| Project/Area Number |
10680617
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Kyushu Institute of Technology |
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Principal Investigator |
SAKAMOTO Junshi KIT, Dept. of Biochemical Engineering and Science, Associate Professor, 情報工学部, 助教授 (80175364)
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| Co-Investigator(Kenkyū-buntansha) |
NOGUCHI Shunsuke KIT, Dept. of Biochemical Engineering and Science, Assistant Professor, 情報工学部, 助手 (30222194)
SONE Nobuhito KIT, Dept. of Biochemical Engineering and Science, Professor, 情報工学部, 教授 (20049034)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | thermophile / Gram-positive bacteria / respiratory chain / cytochrome / oxidase / bioenergetics |
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| Research Abstract |
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.
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