| Co-Investigator(Kenkyū-buntansha) |
OH-OKA Hirozo Graduate School of Science, Department of Biology, Associate Professor, 大学院・理学研究科, 助教授 (30201966)
FUKUYAMA Keiichi Graduate School of Science, Department of Biology, Professor, 大学院・理学研究科, 教授 (80032283)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2001: ¥3,100,000 (Direct Cost: ¥3,100,000)
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| Research Abstract |
Iron-sulfur proteins are present in almost all living organisms and exhibit diverse functions, which include electron transport, redox and non-redox catalysis, and sensing for regulatory processes. They contain clusters of iron and sulfur atoms with vailiable complexity, such as [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters. Still very little is known about the in vivo synthesis of these clusters, which is believed to require a complex machinery encoded us prokaryotes by the isc (iron suffer cluster) operon, iscRSUA-hscBA-fdx-ORF3. We have undertaken genetic and biochemical approaches to elucidate the complex mechanism by which Fe-S dusters are assembled.
1.Essential compornents of the ISC machinery (IscS, IscU, HscB, HscA, and Fdx) have been identified by a systematic mutational analysis of the isc operon in Eseherichia coli.
2.Biochemical characterization of the recombinant IscA led to the identification of labile [2Fe-2S] cluster and specific interaction with Fdx.
3.We determined the crystal structure of Fdx, which suggested the important role of Cys46 located on the molecular susface in the vicinity of [2Fe-2SJ cluster.
4.Network of protein-protein interactions among the components of the ISC machinery has been elucidated by the combination of two-hybrid and pull-down analyses.
5.We have isolated a covalently-bound IscS/IscU complex and identified that Cys328 of IseS and Cys63 of lscU are the sites of disulfide bridge formation.
6.We have analyzed spontaneous psuedorevertants isolated from a mutant shale lacking all the components of the ISC machinery, which led to the identification of the sufABCDSE (suf) operon participating in the ISC-independent pathway for the assembly of Fe-S clusters. The genes homologous to sufBC are present in a wide range of bacteria, archaea and plastids, suggesting that this type of system is almost ubiquitous in nature.
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