权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Structure and function of two novel respiratory chain complex from Gram-positive thermophilic bacteria

革兰氏阳性嗜热菌两种新型呼吸链复合物的结构和功能

基本信息

批准号：
10680617
负责人：
SAKAMOTO Junshi
金额：
$ 1.92万
依托单位：
Kyushu Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680617/
关键词：
thermophile Gram-positive bacteria respiratory chain cytochrome oxidase bioenergetics

项目摘要

The genes encoding cytochrome bd-type quinol oxidase were completely cloned from the Gram-positive thermophilic bacteria Bacillus stearothermophilus. Comparison and alignment with sequences reported for other cytochromes bd show that these oxidases can be divided into two subfamilies and that the B. stearothermophilus enzyme is the first member of the second group which has been isolated and characterized. Hydropathic analysis indicates that the previous structural model for cytochrome bd mainly based on the analysis of the E. coli enzyme needs to be revised.The genes for cytochrome boィイD23ィエD2-type cytochrome c oxidase were ligated to the shuttle vector pSTE12 and introduced in the wild B. stearothermophilus cells to be over-expressed. The produced oxidase was purified, reconstituted into liposomes, and showed HィイD1+ィエD1 pumping activity, although the HィイD1+ィエD1/O ratio was lower than that of cytochrome caaィイD23ィエD2-type oxidase. A strain of mutant cells in which cytochrome boィイD23ィエD2-type oxidase mainly operates was isolated. The HィイD1+ィエD1 pumping activity and steady-state growth extent of the cells were higher than those of the mutant K17, in which cytochromes bd operates as the main terminal oxidase, and lower than those of the wild cells, in which cytochrome caaィイD23ィエD2 mainly operates. This finding suggests that the efficiency of cellular energy metabolism depends on which respiratory enzyme actually works.Furthermore, the ctaA gene, located upstream of the cytochrome caaィイD23ィエD2 operon, was cloned from B. stearothermophilus and over-expressed in E. coli host cells. The CtaA product in the membrane preparation converts heme O into heme A in vitro. This finding is a direct evidence to show that CtaA is the heme A synthase.

从革兰氏阳性嗜热脂肪芽孢杆菌（Bacillus stearothermophilus）中克隆了编码细胞色素bd型醌醇氧化酶的基因。与其他细胞色素bd的序列比较和比对表明，这些氧化酶可分为两个亚家族，B。嗜热脂肪菌酶是已被分离和表征的第二组的第一个成员。亲水性分析表明，以往的细胞色素bd结构模型主要是基于对E。将细胞色素氧化酶D23和D2型细胞色素c氧化酶基因连接到穿梭载体pSTE 12上，并导入野生型B。嗜热脂肪细胞过度表达。所产生的氧化酶被纯化，重组到脂质体中，并显示H β D1+ H β D1泵活性，尽管H β D1+ H β D1/O比低于细胞色素α D23 β D2型氧化酶。分离到一株细胞色素氧化酶D23-D2型氧化酶主要起作用的突变细胞。突变体K17细胞的H β D1+ H β D1泵活性和稳态生长幅度高于以细胞色素bd为主要末端氧化酶的突变体K17，而低于以细胞色素caa β D23 β D2为主要末端氧化酶的野生型细胞。这一发现表明细胞能量代谢的效率取决于哪种呼吸酶实际上起作用。此外，位于细胞色素caa受体D23受体D2操纵子上游的ctaA基因是从B克隆的。stearothermophilus中表达，并在E. coli宿主细胞。膜制剂中的CtaA产物在体外将血红素O转化为血红素A。这一发现是证明CtaA是血红素A合酶的直接证据。