Aflatoxin Biosynthesis and Iterative Type I Polyketide Synthases

描述（由申请人提供）：黄曲霉毒素是霉菌毒素中的一种范例，在环境毒理学中占有中心地位。肝癌是世界上第三大最常见的癌症死亡原因。肝炎感染和饮食中的黄曲霉毒素是导致该病发生的主要危险因素。产黄曲霉的曲霉种，如寄生曲霉、黄曲霉和野曲霉，通常会感染谷物和食品，这些地方有明确的流行病学数据与人类疾病相关。慢性摄入是亚洲、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的相互作用之间已经形成了直接的联系，特别是在p53基因的一个热点上，导致其重要的编码细胞周期调节蛋白发生突变。了解其生物合成将有助于控制这种环境致癌物。黄曲霉毒素是通过异常漫长和复杂的生物合成产生的。关键的分子重排，一些显著的机制，是由细胞色素P450催化的。这些裂解和重排反应的机制将通过化学模拟来研究和模拟，以了解其潜在的化学性质。第二个，也是最主要的目标是利用当前资助期取得的令人兴奋的进展，以了解黄曲霉毒素生物合成和许多其他真菌天然产物的非还原迭代聚酮合酶的功能。在三种主要类型的PKSs中，对迭代I型系统的规划了解最少；也就是说，如何招募起始单元并开始合成，如何确定链长度，如何稳定规范的和内在反应性的聚2-酮中间体，如何在链延伸过程中控制氧化还原状态，以及如何将分子内环化控制为特定的环状结构而不是其他形式。与此叠加的是迭代催化的基本问题，这是一种罕见但令人印象深刻的高效过程，在这种过程中，这些多域酶的活性位点被反复使用，容纳一系列生长的底物，同时忠实地执行合成程序。我们已经在这些结构域中发现了两个以前未被识别的结构域，我们现在认为这类酶是一般的，一个启动单元：酰基载体蛋白转酰基酶（SAT）结构域，这可能解释了启动单元效应的经典观察，以及一个产物模板（PT）结构域，在初步结果中明确在聚酮稳定和产物模板中起作用。本文概述了生化、x射线晶体学和质谱实验，以了解这些结构域各自的功能，并在迭代催化的新图景中共同工作。

DESCRIPTION (provided by applicant): The aflatoxins are a paradigm among mycotoxins and occupy a central place in environmental toxicology. Hepatocarcinomas are the third most common cause of cancer death in the world. Hepatitis infections and dietary aflatoxin are major risk factors contributing to the incidence of this disease. The aflatoxigenic Aspergillus species, A. parasticus, A. flavus and A. nomius, commonly infect grains and food stuffs where clear epidemiological data correlate to human disease. Chronic ingestion is 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 in a hot spot in the p53 gene leading to mutation of its important encoded cell cycle regulating protein. Understanding its biosynthesis will lead to control of this environmental carcinogen. The aflatoxins are created by an unusually long and complex biosynthesis. Key molecular rearrangements, some remarkable mechanistically, are catalyzed by cytochromes P450. The mechanisms of these cleavage and rearrangement reactions will be studied and modeled by chemical mimetics to understand their underlying chemistry. The second, and principal, goal is to capitalize upon exciting progress made in the current grant period to understand the function of non-reducing iterative polyketide synthases central to aflatoxin biosynthesis and many other fungal natural products. Of the three principal types of PKSs, least is known about the programming of iterative Type I systems; that is, how are starter units recruited and synthesis begun, how is chain length determined, how is the canonical and intrinsically reactive poly 2-keto intermediate stabilized, how is redox state controlled during chain elongation, and, finally, how are intramolecular cyclizations controlled to specific ring forms in preference to others. Superimposed on this is the fundamental question of iterative catalysis, a rare but impressively efficient process in which active sites in these polydomainal enzymes are used over and over again, accommodating a sequence of growing substrates yet faithfully executing a synthetic program. We have discovered two previously unrecognized domains in these domains we now believe are general to this class of enzymes, a starter unit:acyl-carrier protein transacylase (SAT) domain, which might account for the classical observation of a starter unit effect, and a product template (PT) domain, which in preliminary results clearly has a role in polyketide stabilization and product templating. Biochemical, X-ray crystallographic and mass spectrometric experiments are outlined to understand the functions of these domains individually and working together in what is already emerging as a new picture of iterative catalysis. PUBLIC HEALTH RELEVANCE The aflatoxins occupy a central place in environmental toxicology. Hepatitis infections and dietary aflatoxin from infected grains and foodstuffs are major risk factors for hepatocarcinoma, the third most common cause of cancer death in the world. Understanding its biosynthesis will lead to its control.