Type III Polyketide Synthases: Structure and Mechanism

天然芳香聚酮类药物，如抗生素四环素和抗癌剂柔红霉素，是一类重要的药物，与它们的半合成衍生物一起，对人类健康起着至关重要的作用。在生物化学和结构水平上对II型聚酮合成酶催化的芳香族聚酮组装的基本了解无疑将增加我们对这些重要生物合成过程的认识，并有助于合理地设计新的化学实体。虽然在过去的十年中，我们对芳香聚酮自然合成的基本知识有了实质性的增长，但今天仍然存在一些基本的差距。因此，在这一竞争性更新申请中，我们建议进一步开展聚酮类抗生素肠霉素的生物合成研究，肠霉素已成为解决芳香聚酮组装早期阶段的重要载体，包括启动单元选择、酮还原反应的时机、环化潜力以及pks后修饰反应。它们简单的基因和蛋白质结构使得它们可以使用各种复杂的方法进行研究，包括异种生物合成，体外和体内生化分析，酶工程的定向和随机方法以及原子分辨率蛋白质

Natural aromatic polyketides such as the antibiotic tetracycline and the anticancer agent daunorubicin represent an important class of pharmaceuticals that, together with their semi-synthetic derivatives, command a vital role in human health. A basic understanding of aromatic polyketide assembly catalyzed by iterative type II polyketide synthases (PKSs) at the biochemical and structural levels will undoubtedly increase our appreciation for these important biosynthetic processes and will aid in the rational engineering of new chemical entities. While the past decade has witnessed substantial growth in our basic knowledge on how aromatic polyketides are naturally synthesized, a number of fundamental gaps still persist today. We thus propose in this competitive renewal application to further our biosynthetic studies on the polyketide antibiotic enterocin, which has emerged as an important vehicle to address the early stages in aromatic polyketide assembly involving starter unit selection, timing of the ketoreduction reaction, and cyclization potential as well as post-PKS modification reactions. Their simple gene and protein architecture makes them amendable for study using a variety of sophisticated approaches including heterologous biosynthesis, in vitro and in vivo biochemical analysis, directed and random approaches towards enzyme engineering, and atomic resolution protein x-ray crystallography. The specific aims for this proposal are thus: (1) to biochemically analyze the proposed acyl carrier protein-independent enterocin PKS priming mechanism with the goal of engineering aromatic polyketide libraries; (2) to biochemically characterize the flavoprotein EncM, which catalyzes an unprecedented series of biosynthetic reactions involving oxidative Favorskii rearrangement, aldol condensation and heterocycle-forming reactions; and (3) to structurally characterize several enterocin biosynthetic enzymes in association with our structural biology collaborators. The outcomes of this research plan will illuminate new biochemical reactions in natural product biosynthesis and will provide new enzymes for the combinatorial biosynthesis of novel chemistry with the potential of leading to new drugs.