Biosynthesis of the Antibiotic Polyketide Enterocin

描述（由申请人提供）：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，连同它们的半合成衍生物，对人类健康起着至关重要的作用。对II型聚酮合成酶（pks）在生物化学和结构水平上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，我们建议在这一竞争性更新申请中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x射线晶体学。因此，本提案的具体目标是：(1)利用最近开发的体外工艺以及高分辨率蛋白质质谱法对肠球菌蛋白PKS启动和延伸反应进行生化表征；(2)对黄蛋白EncM进行生物化学表征，该蛋白催化了一系列前所未有的生物合成反应，包括氧化类favorskii重排、醛醇缩合和杂环形成反应；(3)对苯丙氨酸解氨酶进行机理和结构表征。苯丙氨酸解氨酶是一种罕见的原核酶，首次在肠球蛋白生物合成途径中被发现，目前用于治疗儿童苯丙酮尿症的临床前试验。该研究计划的结果将阐明天然产物生物合成中的新生化反应，并将为开发生物有机化学中的新型生物催化剂以及PAL治疗酶提供机会。

DESCRIPTION (provided by applicant): 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 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, 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 of this proposal are thus: (1) to biochemically characterize the enterocin PKS priming and extension reactions using a recently developed in vitro process together with high-resolution protein mass spectrometry; (2) to biochemically characterize the flavoprotein EncM, which catalyzes an unprecedented series of biosynthetic reactions involving oxidative favorskii-like rearrangement, aldol condensation and heterocycle-forming reactions; and (3) to mechanistically and structurally characterize phenylalanine ammonia-lyase, a rare prokaryotic enzyme first discovered in the enterocin biosynthetic pathway and now in pre-clinical trials to treat the childhood disorder phenylketonuria. The outcome of this research plan will illuminate new biochemical reactions in natural product biosynthesis and will provide the opportunity to develop novel biocatalysts in bioorganic chemistry as well as therapeutic enzymes in the case of PAL.