The specific aims of this program focus on benz[a]anthraquinone- derived antibiotics. Work will be continued on the kinamycin antibiotics produced by Streptomyces murayamaensis, and on antibiotic PD 116740, produced by S. WP 4669. A limited set of experiments will be carried out in S. rimosus, in order to identify one of the key branch-points in the general matrix of benz[a]anthraquinone biosynthesis by examining the precursor/product relationship of tetrangomycin and tetrangulol. The Research Design includes structural studies to identify more biosynthetic intermediates in each pathway. This will be done by a combination of characterizing additional metabolites, and of synthesizing and testing reasonable candidates. In the kinamycin pathway, metabolites that are potentially intermediates will be identified by feeding known intermediates to a current set of 37 blocked mutants. The Research Design will include biochemical studies to detect and characterize key enzymes in these pathways (e.g. for amino- and amidotransferases, aryl epoxidases and arylepoxide hydrolases, hydroquinone epoxidases, and aromatic ringcleaving enzymes), and molecular genetics studies to clone, sequence, and characterize selected genes. The broad objective of this research program is to understand which chemical reactions are available in Nature and how they are linked together to produce complex structures with significant bio- medically relevant activity, in particular, antibiosis. This objective will be attained through the described multi-faceted approach that blends carefully chosen experiments in synthesis, biosynthesis, enzymology and molecular genetics. Such an interdisciplinary approach has become more and more essential for the modern bioorganic chemist who desires to study metabolism, its regulation and its consequences. It will lay the groundwork for applications such as modifying bioactivity by metabolic and genetic engineering to generate new molecular structures. This interdisciplinary approach is also an effective vehicle to train new chemists with an understanding of biology and a broad range of skills for solving important biochemical problems.
