DESULFOVIBRIO HYDROGENASE--MOLECULAR BIOLOGY

脱硫弧菌加氢酶--分子生物学

• 批准号: 3286736
• 负责人: HARRY D PECK
• 金额: $ 16.7万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-05 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3286736
• 关键词: Desulfovibrio; Escherichia coli; anaerobic bacteria; bacterial genetics; cytoplasm; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme inhibitors; enzyme mechanism; gene expression; genetic manipulation; genetic mapping; genetic strain; hydrogen transport; hydrogenase; iron sulfur protein; membrane permeability; microorganism metabolism; molecular cloning; mutant; nucleic acid probes; oxidation reduction reaction; protein biosynthesis; protein sequence; radiotracer; sulfur metabolizing bacteria

The specific aims of the proposed research are: 1) to finalize the structure of the functionalized non-heme iron center (H cluster) that is uniquely involved in both substrate (H2) activation and electron transfer; 2) to analyze the function of the H cluster and two ferredoxin- type clusters by specific probes utilizing biophysical techniques; 3) to attempt to isolate the gene product of the putative hydrogenase gene, hyd lambda from Desulfovibrio vulgaris which is suspected to be a new-type of (Fe) hydrogenase in Desulfovibrio and may resolve the problem of the number and types of (Fe) hydrogenases; 4) to establish a DNA transfer system with D. vulgaris using a wide host range plasmid and generate (Fe) hydrogenase negative mutants in order to obtain biosynthesis of active (Fe) hydrogenase and 5) to investigate the role of the small subunit and its putative signal peptide in the periplasmic localization of the (Fe) hydrogenase with the goal of determining how the large subunit that lacks a signal peptide is translocated across the cytoplasmic membrane.

建议研究的具体目标是：1）完成 功能化的非血红素铁中心（H簇）的结构， 独特地参与底物（H2）活化和电子 2）分析H簇和两个铁氧还蛋白的功能， 利用生物物理技术通过特异性探针对簇进行分型; 3） 尝试分离推定的氢化酶基因hyd的基因产物 从疑似为一种新型的 (Fe)氢化酶，并可能解决 （Fe）氢化酶的数量和类型; 4）建立DNA转移 系统，D. vulgaris使用广泛宿主范围的质粒并产生（Fe） 氢化酶阴性突变体，以获得生物合成活性 (Fe)氢化酶和5）研究小亚基的作用， 其假定的信号肽在周质定位的（铁） 氢化酶的目标是确定缺乏的大亚基 信号肽跨细胞质膜易位。