MUTASYNTHETIC & BIO-ORGANIC STUDIES OF ANTIBIOTICS

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We propose to continue our work on the biosynthesis of quinone-containing antibiotics, and will further develop our studies on sarubicin A, kinamycin D, streptonigrin, and naphthyridinomycin, as well as initiate work on antibiotic G7063-2 which we believe may be biogenetically related to sarubicin A. With all four of our ongoing projects we will synthesize and test a variety of structures that we believe to be key intermediates in each biosynthetic pathway. These as well as the study of G7063-2, will primarily use the stable isotopes 13C, 2H, 15N, and 18-0, and they will be detected by high field NMR spectroscopy, often using spin-coupling or isotope-induced shift techniques. Our work has already revealed the presence of new aromatic amino acids in three of these pathways, and we will prepare cell-free extracts of each relevant microorganism in order to study each of the responsible enzymes and the mechanisms of the reactions that they catalyze. These include the enzymes that convert shikimic acid to 6-hydroxyanthranilic acid in sarubicin A biosynthesis, that convert shikimic acid to 4-aminoanthranilic acid and/or 4-amino-3-hydroxyanthranilic acid in streptonigrin biosynthesis, and that convert acetate to 3-amino-2-hydroxy-6-methylbenzoic acid in kinamycin D biosynthesis. We will also mutagenize the microorganisms that produce naphthyridinomycin, streptonigrin, sarubicin A, and kinamycin D, in order to develop mutants blocked in each of these pathways. We anticipate that these mutants will accumulate new metabolites that are intermediates in the antibiotic biosynthesis, and each of these compounds will be isolated and characterized to provide information about the pathway that would otherwise be unobtainable. In the more distant future, these mutants will provide vehicles for studying the regulation of these pathways and understanding the way they fit into the development of the producing organism.