Penicillin and related beta-lactam antibiotics have been a mainstay in the treatment of infections for 50 years. However, their effectiveness, like other known classes of antibiotics, has come under increasing challenge from the rise of multiply drug-resistant pathogenic bacteria. Efforts have intensified to understand mechanisms of resistance and to overcome them. Structural modification through genetic manipulation of their biosynthetic pathways is a promising approach to produce variants of known antibiotics by cost-effective fermentation and semi-synthetic methods. Continuation of a program to investigate beta-lactam antibiotic biosynthesis is proposed in this application. Three of the four known classes of these antibiotics will be studied: (1) clavulanic acid, a potent inhibitor/inactivator of beta-lactamase enzymes and a wide-spread source of resistance, (2) the nocardicins, a family of monocyclic beta-lactams, and the metabolically related monobactams, and (3) the carbapenems, represented clinically by thienamycin and its derivatives, but most simply by carbapen-2-em-3-carboxylic acid. With the recent isolation of biosynthetic gene clusters for at least one member of each of these four principal groups, work is poised to advance rapidly in an integrated research plan using techniques ranging from organic synthesis and enzymology to molecular biology and macromolecular structural methods. Clavaminate synthase will be examined in mutagenesis experiments to identify ligands to the catalytic iron, substrate analogues will be prepared to elucidate the controlling factors in hydroxylation vs. oxidative cyclization chemistry mediated by this protein and as probes in spectroscopic studies of the iron binding site. The newly discovered beta-lactam synthetase active in this pathway will be examined in detail kinetically and for its substrate tolerance, and the poorly understood transformations at the end and beginning of the pathway will be investigated in both gene disruption, and over- expression experiments. The recently discovered nocardicin gene cluster will be studied with emphasis on determining how the presumed precursor peptide is assembled and the beta-lactam formed. Possible evolutionary cross-over between clavulanic acid and the carbpenems will be examined particularly with respect to an apparent similarity of beta-lactam synthesis. These insights will be extended to examination of beta- lactam formation in the prephytotoxin tabtoxin.
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