Article Text

  1. D. Dodd,
  2. J. G. Reese,
  3. C. R. Louer,
  4. J. D. Ballard,
  5. M. A. Spies,
  6. S. R. Blanke
  1. University of Illinois, Urbana-Champaign, Urbana, IL; Oklahoma City, OK.


Bacillus anthracis synthesizes two complex structures, a peptidoglycan cell wall and poly-γ-D-glutamic acid (PDGA) capsule, which require an accessible pool of D-glutamate. The mechanisms, however, underlying the establishment of accessible pools of D-glutamate for B. anthracis are poorly understood. B. anthracis harbors two genes, racE1 and racE2, which are each predicted to encode a glutamate racemase capable of converting L-glutamate to D-glutamate. However, the respective roles, if any, of RacE1 or RacE2 in catalyzing the racemization of D-glutamate to L-glutamate have not been investigated. The objective of this study was to compare the in vitro properties of the racE1 and racE2 gene products, with the explicit purpose of establishing whether either or both of these proteins have the capacity to catalyze the conversion of L-glutamate to D-glutamate. racE1 or racE2 were cloned from B. anthracis Sterne 7702 and expressed as recombinant proteins in Escherichia coli. Each protein was purified to homogeneity. We developed an assay based on circular dichroism for directly monitoring glutamate racemase activity. Both RacE1 and RacE2 exhibited detectable glutamate racemase activity. Characterization of both enzymes revealed similar pH profiles and a lack of dependency for various metal cofactors. However, the catalytic efficiency (kcat /KM) of RacE2 was twice that of RacE1. In addition, RacE2 exists in solution as a dimer, whereas RacE1 exists primarily as a monomer. RacE1 forms a higher-ordered complex in the presence of L-glutamate, whereas the quaternary structure of RacE2 is largely independent of substrate. Collectively, these data indicate that although both racE1 and racE2 encode proteins that catalyze the racemization of L-glutamate to D-glutamate in vitro, differences in the properties of these two enzymes suggest that these two enzymes may have distinct cellular roles.

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