The biosynthesis of the potent environmental carcinogen aflatoxin B1 is to be investigated. This mycotoxin enters the food supply through contamination of grains by the molds Aspergillus flavus, A. Parasiticus and A. Nomius which produce this compound as a normal metabolite. Chronic ingestion leads to liver tumors that are a major cause of premature death in Asia, Africa and Central America. A direct link has been forged between the interaction of the metabolically-activated form of the toxin and DNA, particularly at a """"""""hot spot"""""""" in the p53 gene leading to mutation of the encoded protein. Alterations in p53 are associated with ca. 50% of human cancers. An understanding of its biosynthesis presents problems of fundamental interest in bioorganic chemistry and may afford mean to control the occurrence of this environmental hazard. Described in this application is a set of interlocking experimental approaches to define in detail the biosynthesis of aflatoxin B1. (1) The syntheses of several potential intermediates are proposed in 13C- labeled form for whole-cell incorporation experiments. (2) These intermediates will be examined for their production in vivo and active conversion in cell-free systems. (3) Specific gene disruption and cotransformation experiments are outlined to help determine the function of individual encoded proteins of the aflatoxin and sterigmatocystin biosynthetic clusters. These will be carried out both at Hopkins and in collaboration with groups working in a.parasiticus and A. Nidulans, the sterigmatocystin producer. (4) As chemical transformations are established for the remarkable molecular rearrangements that characterize this pathway, the responsible genes will be over-expressed for detailed mechanistic study. (5) Aggregation and its effect on catalysis of the component biosynthetic enzymes will be probed by a variety of assays sensitive to protein association. (6) Selective inactivators and substrate analogues will be synthesized to elucidate the details of these enzymic reactions-notably of the polyketide synthase and the fatty acid synthases.
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