Aflatoxin Biosynthesis and Type I Polyketide Synthesis

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DESCRIPTION (provided by applicant): Studies of the potent environmental carcinogen aflatoxin B1 will be continued. This mycotoxin enters the food supply through contamination of grains by the molds Aspergitlus fiavus, 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 means to control the occurrence of this environmental hazard. The investigation has two major goals. The first seeks understanding of fungal Type t polyketide synthases (PKSs)-how iterative function is accomplished, how "programming" is achieved to control polyketide chain length, cyclization geometry and oxidation state. A particularly interesting example of this class lies at the heart of aflatoxin biosynthesis in a complex of the PKS with two specialized yeast-like fatty acid synthase (FAS) subunits in a catalytic complex, NorS. The function of NorS will be examined both in "dissection" experiments using expressed domains of the PKS and FASs, and with the intact recombinant subunits themselves. A newly developed algorithm (UMA) to identify linker regions in multidomainal proteins will guide the experiments to examine function, physical association and structure of domains individually and in groups. The second goal focuses on the three principal oxidative skeletal rearrangement steps that characterize the progression of the biosynthetic pathway from the initially formed anthraquinone to the final substituted coumarin of aflatoxin B1. These are the rearrangement of averufin to l'-hydroxyversicolorone, versicolorin A to demethylsterigmatocystin, and O-methylsterigmatocystin to aflatoxin. Gene disruption experiments have identified as many as 3 proteins mediating a single transformation, but in each case a particular cytochrome P450 has been identified. The mechanisms of these reactions are of interest in themselves, and in the broader context of acetogenin biosynthesis.