Characterization of the alanine racemases from two mycobacteria

D-Alanine is a necessary precursor in the biosynthesis of the bacterial peptidoglycan. The naturally occurring L-alanine isomer is racemized to its D-form through the action of a class of enzymes called alanine racemases. These enzymes are ubiquitous among prokaryotes, and with very few exceptions are absent in eukaryotes, making them a logical target for the development of novel antibiotics. The alanine racemase gene from both Mycobacterium tuberculosis and M. avium was amplified by PCR and cloned in Escherichia coli. Overexpression of the proteins in the E. coli BL21 system, both as native and as His-tagged recombinant products, has been achieved. The proteins have been purified to electrophoretic homogeneity and analyzed biochemically. A D-alanine requiring double knock-out mutant of E. coli (alr, dadX) was constructed and the cloned genes were able to complement its deficiencies.