Mass Spectrometric Studies of Thiotemplate Biosynthesis

硫模板生物合成的质谱研究

• 批准号: 8494636
• 负责人: NEIL L KELLEHER
• 金额: $ 28.25万
• 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 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8494636
• 关键词: 4' -phosphopantetheine; Actinobacteria class; 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.

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）和聚酮化合物合酶（PKSS）合成的，但是大多数微生物并未表达其大多数NRPS或PKS enzymes。在此R01的第一个授予期间，我们将质谱（MS）开发为一种轻松的方法，以直接检测约30个已在体外研究的系统中的共价中间体。我们建议继续为肽抗生素和迭代PKS系统（特定目标1）继续这种活动。在特定目标2和3中，我们还建议使用高吞吐量LC-MS简化NRP/PKS分析。这将酶学的各个方面与本地生产者的微生物蛋白质组学融为一体，成为一种全新的自然产品发现方法和NRPS和PKS系统的研究。 由于它们的尺寸巨大（通常> 2000氨基酸）和独特的标志物离子，这些离子是从其共同辅因子（磷酸耐药）衍生而来的，因此我们显示了基于质谱的蛋白质组学的概念证明，并高度选择性地检测出表达的NRPS/PPKS基因簇的天然蛋白质组中的本机蛋白质组织中，而无需基因组序列，而不是需要基因组序列。这种方法（称为“棱镜”）将允许在杆菌，链霉菌甚至真菌中明确检测NRPS/PKS系统。因此，我们主张一种新的“蛋白质第一”策略，以找到实际表达并有效地将其与其生产的天然产品联系起来的基因簇。 在一个新型类型的大型酶“组装线”中，这种新型方法尤其及时，以加速速度发现了反抗分类和克隆的时代，但相对较少的新方法可用于研究它们。在强调自然产品扩展的这一领域，我们的活动将有助于对涉及的化学过程的基本理解，这些化学过程与我们提供了许多社会的新天然产物的生物发生有用，以抗菌细菌感染和癌症。