The importance of unusual ring structures in a diverse array of therapeutic agents suggests that the ability to engineer compounds containing these moieties could lead to new, medically useful drugs. The ribosomally synthesized antibacterial agent, Microcin B17, is generated following post-translational modifications that produce both oxazole and thiazole rings in the polypeptide precursor. The enzyme responsible for this activity, Microcin B17 synthetase, provides an ideal system to study heterocycle formation because all of the genes for the multi-protein complex have been cloned and biochemical assays to monitor substrate modification have been established. This proposal describes studies that explore the mechanism of action of one enzymatic component, McbB, which may employ zinc as a cofactor. First, the stoichiometry of zinc ions bound by McbB and the metal requirements for catalytic activity will be established. The second set of experiments, which include several types of spectroscopy and site-directed mutagenesis, will examine the metal coordination site(s) and the influence of the metal ligands on substrate modification. Finally, McbB mutants, as well as modified substrates, will be used to elucidate the details of the enzymatic mechanism of oxazole and thiazole synthesis.
