Catabolic alanine racemase from Salmonella typhimurium: DNA sequence, enzyme purification, and characterization

Abstract

The alanine racemase encoded by the S. typhimurium dadB gene was purified to 90% homogeneity from an overproducing strain. At 37.degree. C the enzyme has a specific activity of 1400 U/mg (Vmax, L-toD-alanine). Active enzyme molecules are monomers of MW 39,000 with 1 molecule of pyridoxal 5''-phosphate bound per subunit. The Km for L- and D-alanine are 8.2 and 2.1 mM, respectively. Measurement of turnover numbers yielded the expected Keq value of 1.0. Determination of 22 of the 25 N-terminal amino acid residues of the purified polypeptide allowed localization of cloned DNA encoding the structural gene. Sequencing of subcloned DNA revealed that the dadB gene encodes a polypeptide of 356 amino acids whose calculated MW (apoenzyme) was 39,044.