Expression, purification and preliminary X-ray analysis of crystals of<i>Bacillus subtilis</i>glutamate racemase

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

doi:10.1107/s0907444904021134

Cited by 15 publications

(16 citation statements)

References 20 publications

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“…8,9,18,[24][25][26] However, RacE from B. subtilis forms a dimer in solution in the presence of saturating concentrations of either D or L-glutamate or D or L-glutamine, but it remains a monomer in the presence of aspartate. 26 The RacE enzyme from A. pyrophilus, a monomer under low salt concentrations, has also been reported to form a dimer under high salt concentrations. 25 The formation of a dimeric structure for the A. pyrophilus enzyme was also inferred from the X-ray crystal structure.…”

Section: Oligomeric State Of Race1 and Race2 Enzymes In Solutionmentioning

confidence: 97%

Structural and Functional Analysis of Two Glutamate Racemase Isozymes from Bacillus anthracis and Implications for Inhibitor Design

May

Mehboob

Mulhearn

et al. 2007

Journal of Molecular Biology

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

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Section: Oligomeric State Of Race1 and Race2 Enzymes In Solutionmentioning

confidence: 97%

Structural and Functional Analysis of Two Glutamate Racemase Isozymes from Bacillus anthracis and Implications for Inhibitor Design

May

Mehboob

Mulhearn

et al. 2007

Journal of Molecular Biology

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“…Notably, within the family of glutamate racemases, quaternary structure differences apparently exist, as enzymes from B. subtilis (YrpC) (1), B. pumilus (33), L. fermentum (13,19), and Pediococcus pentosaceus (37) have been reported to be monomers, while a dimeric form has been reported for A. pyrophilus (29) and there are conflicting reports about the quaternary structure of the E. coli enzyme (15,62). Furthermore, RacE from B. subtilis has been reported to exist in equilibrium between a monomer and a dimer (57). Studies are currently under way in our laboratory to further explore the cellular roles of RacE1 and RacE2 and to elucidate the basis of regulation of their cellular activities.…”

Section: Vol 189 2007 Glutamate Racemases From B Anthracis 5273mentioning

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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“…However, the gel filtration of Alr1 was carried out in the absence of L-alanine, in 50 mM sodiumphosphate buffer (pH 7.2) containing 50 μM PLP and 200 mM NaCl. At higher salt concentrations, molecules tend to be monomeric, as shown by glutamate racemase from B. subtilis (Taal et al, 2004). Hence, it appears likely that no dimeric Alr1 resided in the gel-filtration column.…”

Section: Discussionmentioning

confidence: 96%

Alanine racemase from Helicobacter pylori NCTC 11637:Purification, characterization and gene cloning

et al. 2007

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

Cited by 15 publications

References 20 publications

Structural and Functional Analysis of Two Glutamate Racemase Isozymes from Bacillus anthracis and Implications for Inhibitor Design

Structural and Functional Analysis of Two Glutamate Racemase Isozymes from Bacillus anthracis and Implications for Inhibitor Design

Functional Comparison of the Two Bacillus anthracis Glutamate Racemases

Alanine racemase from Helicobacter pylori NCTC 11637:Purification, characterization and gene cloning

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