Mass Spectrometric Studies of Thiotemplate Biosynthesis

硫模板生物合成的质谱研究

• 批准号: 7783096
• 负责人: NEIL L KELLEHER
• 金额: $ 52.95万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7783096
• 关键词: 4' -phosphopantetheine; Actinobacteria class; Actinomyces; Advocate; Aflatoxins; Amino Acid Sequence; Amino Acids; Anabolism; Antibiotics; Antifungal Agents; Area; Bacillus (bacterium); Bacterial Infections; Biogenesis; Biological Assay; Biological Factors; Chemicals; Classification; Cloning; Complex; DNA Sequence; Detection; Development; Enzymatic Biochemistry; Enzymes; Gene Cluster; Genetic; Genome; Goals; Grant; In Vitro; Ions; Link; Logic; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Microbe; Peptide Antibiotics; Peptide Sequence Determination; Peptides; Pharmaceutical Preparations; Process; Property; Proteins; Proteome; Proteomics; Societies; Streptomyces; System; Technology; Translating; base; cofactor; enzyme reconstitution; fighting; fungus; genome sequencing; in vivo; microbial; microbial genome; novel; novel strategies; overexpression; peptide synthase; polyketide synthase; public health relevance; small molecule; yersiniabactin

DESCRIPTION (provided by applicant): Many natural products with antibiotic, anticancer and antifungal properties are synthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), yet most microbes do not express the great majority of their NRPS or PKS enzymes. In the first granting period of this R01, we developed mass spectrometry (MS) into a facile approach to directly detect covalent intermediates in ~30 systems that have been studied in vitro. We propose to continue this activity for peptide antibiotics and iterative PKS systems (Specific Aim 1). In Specific Aims 2 and 3, we also propose to streamline NRPS/PKS assays using high throughput LC-MS. This blends aspects of enzymology with microbial proteomics of native producers into an entirely new approach to natural product discovery and the study of NRPS and PKS systems. By virtue of their huge size (often >2000 amino acids) and unique marker ions deriving from their common cofactor (phosphopantetheine), we show proof-of-concept for mass spectrometry-based proteomics with highly selective detection of expressed NRPS/PKS gene clusters in native proteomes - without requiring genome sequence information a priori. This method (dubbed "PrISM") will allow unambiguous detection of NRPS/PKS systems in Bacilli, Streptomyces, and even fungi. We therefore advocate a new "proteome-first" strategy to find gene clusters that are actually expressed and efficiently link them to the natural products they produce. The new approaches proposed here are especially timely in an era when new types of mega-enzyme "assembly lines" that defy classification and cloning are being discovered at an accelerating rate, but relatively few new approaches are available to study them. With emphasis on this area of natural products expanding, our activities will assist in the fundamental understanding of chemical processes involved in biogenesis of new natural products of the types that have provided so many of our societies useful drugs to fight both bacterial infection and cancer. PUBLIC HEALTH RELEVANCE: The discovery of new and novel natural products is burdened by the rediscovery of common antibiotics and antiproliferative compounds. Compounds made by NRPS/PKS enzymes have proven to provide diverse modes of action toward myriad different targets. The use of proteomics technology to discover natural products is a novel approach to this difficult problem.

描述（由申请人提供）：许多具有抗生素、抗癌和抗真菌特性的天然产物是由非核糖体肽合成酶（NRPS）和聚酮化合物合成酶（PKS）合成的，但大多数微生物并不表达绝大多数的 NRPS 或 PKS 酶。在该 R01 的第一个授权期间，我们将质谱 (MS) 开发为一种简便的方法，可直接检测已在体外研究的约 30 个系统中的共价中间体。我们建议继续开展肽抗生素和迭代 PKS 系统的这项活动（具体目标 1）。在具体目标 2 和 3 中，我们还建议使用高通量 LC-MS 简化 NRPS/PKS 测定。这将酶学与本地生产者的微生物蛋白质组学相结合，形成一种全新的天然产物发现以及 NRPS 和 PKS 系统研究方法。 凭借其巨大的尺寸（通常> 2000个氨基酸）和源自其共同辅因子（磷酸泛酰乙胺）的独特标记离子，我们展示了基于质谱的蛋白质组学的概念验证，可高度选择性地检测表达的NRPS/PKS基因簇在天然蛋白质组中 - 不需要先验的基因组序列信息。这种方法（称为“PrISM”）将能够明确检测杆菌、链霉菌甚至真菌中的 NRPS/PKS 系统。因此，我们提倡一种新的“蛋白质组优先”策略，以寻找实际表达的基因簇，并将它们有效地连接到它们产生的天然产物。 在这样一个时代，这里提出的新方法尤其及时：无法分类和克隆的新型巨型酶“装配线”正在加速被发现，但可用于研究它们的新方法相对较少。随着对天然产物这一领域的扩展，我们的活动将有助于对涉及新天然产物生物发生的化学过程的基本理解，这些类型为我们社会的许多人提供了对抗细菌感染和癌症的有用药物。 公共卫生相关性：常见抗生素和抗增殖化合物的重新发现给新型天然产物的发现带来了负担。 NRPS/PKS 酶制成的化合物已被证明可以针对无数不同的目标提供多种作用模式。利用蛋白质组学技术发现天然产物是解决这一难题的新方法。