Functional comparison of the two Bacillus anthracis glutamate racemases

From the Department of Microbiology and the Institute for Genomic Biology and the Department of Biochemistry, University of Illinois, Urbana, Illinois, USA and the Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA

^* To whom correspondence should be addressed. Email: aspies{at}life.uiuc.edu. sblanke{at}life.uiuc.edu.

	Abstract

Glutamate racemase activity in B. 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 that harbor a single glutamate racemase gene, the genomic sequence of Bacillus 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 E. 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-ordered species. Analysis of purified recombinant RacE1 and RacE2 revealed that both 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 enzymatic activities in 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 both encode functional glutamate racemases with similar, but not identical, active site features.