Gene cloning and characterization of alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei

Alanine racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Sh igella flexneri, and Shigella sonnei were cloned and expressed in Escherich ia coli JM109. All genes encoded a polypeptide of 359 amino acids, and show ed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two resid ues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile 225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homog eneity has an apparent molecular mass about 43,000 by SDS-gel electrophores is, and about 46,000 by gel filtration. However, all enzymes showed an appa rent molecular mass about 60,000 by gel filtration in the presence of a sub strate, 0.1 M L-alanine. These results suggest that the Shigella alr enzyme s having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymo logical. properties, and showed lower catalytic activities (about 210 units /mg) than those of homodimeric alanine racemases reported. (C) 2001 Academi c Press.