Assembly Line Biosynthesis of Bacterial Siderophores

细菌铁载体的装配线生物合成

• 批准号: 6848700
• 负责人: JORGE H CROSA
• 金额: $ 29.5万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6848700
• 关键词: Vibrio; acid aminoacid ligase; bacteria infection mechanism; bacterial genetics; bacterial proteins; biosynthesis; enzyme mechanism; enzyme model; enzyme reconstitution; intermolecular interaction; iron metabolism; microorganism metabolism; model design /development; molecular assembly /self assembly; molecular cloning; molecular site; protein purification; protein structure function; siderophores; site directed mutagenesis; transcription factor

DESCRIPTION (provided by the applicant): The goal of this research is to understand the mechanism of biosynthesis of bacterial peptide siderophores. We use as a paradigm the peptide siderophore anguibactin that is produced by the pathogenic bacterium Vibrio anguillarum. Anguibactin is an important component of the pJM1 plasmid-mediated iron uptake system that is essential for virulence of these pathogenic vibrios. Genetic and physiological analysis led us to the identification and cloning of genes encoded on the pJM1 plasmid that play an essential role in anguibactin biosynthesis. DNA sequence and protein analysis revealed that these genes encode polypeptides that possess domains found in nonribosomal peptide synthetases (NRPSs), originally identified as components of the biosynthetic machinery for the synthesis of antibiotics in gram-positive bacteria. These proteins have been named AngB, AngM, AngN, and AngR and possess modules that could be involved in one or more of the following reactions during the biosynthesis of anguibactin: peptidyl carrier protein (PCP), involved in thioester formation; condensation (C), intervening in peptide bond formation; cyclization (Cy), involved in both condensation and heterocycle formation, and adenylation (A), which is responsible for substrate activation. AngB is an isochorismate lyase that also operates as an aryl carrier (ArCP) protein during siderophore assembly. Other proteins encoded by plasmid-mediated genes, such as AngH and AngU, possess enzymatic activity for the synthesis of histamine from histidine, and for the further oxidation of this compound to hydroxy-histamine, which is a basic building block of anguibactin. Our present efforts are thus directed to elucidate the role of the specific modules of the NRPSs in siderophore biosynthesis. The specific aims to achieve this goal are: 1. Dissection of the mechanisms of assembly line enzymology of anguibactin biosynthesis. We have purified these NRPSs proteins and have obtained antibodies which will be used in the assessment of the role of these polypeptides by using in vitro synthesis reactions including swapping of equivalent NRPS modules intervening in siderophore biosynthesis in V. anguillarum, Vibrio cholerae, and other pathogens. 2. Mutational analysis of the NRPSs genes that will include random mutagenesis and site-directed mutagenesis of the specific modules. We will also purify selected mutant proteins to be used in in vitro synthesis reactions to identify single steps during anguibactin assembly. 3. Identification of chromosomal-encoded proteins intervening in anguibactin biosynthesis. We will use a combination of transposon-directed cloning, genetic, immunological and biochemical approaches to characterize these genes. The combination of the in vivo genetic and the in vitro biochemical approaches will likely lead to the dissection of the mechanisms of siderophore biosynthesis and, in turn, to the exploration of new avenues to understand this contribution to bacterial virulence.

描述(由申请人提供)：本研究的目的是 了解细菌多肽铁载体的生物合成机制。我们 使用含铁载体angibactin多肽作为范例，该多肽由 致病细菌鳗弧菌。Anguibactin是一个重要的成分 PJM1质粒介导的铁摄取系统对毒力是必不可少的 这些致病弧菌中。遗传和生理分析将我们带到了 PJM1载体上编码的基因的鉴定和克隆 在angibactin生物合成中的重要作用。DNA序列和蛋白质分析 发现这些基因编码的多肽具有在 非核糖体多肽合成酶(NRPS)，最初被确定为成分 革兰氏阳性菌合成抗生素的生物合成机制 细菌。这些蛋白被命名为AngB、AngM、AngN和AngR，并拥有 可能参与以下一个或多个反应的模块 肌动蛋白：肽基载体蛋白(PCP)的生物合成 硫酯的形成；缩合(C)，介入多肽键的形成； 环化(Cy)，参与缩合和杂环形成，以及 腺化(A)，负责底物激活。AngB是一个 异分支酸裂解酶，也作为芳基载体(ARCP)蛋白在 铁载体组件。其他由质粒介导的基因编码的蛋白质，如 AngH和Angu具有合成组胺的酶活性 组氨酸，并进一步将该化合物氧化为羟基组胺， 它是angibactin的基本组成部分。我们目前的努力是这样的 旨在阐明NRPS的特定模块在 铁载体生物合成。实现这一目标的具体目标是：1. Angibactin流水线酶学机理剖析 生物合成。我们已经纯化了这些NRPSs蛋白，并获得了 将用于评估这些抗体的作用 多肽的体外合成反应，包括交换 等同的NRPS模块参与铁载体的生物合成。 鳗鱼、霍乱弧菌和其他病原体。2.基因突变分析 将包括随机突变和定点定向的NRPS基因 特定模块的诱变。我们还将提纯精选的突变体 用于体外合成反应以鉴定单步反应的蛋白质 在angibactin组装期间。3.染色体编码蛋白的鉴定 干预肌动蛋白的生物合成。我们将结合使用 转座子定向克隆、遗传、免疫学和生化方法 来确定这些基因的特征。 体内遗传和体外生化方法的结合 可能会导致对铁载体机制的剖析 生物合成，进而有助于探索理解这一点的新途径 对细菌致病力的贡献。