Bacterial resistance to antibiotics is a clinically significant problem that threatens current paradigms of antibacterial chemotherapy. This is particularly true for the treatment of tuberculosis, where singly- and multiply-drug resistant clinical strains of Mycobacterium tuberculosis have been identified with increasing frequency. Very few compounds are selective antimycobacterial agents, and mycobacteria are intrinsically resistant to many antibiotics either because of the constitutive expression of degrading enzymes (e.g., Beta-lactamases) or the inability of antibiotics to penetrate the uniquely hydrophobic outer cell wall. The principal investigator and his group have previously focused their attention on the DAP/L-lysine biosynthetic pathway, and will use similar approaches to explore the three-dimensional structures and chemical mechanisms of M. tuberculosis enzymes involved in the biosynthesis of pantothenate, a vitamin in mammals, and an essential pathway in M. tuberculosis. Specifically, they will clone, express and purify the M. tuberculosis ilvGM, ilvC and ilvD gene products involved in branched chain and pantothenate biosynthesis, and the panB gene product involved in pantothenate biosynthesis. They will clone, express and purify the M. tuberculosis dxs- and dxr-encoded deoxyxylulose-5-phosphate synthase and isomeroreductase for mechanistic comparison to the ilvC- and ilvGM-encoded enzymes. They will also determine the structures and chemical mechanisms of aminoglycoside N-acetyltransferases, enzymes that are primarily responsible for clinical resistance to aminoglycosides in both Gram-negative and Gram-positive bacterial pathogens. These enzymes catalyze a rich variety of chemistries, and plausible chemical mechanisms suggest that mechanism-based, and bi- and tri-substrate analogue inhibitors might be found. They will continue to use an integrated approach involving kinetic and chemical mechanism studies, three-dimensional structural studies, and site-directed mutagenesis to identify active site residues important for substrate and inhibitor recognition, and fulfill their long term goal of characterizing unique bacterial multi-step biosynthetic pathways that are absent in humans.
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