We propose to continue our studies of antibiotic biosynthesis and will focus on the secondary metabolism of amino acids and of glucose. We will be concerned with the generation of w-N-hydroxy- , 3-hydroxy-, 2,3-dehydro-, and B-amino acids and with the formation and further transformation of nucleosides involved in antibiotic biosynthesis. Key enzymes will be isolated and characterized. The genome for acivicin biosynthesis will be cloned. Experiments are planned to further clarify the biosynthesis of streptothricin F and to determine the biogenetic relationship of capreomycidine (capreomycin biosynthesis) and 3-epicapromycidine (elastatinal biosynthesis). Potential nucleoside intermediates in streptothricin F biosynthesis and in blasticidin S biosynthesis will be synthesized and tested. Arginine-2,3-aminomutase will be isolated from Streptomyces qriseochromogenes (blasticidin S biosynthesis) and its reaction mechanism studied. L-Ornithine N- monooxygenase and acivicin 4-monooxygenase will be isolated from S. sviceus, and the intermediates in acivicin biosynthesis beyond N-hydroxyornithine determined. Studies on the conversion of p- aminophenylalanine to p-aminophenylserine in chloramphenicol biosynthesis (S. 3022a) will be completed. The biosynthesis of cis and trans-L-3-hydroxyproline, of erythro-L-3-hydroxyleucine, of 2,3-dehydrotryptophan, and of threo-L-3-methyltryptophan residues of telomycin will be studied in S. canus. The biosynthesis of threo-D-3-hydroxyaspartic acid and of the N-hydroxy-D-ornithine lactam of the siderophore pseudobactin will be elucidated, as will that of the unusual chromophore of this pseudomonas metabolite. Most of the labeled compounds needed for these studies will be synthesized in our laboratories. We will primarily use stable isotopes with analysis by high field NMR spectroscopic techniques. Our 15N/13C NMR spin coupling technique will continue to be essential for mapping out complex biosynthetic pathways. It is hoped that our investigations will continue to reveal previously obscured biogenetic relationships amongst seemingly diverse structures, and will expand our understanding of organic reactions and reactivity. Our results should lay the groundwork for cloning the genes responsible for production of these antibiotics, and may lead to new, hybrid antibiotics with improved characteristics.
