Cloning and overexpression of the enolpyruvyltransferases from Staphylococcus aureus.

Abstract

Numerous antimicrobial compounds, which once controlled pathogenic organisms, are largely ineffective primarily due to the evolution of antibiotic resistance genes acquired by these pathogenic bacteria. This situation constitutes a serious public health threat that urgently calls for the development of new antibacterial agents to avoid a post-antibiotic era. The search and development of new antimicrobials must be an on-going process in order to maintain a safe and effective drug library. Ideally, the novel agent would target the microbe exclusively by inhibiting a critical cellular process, with little or no toxicity to the host. One possible treatment could be a regime that inhibits enolpyruvyltransferases. These enzymes are involved in enzymatic pathways that are absent from animals and thus, are attractive targets for the development of novel antimicrobials. Currently, MurA (UDP-N-acetylglucosamine enolpyruvyltransferase), encoded by the murA 1 and murA2 genes, and EPSP synthase (5-eno1pyruvy1 shikimate-3phosphate synthase), encoded by the aroA gene, are the only known examples of enolpyruvyltransferases. In this study we have accomplished 2 specific objectives. First, we have cloned aroA, murAL, and murA2 from Staphylococcus aureus. Secondly, we have overexpresed aroA, murAL, and murA2 in Escherichia coli. Accomplishing these two objectives will help to better understand the eno1pyruvyltransferases, both stmcturally and functionally, with the ultimate goal of developing novel antibiotics to combat the almost unabated resurgence of infectious pathogens.