Type III Polyketide Synthases: Structure and Mechanism

天然芳香族聚酮化合物，如抗生素四环素和抗癌剂柔红霉素，代表了一类重要的药物，与它们的半合成衍生物一起，在人类健康中起着至关重要的作用。一个基本的了解芳香族聚酮组装催化迭代II型聚酮酶（PKS）在生物化学和结构水平无疑将增加我们对这些重要的生物合成过程的赞赏，并将有助于合理的工程新的化学实体。虽然在过去的十年里，我们对芳香族聚酮化合物是如何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根本性的差距。因此，我们建议在这个竞争性的更新申请，以进一步我们的生物合成研究的聚酮抗生素肠菌素，这已成为一个重要的车辆，以解决在芳香族聚酮组装的早期阶段，涉及起始单元的选择，酮还原反应的时间，和环化潜力，以及后PKS修饰反应。它们简单的基因和蛋白质结构使其可用于各种复杂方法的研究，包括异源生物合成，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 X射线晶体学因此，该提议的具体目标是：（1）生化分析所提出的酰基载体蛋白非依赖性肠菌素PKS引发机制，目标是 工程化芳香族聚酮化合物文库;（2）对黄素蛋白EncM进行生物化学表征， 它催化了一系列前所未有的生物合成反应， 重排、羟醛缩合和杂环形成反应;和（3）结构上 表征与我们的结构生物学相关的几种肠菌素生物合成酶 合作者该研究计划的成果将阐明天然产物生物合成中的新生化反应，并将为新化学的组合生物合成提供新的酶，具有开发新药的潜力。

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.