Biosynthesis of the Antibiotic Polyketide Enterocin

抗生素聚酮肠毒素的生物合成

基本信息

批准号：
7364670
负责人：
BRADLEY S MOORE
金额：
$ 25.72万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-08-01 至 2012-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7364670
关键词：
3-hydroxybutanal Address Anabolism Antibiotics Antineoplastic Agents Architecture Bacterial Typing Binding Biochemical Biochemical Reaction Biological Factors Chemicals Chemistry Childhood Class Coupled Crystallography Cyanobacterium Cyclization DNA Sequence Analysis DNA Sequence Rearrangement Daunorubicin Disease Engineering Enzymatic Biochemistry Enzymes Family Flavins Flavoproteins Fourier Transform Gene Proteins Generations Genes Growth Health Histidine Human In Vitro Knowledge Label Libraries Mass Spectrum Analysis Metabolic Modeling Modification Mutagenesis Nature Nostoc Numbers Outcomes Research Pathway interactions Pharmacologic Substance Phenylalanine Ammonia-Lyase Physical condensation Process Proteins Reaction Recombinants Research Personnel Resolution Role Series Site-Directed Mutagenesis Specificity Staging Structure Substrate Specificity Testing Tetracyclines Therapeutic Agents Time Today Translating base chemical synthesis enterocin in vivo member novel polyketide synthase preclinical study programs therapeutic enzyme thioester three dimensional structure

项目摘要

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.

描述（由申请人提供）：天然芳族聚酮化合物，例如抗生素四环素和抗癌剂Daunorubicin代表了一类重要类别的药物，它们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。对在生化和结构水平上通过II型聚酮化合物合酶（PKS）催化的芳族聚酮化组装的基本理解无疑会增加我们对这些重要的生物合成过程的欣赏，并将有助于新化学实体的合理工程。尽管过去十年来，我们关于芳族聚酮化合物如何自然合成的基本知识已经实现了实质性的增长，但如今仍然存在许多基本差距。因此，我们在这种竞争性更新应用中提出，以进一步对Polyketide抗生素肠球菌蛋白进行生物合成研究，该研究已成为解决芳族聚酮化合物组装的早期阶段的重要工具，涉及启动单位选择的芳香族聚酯组装，酮分泌反应的时机，环化反应，环化电位以及后PKS的反应。它们的简单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，包括异源生物合成，体外和体内生物化学分析，酶工程和原子分辨率分辨率蛋白质X射线晶体学的定向和随机方法。因此，该提案的具体目的是：（1）使用最近开发的体外过程以及高分辨率蛋白质质谱法，以生化为特征肠球素PKS启动和延伸反应；（2）生化表征了黄蛋蛋白ENCM，该ENCM催化了涉及涉及氧化favorskii的重排，醛醇的凝结和杂环形成反应的一系列前所未有的生物合成反应；（3）在机械和结构上表征苯丙氨酸氨 - 裂解酶，这是一种罕见的原核酶，最初是在肠肠蛋白生物合成途径中发现的，现在是在临床前试验中治疗儿童疾病苯酮苯尿尿症的。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的新型生物催化剂以及在PAL的治疗酶中提供机会。