Biosynthesis of the Antibiotic Polyketide Enterocin

描述（由申请人提供）：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，是一类重要的药物，与其半合成衍生物一起，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，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它们能够使用各种复杂的方法进行研究，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，该建议的具体目标是：（1）使用最近开发的体外方法以及高分辨率蛋白质质谱法来生物化学地表征肠菌素PKS引发和延伸反应;（2）生物化学地表征黄素蛋白EncM，其催化一系列前所未有的生物合成反应，包括氧化类Repleskii重排、羟醛缩合和杂环形成反应;和（3）从机理和结构上表征苯丙氨酸解氨酶，其是一种罕见的原核酶，首次在肠菌素生物合成途径中发现，现在在临床前试验中用于治疗儿童疾病苯丙酮尿症。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.