NADH-UBIQUINONE REDUCTASE OF PARACOCCUS DENITRIFICANS

脱氮副球菌的 NADH-泛醌还原酶

• 批准号: 3283649
• 负责人: TAKAO YAGI
• 金额: $ 24.29万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283649
• 关键词: NAD(P)H dehydrogenase; active sites; affinity labeling; bacterial genetics; bioenergetics; biological transport; electron transport; enzyme complex; enzyme mechanism; enzyme reconstitution; enzyme structure; gene deletion mutation; genetic manipulation; gram negative bacteria; hydrogen transport; iron sulfur protein; laboratory rabbit; open reading frames; operon; structural genes; ubiquinone

The overall goal of this grant application is to investigate the structure and the function of the subunits of the Paracoccus NADH dehydrogenase complex. During the current granting period (2 years), we have identified the NADH-binding subunit of this enzyme complex utilizing direct photoaffinity labeling in the presence of [32 P]NADH. The labeled polypeptide was isolated by SDS gel electrophoresis and was shown to crossreact with antiserum to the NADH-binding subunit of the bovine NADH-ubiquinone oxidoreductase. In addition, we have cloned the structural gene of the NADH-binding subunit of the Paracoccus NADH dehydrogenase complex. The NADH-binding subunit has 431 amino acid residues and a calculated molecular weight of 47,191. The encoded protein contains a putative NAD(H)-binding and an iron-sulfur cluster-binding consensus sequence. Furthermore, when partial DNA sequencing of the nearby regions was performed, sequences homologous to the 24 kDa, the 49 kDa and the 75 kDa polypeptides of bovine complex I were found, suggesting that the structural genes of the Paracoccus NADH dehydrogenase complex construct at least one operon. Studies planned for this grant period are as follows: (i) Determination of the DNA sequence of the operon for the Paracoccus NADH dehydrogenase complex. When an unidentified open reading frame(s) is present in this operon, the presence of possible polypeptide(s) will be assessed using antiserum to an oligopeptide based on the deduced primary structure of the possible protein. (ii) Identification of the DCCD-binding and the rotenone-binding subunit(s) of the Paracoccus NADH dehydrogenase complex. (iii) Construction and characterization of NADH-binding subunit gene deletion mutants for the Paracoccus NADH dehydrogenase complex using gene replacement techniques.

该赠款申请的总体目标是调查 nadh和daracoccus the的结构和功能 脱氢酶复合物。 在当前授予期（2年）中，我们 已经确定了使用该酶复合物的NADH结合亚基 在[32 p] NADH存在下直接的光性标记。 标签 通过SDS凝胶电泳分离多肽，并显示为 与牛的NADH结合亚基交叉反应 NADH-偶联氧化还原酶。 另外，我们克隆了NADH结合的结构基因 NADH脱氢酶复合物的亚基。 纳德结合 亚基具有431个氨基酸残基和计算的分子量 47,191。 编码的蛋白质包含假定的NAD（H）结合和一个 铁硫簇结合共识序列。 此外，什么时候 进行了附近区域的部分DNA测序，序列 与24 kDa，49 kDa和75 kDa多肽同源 发现了牛络合物I，表明该结构基因 NADH降解脱氢酶复合构建体至少一个操纵子。 计划期间计划的研究如下： （i）测定旁孢子的操纵子的DNA序列 NADH脱氢酶复合物。 当一个身份不明的开放阅读框架 存在于该操纵子中，可能的多肽的存在将 根据推论，使用抗血清对寡肽进行评估 可能蛋白质的主要结构。 （ii）鉴定DCCD结合和烤面包酮结合 NADH脱氢酶复合物的亚基亚基。 （iii）NADH结合亚基基因的结构和表征 使用基因的daracoccus NADH脱氢酶复合物的缺失突变体 替代技术。