Bacterial resistance to antibacterial compounds is an important and emerging threat to current paradigms of antibiotic therapy. This is particularly true of treatment regimens for tuberculosis, where both singly-and multiply-drug resistant Mycobacterium tuberculosis clinical isolates have been identified with increasing frequency. The mycobacterial biosynthetic pathway for diaminopimelate/lysine is explored with the hope of providing new therapeutically inhibitable target enzymes. It has been shown that this pathway is essential for mycobacterial growth. An enzymatic and structural characterization of the eight enzymes in the pathway has been initiated. The current application proposes to continue those studies, and to sequentially characterize all of the remaining enzymes in the pathway. Specifically, the applicant's laboratory will clone, sequence, express, purify and mechanistically and structurally characterize the dapC-encoded transaminase, the dapE-encoded desuccinylase, the dapF-encoded epimerase and the lysA-encoded decarboxylase. These will complement ongoing studies with the dapA-encoded synthase, dapB-encoded reductase, dapD-encoded succinylase and ddh-encoded dehydrogenase. Plausible chemical mechanisms for many of these enzymes suggest that a mechanism-based approach would provide inhibitors with high selectivity. It is anticipated that the combination of molecular genetic, kinetic, mechanistic and three-dimensional structural approaches described will provide relevant information to a variety of potential inhibitor design alternatives, including structure-based and high throughput screening approaches. The long term goals of the research program are to obtain detailed mechanistic and structural information about an important multi-step bacterial biosynthetic pathway which is absent in mammals.
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