2001 Fiscal Year Final Research Report Summary
Studies on a Novel Energy-Coupling Enzyme Family Rnf That Functions at Very Low Redox Potential
| Project/Area Number |
11440236
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
植物生理
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| Research Institution | Osaka University |
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Principal Investigator |
SAEKI Kazuhiko Osaka University, Department of Biology, Graduate School of Science, Associate Professor, 大学院・理学研究科, 助教授 (40201511)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Yasuhiro Osaka University, Department of Biology, Graduate School of Science, Lecturer, 大学院・理学研究科, 講師 (10154874)
FUKUYAMA Keiichi Osaka University, Department of Biology, Graduate School of Science, Professor, 大学院・理学研究科, 教授 (80032283)
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| Project Period (FY) |
1999 – 2001
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| Keywords | RHODOBACTER / FERREDOXIN / ELECTRON-TRANSPORT / NITROGEN-FIXATION / NADH / MEMBANE-PROTEIN-COMPLEX |
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| Research Abstract |
The rnf(rhodobacter nitrogen fixation) operon in Rhodobacter capsulatus is essential for nitrogen fixation under light. The genes encode a protein complex in chromatophore membrane that might function as an energy-consuming NADH-ferredoxin oxidoreductase.
1) We have partially purified the Rnf complex using Ni-NTA affinity column with hexahistidine-tagged RnfB expressed the cells of non-polar rnfB mutant strain KF11l containing the corresponding modified rnfB gene. However, the iron-sulfur center in RnfB protein was so unstable that complete purification was unsuccessful.
2) Analysis by phoA-fusion method revealed that RnfA and RnfE both span membrane six times with opposite Membrane topology of transmembrane subunits RnfA, RnfD and RnfE was analyzed by the. It was revealed that RnfA and RnfE span membrane 6 times with an opposite orientation and that RnfD protein spans membrane 8 times with its central hydrophilic region exposed to periplasm.
3) Downstream analysis of the known rnf genomic region revealed there is another gene (rnfI) that is essential for nitrogen fixation. RnfI protein shows similarity to the FADbinding subunit of periplasmic flavocytochrome c sulfide dehydrogenase.
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