Characterization of the alanine racemases from two Mycobacteria

D-Alanine is a necessary precursor in the biosynthesis of the bacterial pep tidoglycan. The naturally occurring L-alanine isomer is racemized to its D- form through the action of a class of enzymes called alanine racemases. The se enzymes are ubiquitous among prokaryotes, and with very few exceptions a re absent in eukaryotes, making them a logical target for the development o f novel antibiotics. The alanine racemase gene from both Mycobacterium tube rculosis and M. avium was amplified by PCR and cloned in Escherichia coli. Overexpression of the proteins in the E, coil BL21 system, both as native a nd 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 lair, dadX) was con structed and the cloned genes were able to complement its deficiencies. (C) 2001 Federation of European Microbiological Societies. Published by Elsevi er Science B.V. All rights reserved.