| Project/Area Number |
12480180
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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 |
Structural biochemistry
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| Research Institution | Osaka University |
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Principal Investigator |
TANIZAWA Katsuyuki Osaka University, Institute of Scientific and Industrial Research, Professor, 産業科学研究所, 教授 (20133134)
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| Co-Investigator(Kenkyū-buntansha) |
OKAJIMA Toshihide Osaka University, Institute of Scientific and Industrial Research, Associate Professor, 産業科学研究所, 助教授 (10247968)
黒田 俊一 大阪大学, 産業科学研究所, 助教授 (60263406)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥15,700,000 (Direct Cost: ¥15,700,000)
Fiscal Year 2002: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2001: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 2000: ¥8,200,000 (Direct Cost: ¥8,200,000)
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| Keywords | quinone cofactor / copper amine oxidase / amine dehydrogenase / topa quinone / built-in cofactor / X-ray crystallography / quinohemoprotein / post-translational modification / 銅イオン |
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| Research Abstract |
Over the recent decade, a number of unique cofactors derived from amino acid residues have been discovered in various enzyme proteins, including 2,4,5-trihydroxyphenylalanine quinone (topa quinone; TPQ) of copper amine oxidase and tryptophan tryptophylquinone (TTQ) of bacterial amine dehydrogenase. These quinone co factors temporarily store the reducing equivalents derived from the substrate before passing them on to exogenous 1- or 2-electron acceptors such as a cytochrome, a cupredoxin, or molecular oxygen. Both of TPQ and TTQ are encoded as ordinary amino acids (Tyr or Trp) in the enzyme genes and thus are synthesized by post-translational modification. Using the inactive precursor forms of recombinant copper amine oxidases from Arthrobacter globiformis overproduced in Escherichia coli, we demonstrated that TPQ is generated through self-processing of the enzyme proteins with the participation of the bound copper ion. X-ray crystallographic studies of the Cu/TPQ-less inactive enzyme
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(apoenzyme) as well as the Cu/TPQ-containing active form (holoenzyme) revealed the structural difference only in the active site. Using X-ray crystallography, we have probed the copper-dependent autooxidation process of TPQ. Apo enzyme crystals were anaerobically soaked with copper; the structure determined from this crystal provided a view of the initial state: the unmodified tyrosine coordinated to the bound copper. Exposure of the copper-bound crystals to oxygen led to the formation of freeze-trapped intermediates; structural analyses indicated that these intermediates contain dihydroxyphenylalanine quinone and trihydroxyphenylalanine. These are the first visualized intermediates during TPQ biogenesis in copper amine oxidase. Furthermore, we have recently identified a novel quinone co factor, cysteine tryptophylquinone (CTQ) in quinohemoprotein amine dehydrogenases from Paracoccus denitrificans and Pseudomonas putida. CTQ is the forth quinone co factor derived from amino acids, following TPQ, TTQ, and lysine tyrosylquinone (LTQ) found in lysyl oxidase. Less
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