Cloning and Expression of E. Coli Glutamate Racemase for Design of Antimicrobial Therapeutics
Glutamate racemase, the gene product of murI, is an enzyme that interconverts L-glutamate (L-glu) and D-glutamate (D-glu). D-glu is an essential component of the polypeptide chains in the peptidoglycan structure found in bacteria cell walls. The conversion of L-glu to D-glu catalyzed by Glutamate racemase is vital peptidoglycan biosynthesis. Glutamate racemase is found in pathogenic bacteria, such as Escherichia Coli (E. coli), S. Pneumonia and Staphylococci aureus. The peptidoglycan cell wall is one common target of antibiotics. Inhibition of glutamate racemase blocks production of D-glu thereby inhibiting biosynthesis of the peptidoglycan cell wall, leaving bacteria vulnerable. Compounds designed to inhibit S. pnemoniae Glutamate racemase have shown effective antimicrobial activities in whole cell assays. The sequence similarities between glutamate racemase from various pathogenic organisms vary considerably and the structures for each bacterial protein has not been solved. This project will examine the inhibitory ability of compounds selected based on computer modeling of various active sites. In order to allow testing of such compounds, an effective and safe method of obtaining enzyme is required. Currently, we are working to amplify and clone the murI gene from E.Coli. Once cloned the enzyme will be expressed, purified and used to examine potential inhibitors.