Aflatoxin Biosynthesis and Type I Polyketide Synthesis

描述（由申请人提供）：将继续对强效环境致癌物黄曲霉毒素B1的研究。这种真菌毒素通过真菌曲霉、寄生曲霉和诺米乌斯曲霉污染谷物进入食物供应，这些霉菌产生这种化合物作为正常的代谢物。慢性摄入导致肝脏肿瘤，这是亚洲、非洲和中美洲过早死亡的一个主要原因。这种毒素的代谢激活形式与DNA之间的相互作用已经形成了直接的联系，特别是在p53基因的一个“热点”上，导致编码蛋白质的突变。大约50%的人类癌症与p53基因的改变有关。对其生物合成的理解提出了生物有机化学的基本问题，并可能提供控制这种环境危害发生的方法。调查有两个主要目标。第一个是寻求对真菌t型聚酮合成酶（pks）的理解——迭代功能是如何完成的，如何实现“编程”来控制聚酮链长度、环化几何形状和氧化态。这类中一个特别有趣的例子是黄曲霉毒素生物合成的核心，它是PKS与催化络合物NorS中两个专门的酵母样脂肪酸合成酶（FAS）亚基的复合体。NorS的功能将通过PKS和FASs表达域的“解剖”实验以及完整的重组亚基本身来检验。一种新开发的识别多结构域蛋白连接区域的算法（UMA）将指导实验研究单个和群体结构域的功能、物理关联和结构。

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.