| Project/Area Number |
13125204
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Science and Engineering
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| Research Institution | OSAKA UNIVERSITY |
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Principal Investigator |
TANIZAWA Katsuyuki Osaka Univ., 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) |
YAMAGUCHI Hiroshi Kwansei Gakuin University, School of Science and Technology, Lecturer, 理工学部, 専任講師 (10252719)
OKAJIMA Toshihide Osaka Univ., Institute of Scientific and Industrial Research, Assoc, Prof., 産業科学研究所, 助教授 (10247968)
SASAI Hiroaki Osaka Univ., Institute of Scientific and Industrial Research, Professor, 産業科学研究所, 教授 (90205831)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥41,300,000 (Direct Cost: ¥41,300,000)
Fiscal Year 2003: ¥8,100,000 (Direct Cost: ¥8,100,000)
Fiscal Year 2002: ¥8,100,000 (Direct Cost: ¥8,100,000)
Fiscal Year 2001: ¥25,100,000 (Direct Cost: ¥25,100,000)
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| Keywords | quinone cofactor / copper amine oxidase / amine dehydrogenase / topa quinone / built-in cofactor / X-ray crystallography / quinohemoprotein / post-translational modification |
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| Research Abstract |
Over the recent decade, a number of unique cofactors derived from amino acid residues and thereby called built-in cofactors 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. 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. The present studies aimed at elucidating the structure and function of composite biocatalysts containing built-in quinone cofactors and metals for their molecular design. Several representative achievements are as follows.
1)We have identified a novel quinone cofactor, cysteine tryptophylquinone (CTQ) in quinohemoprotein amine dehydrogenases from Paracoccus denitrificans and Pseudomonas putida. CTQ is the forth quinone cofactor derived from amino acids, following TPQ, TTQ, and lysine tyrosylquinone (LTQ) fo
… More
und in lysyl oxidase.
2)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.
3)X-ray crystallographic studies of the Cu/TPQ-less inactive enzyme (apoenzyme) as well as the Cu/TPQ-containing active form (holoenzyme) revealed the structural difference only in the active site.
4)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.
5)A detailed reaction mechanism of copper amine oxidase has been proposed based on X-ray crystallographic and kinetic analyses of a mutant enzyme for the invariant active site base, Asp298.
6)A putative [Fe-S]-binding protein has been shown to be essential for the biosynthesis of active quinohemoprotein amine dehydrogenase containing CTQ. Less
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