Functional Comparison of the Two Bacillus anthracis Glutamate Racemases

Dylan Dodd; Joseph G. Reese; Craig R. Louer; Jimmy D. Ballard; M. Ashley Spies; Steven R. Blanke

doi:10.1128/JB.00352-07

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Functional Comparison of the Two Bacillus anthracis Glutamate Racemases

Journal article

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Functional Comparison of the Two Bacillus anthracis Glutamate Racemases

Dylan Dodd, Joseph G. Reese, Craig R. Louer, Jimmy D. Ballard, M. Ashley Spies and Steven R. Blanke

Journal of bacteriology, Vol.189(14), pp.5265-5275

05/11/2007

DOI: 10.1128/JB.00352-07

PMCID: PMC1951872

PMID: 17496086

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Abstract

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.

Enzymes and Proteins

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Title: Subtitle: Functional Comparison of the Two Bacillus anthracis Glutamate Racemases
Creators: Dylan Dodd - University of Illinois Urbana-Champaign
Joseph G. Reese - University of Illinois Urbana-Champaign
Craig R. Louer - University of Illinois Urbana-Champaign
Jimmy D. Ballard - University of Oklahoma Health Sciences Center
M. Ashley Spies - University of Illinois Urbana-Champaign
Steven R. Blanke - University of Illinois Urbana-Champaign
Resource Type: Journal article
Publication Details: Journal of bacteriology, Vol.189(14), pp.5265-5275
DOI: 10.1128/JB.00352-07
PMID: 17496086
PMCID: PMC1951872
NLM abbreviation: J Bacteriol
ISSN: 0021-9193
eISSN: 1098-5530
Publisher: American Society for Microbiology
Language: English
Date published: 05/11/2007
Academic Unit: Pharmaceutical Sciences and Experimental Therapeutics; Biochemistry and Molecular Biology; Medicinal and Natural Products Chemistry
Record Identifier: 9984288729102771

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