The research outlined in this proposal is aimed at elucidating the mode of biosynthesis of a number of antibiotics. Compounds and problems to be studied include the enzymology of formation of pyrrolnitrin and dihydrophenylalanine, the formation of cyclohexane rings in ansatrienin and asukamycin, the formation of mC7N units in ansatrienin, naphthomycin and/or rifamycin on the one hand, and in asukamycin and manumycin on the other, the mode of polyketide assembly in lysolipin and in the macrodiolide aplasmomycin, as well as the biosynthesis of reductiomycin, the novel phenazine antibiotics, the esmeraldins and saphenamycins, and the modified peptide antibiotics nosiheptide and thiostrepton. The results are expected to answer fundamental questions in antibiotic biosynthesis and lay the ground work for biological structure modification of complex antibiotics based on biosynthetic information. The methods to be used include (a) feeding experiments with precursors labeled with radioactive (14C, 3H) and/or stable (13C, 15N, 18O, 2H) isotopes and extensive use of Fourier- transform NMR spectroscopy for product analysis, (b) synthesis of potential pathway intermediates and their evaluation in whole cell or cell-free experiments, (c) isolation of enzymes carrying out some of these transformations and (d) synthesis and use of stereospecifically labeled substrates to probe the stereochemistry and mechanism of some of the biosynthetic reactions.
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