Characterization of yrpC gene product of Bacillus subtilis IFO 3336 as glutamate racemase isozyme

Glr, the glutamate racemase of Bacillus subtilis (formerly Bacillus natto) IFO 3336 encoded by the glr gene, and YrpC, a protein encoded by the yrpC g ene, which is located at a different locus from that of the glr gene in the B. subtilis genome, share a high sequence similarity. The yrpC gene comple mented the D-glutamate auxotrophy of Escherichia coli WM335 cells defective in the glutamate racemase gene. Glutamate racemase activity was found in t he extracts of E. coli WM335 clone cells harboring a plasmid, pYRPC1, carry ing its gene. Thus, the yrpC gene encodes an isozyme of glutamate racemase of B. subtilis IFO 3336. YrpC is mostly found in an inactive inclusion body in E. coil JM109/pYRPC1 cells. YrpC was solubilized readily, but glutamate racemase activity was only slightly restored. We purified YrpC from the ex tracts of E, coil JM109/pYRPC2 cells using a Glutathione S-transferase Gene Fusion System to characterize it. YrpC is a monomeric protein and contains no cofactors, like Glr. Enzymological properties of YrpC, such as the subs trate specificity and optimum pH, are also similar to those of Glr. The the rmostability of YrpC, however, is considerably lower than that of Glr. In a ddition, YrpC showed higher affinity and lower catalytic efficiency for L-g lutamate than Glr. This is the first example showing the occurrence and pro perties of a glutamate racemase isozyme.