Overexpression and analysis of murA from extremophiles.

Abstract

Antibiotic Resistance is a steadily growing problem in today's society. The problem has become so pronounced that many scientists feel public health may soon enter a post-antimicrobial era. Worldwide, many laboratories are addressing this issue by developing novel inhibitors that target essential proteins in the cell. Since the bacterial cell wall is required for survival, enzymes involved in its synthesis are potential targets for the development of novel inhibitors. The enzyme UDP-N-acetyl glucosamine enolpyruvyltransferase (MurA) is a valid target as it is involved in the first committed step of bacterial cell wall synthesis. In this study, we have cloned and overexpressed murA from the extremophiles Oceanobacillus iheyensis and Thermatoga maritima, and the mesophile Escherichia coli. Since low guanine plus cytosine Gram-positive bacteria also contain an additional enzyme with UDP-N-acetyl glucosamine enolpyruvyltransferase activity tenned MurZ, the gene encoding this protein was also cloned and overexpressed from 0. iheyensis. Using a whole-cell assay, the overexpression of soluble, enzymatically active MurA (and MurZ) was demonstrated.