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: 6180313
• 负责人: Robert G. Kranz
• 金额: $ 22.38万
• 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/6180313
• 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：许多微生物使用 c 型细胞色素进行电子传递 对增长至关重要的运输系统。 这些的组装 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 该途径存在于许多原核生物、原生动物和植物中，但在 酵母，可能还有脊椎动物和无脊椎动物。 此应用程序适用于 支持在模型细菌红细菌中研究这一新途径 capsulatus，其中许多生物合成基因首先被发现。 使用遗传学、免疫学和生化方法，每种方法的作用 将研究生物发生系统中的蛋白质。 所有蛋白质 途径被束缚在细胞质膜的表面；这是 提出减少血红素和减少细胞色素的半胱氨酸残基 这些蛋白质在表面聚集在一起。 因为这个 细菌的组装发生在细胞质的外表面 膜，所有 c 型细胞色素都位于该膜上，该途径包括 抗菌化疗药物的新的、可及的靶标。 然而一个 更全面地了解单个组装蛋白的功能 并且需要涉及生物发生的潜在机制。 试剂 导致系统的重建将被开发。 具体而言，将进行以下研究： 1）分析 蛋白质 HelABCD 和 Ccl1，参与血红素向周质的递送 表面，包括特定的氨基酸残基和结构域 预计与血红素相互作用。 2）分析两者的过程 脱辅基细胞色素 c 半胱氨酸残基在共价之前被特异性还原 与血红素连接，这是所有 c 型细胞色素的标志。 确切的角色 Ccl2 和 HelX（两种周质硫反应蛋白）的含量将被测定， 有助于我们对硫醇氧化还原途径的总体理解。 3） 进一步研究该途径的体内要求并开发关键 体外重建所需的试剂。