1). This process is not trivial, because the pK a of the ␣-proton is Ϸ21 for the fully protonated fo...

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Section: Concomitant Orientation Of the Carboxylate To Maximize Orbitmentioning

confidence: 57%

Section: Concomitant Orientation Of the Carboxylate To Maximize Orbitmentioning

confidence: 90%

Section: Concomitant Orientation Of the Carboxylate To Maximize Orbitmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Pillai

Cherney

Diaper

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

113

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“…5.1.1.3), which catalyzes the reversible stereoinversion of L-glutamate (5,14,31). Insights into the cofactor-independent amino acid racemases have begun to emerge from biochemical studies of enzymes isolated from several organisms, including Bacillus subtilis, Bacillus pumilus, Bacillus sphaericus, Escherichia coli, Lactobacillus fermentum, Lactobacillus brevis, Aquifex pyrophilus, Staphylococcus haemolyticus, Brevibacterium lactofermentum, and Mycobacterium tuberculosis (1,2,5,10,14,18,19,21,28,29,33,35,40,47,58,61), as well as the recently described crystal structure of RacE-D-glutamate from B. subtilis (43). Several studies have identified glutamate racemase as an essential gene in B. subtilis and E. coli, which has led to the prediction that glutamate racemase activity is important for peptidoglycan biosynthesis in these organisms (14,31).…”

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confidence: 99%

Functional Comparison of the Two Bacillus anthracis Glutamate Racemases

Dodd¹,

Reese

Louer³

et al. 2007

J Bacteriol

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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.

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Changes in quaternary structure cause a kinetic asymmetry of glutamate racemase‐catalyzed homocysteic acid racemization

2018

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Glutamate racemases (GR) catalyze the racemization of d- and l-glutamate and are targets for the development of antibiotics. We demonstrate that GR from the periodontal pathogen Fusobacterium nucleatum (FnGR) catalyzes the racemization of d-homocysteic acid (d-HCA), while l-HCA is a poor substrate. This enantioselectivity arises because l-HCA perturbs FnGR's monomer-dimer equilibrium toward inactive monomer. The inhibitory effect of l-HCA may be overcome by increasing the total FnGR concentration or by adding glutamate, but not by blocking access to the active site through site-directed mutagenesis, suggesting that l-HCA binds at an allosteric site. This phenomenon is also exhibited by GR from Bacillus subtilis, suggesting that enantiospecific, "substrate"-induced dissociation of oligomers to form inactive monomers may furnish a new inhibition strategy.

show abstract

Substrate-Induced Conformational Changes in Bacillus subtilis Glutamate Racemase and Their Implications for Drug Discovery

Cited by 52 publications

References 31 publications

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

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

Functional Comparison of the Two Bacillus anthracis Glutamate Racemases

Changes in quaternary structure cause a kinetic asymmetry of glutamate racemase‐catalyzed homocysteic acid racemization

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