Characterization of the alanine racemases from Pseudomonas aeruginosa PAO1

Alanine racemases are ubiquitous, almost uniquely prokaryotic enzymes catal yzing the racemization between L- and D-alanine. The requirement for D-alan ine as a necessary component of the bacterial cell wall makes this class of enzymes a logical target for the development of novel antibiotics. In an e ffort to better understand the structure and mechanism of these enzymes, we have cloned the two independent alanine racemases from Pseudomonas aerugin osa, an important opportunistic bacterial pathogen of humans and animals. T he dadX(PA) and alr(PA) genes have been sequenced, overexpressed, and their activity was demonstrated by complementing D-alanine auxotrophs of Escheri chia coli. Both gene products were purified to electrophoretic homogeneity, the enzymes were characterized biochemically, and preliminary crystals wer e obtained.