CYTOCHROME C BIOGENESIS--A NEW BIOSYNTHETIC PATHWAY

细胞色素C生物合成--新的生物合成途径

• 批准号: 2910107
• 负责人: Robert G. Kranz
• 金额: $ 21.74万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910107
• 关键词: Rhodospirillales; bacterial genetics; bacterial proteins; biological transport; cytochrome c; hemoprotein biosynthesis; immunoelectron microscopy; immunoprecipitation; laboratory rabbit; monoclonal antibody; oxidation reduction reaction; protein reconstitution; protein structure function; protoplast /spheroplast; site directed mutagenesis; transport proteins

DESCRIPTION: Many microorganisms use c-type cytochromes for electron transport systems that are essential for growth. The assembly of these heme-proteins has recently been shown to require at least eight specific genes in gram-negative bacteria (called helABCDX, ccl1, ccl2, and cycH). Important genomic studies now indicate that although this complex biogenesis pathway is present in many prokaryotes, protozoa and plants, it is absent in yeast and probably vertebrates and invertebrates. This application is for support to study this new pathway in the model bacterium, Rhodobacter capsulatus, in which many of the biosynthetic genes were first discovered. Using genetics, immunological and biochemical approaches, the role of each protein in the biogenesis system will be investigated. All proteins of the pathway are tethered to the surface of the cytoplasmic membrane; it is proposed that reduced heme and reduced cysteine residues of the cytochromes are brought together at the surface by these proteins. Because this assembly in bacteria takes place at the outer surface of the cytoplasmic membrane, where all c-type cytochromes are located, the pathway comprises new and accessible targets for antimicrobial chemotherapeutics. However a more thorough understanding of the functions of individual assembly proteins and the underlying mechanisms involved in biogenesis are required. Reagents leading to the reconstitution of the system will be developed. Specifically, the following studies will be carried out: 1) Analyze proteins HelABCD and Ccl1, involved in heme delivery to the periplasmic surface, including specific amino acid residues and domains which are predicted to interact with heme. 2) Analyze the process whereby the two apocytochrome c cysteine residues are specifically reduced prior to covalent ligation to heme, the hallmark of all c-type cytochromes. The exact roles of Ccl2 and HelX, two periplasmic thioreactive proteins, will be determined, contributing to our understanding of thiol redox pathways in general. 3) Investigate further in vivo requirements for the pathway and develop key reagents required for in vitro reconstitution.

描述：许多微生物将c型细胞色素用于电子。 对增长至关重要的运输系统。这些东西的集合 血红素蛋白最近被证明需要至少八种特定的 革兰氏阴性细菌中的基因(称为helABCDX、ccl1、ccl2和cycH)。 重要的基因组研究现在表明，尽管这种复杂的生物发生 途径存在于许多原核生物、原生动物和植物中，而在 酵母，可能还有脊椎动物和无脊椎动物。此应用程序用于 支持在模式细菌--红细菌中研究这一新途径 在胶囊中，许多生物合成基因首次被发现。 利用遗传学、免疫学和生化方法，每种方法的作用 生物发生系统中的蛋白质将被研究。所有的蛋白质都是 途径被拴在细胞膜的表面；它是 提出了还原血红素和减少半胱氨酸残基的细胞色素 由这些蛋白质在表面聚集在一起。因为这件事 细菌中的组装发生在细胞质的外表面 膜，所有c型细胞色素都位于膜上，该途径包括 新的和可获得的抗微生物化疗药物靶点。然而，一个 对单个组装蛋白的功能有更深入的了解 而涉及生物发生的潜在机制是必需的。试剂 导致该系统的重组将得到发展。 具体而言，将开展以下研究：1)分析 HelABCD和Ccl1蛋白，参与血红素向周质的运输 表面，包括特定的氨基酸残基和结构域，它们是 预计会与亚铁血红素相互作用。2)分析两者之间的过程 细胞色素c半胱氨酸残基在共价之前被特定地还原。 连接到血红素，这是所有c型细胞色素的标志。确切的角色 将测定两种周质硫代反应蛋白CCL2和HelX的含量， 有助于我们从总体上理解硫醇氧化还原途径。3) 进一步研究体内对该途径的需求并开发关键 体外重建所需的试剂。