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 and A. parasiticus 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. An understanding of its biosynthesis presents problems of fundamental interest in bioorganic chemistry and may afford means to control the occurrence of this environmental hazard. Described in the renewal application is a set of interlocking experimental approaches to define in detail the biosynthesis of aflatoxin B1. (1) Cell- free experiments are proposed to investigate the post-bisfuran biosynthetic steps in which the anthraquinone nucleus of versicolorin A is cleaved, deoxygenated, decarboxylated and rearranged to the xanthone of sterigmatocystin. Similar experiments will be undertaken to examine the further conversion of sterigmatocystin to aflatoxin. Intermediates observed will be compared to materials prepared by chemical synthesis. (2) The stereochemistry of oxidative desaturation of the tetra- to dihydrobisfuran will be determined. (3) Mechanism-based inactivators of three pre-bisfuran biosynthetic enzymes will be synthesized and tested for their irreversible inhibition of these enzymes and their ability to function in vivo to inhibit or block aflatoxin production. (4) Purification of versicolorin B synthase (VBS) will be completed and fully documented, and its mechanism of action studied. (5) A recently detected polyketide synthase (NAS) producing norsolorinc acid will be further purified and its catalytic cycle examined, particularly with regard to apparent initiation by hexanoate/hexanoylCoA. (7) The genes coding for VBS and NAS will be cloned and sequenced, and comparisons will be made to other known protein sequences as a prelude to a longer term objective of mapping the aflatoxin pathway structural genes.
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