2007 Fiscal Year Final Research Report Summary
Four-dimensional structure analysis of Ndx family enzyme utilizing a new pH-temperature jump trigger
| Project/Area Number |
18370047
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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 City University |
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Principal Investigator |
KAMIYA Nobuo Osaka City University, Faculty of Science, Professor (60152865)
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| Co-Investigator(Kenkyū-buntansha) |
MIYAHARA Ikuko Osaka City Univ., Faculty of Science, Associate Professor (40271176)
NAKAGAWA Noriko Osaka University, Faculty of Science, Assistant Professor (50379278)
KURAMITSU Seiki Osaka University, Faculty of Science, Professor (60153368)
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| Project Period (FY) |
2006 – 2007
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| Keywords | 4-D structure analysis / pH jump / temperature jump / Ndx family / functional genomics |
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| Research Abstract |
Before starting our research, we tried to improve crystal quality of ADP-ribose pyro-phosphatase (ADPRase) involved in Ndx family, because we needed high resolution analysis to resolve substrates and products which would co-exist in the reaction cavity of ADPRase in time course of enzyme reaction. Many crystallization conditions were tested, and the observable diffraction limit was expanded to 1.1A resolution from 1.7A, previously reported. In order to start the crystalline state reaction of ADPRase, Zn(II) ions were soaked into the crystals, in which the substrate ADPR was introduced in advance. The reaction was stopped by a temperature jump from 295K to 90K. Crystal structures were determined at 9 points of reaction time, 0, 3, 6, 10, 15, 20, 30, 40, 60min, and the in situ observation of ADPRase reaction was succeeded. Our results showed that the enzyme reaction progressed in crystal after the Zn(II) ion soaking as following. (1) The first Zn(II) ion introduced into reaction cavity changed ADPR conformation to an intermediate state, designated as ADPR^*.
(2) The second Zn(II) ion ligated ADPR^*, gultamate side chain of E82 and E86, and two water molecules making a five-ligands coordination structure. (3) One of two water molecules was activated by the second Zn(II) ion and E82 to hydroxide anion, and (4) the anion attacked nucleophilically the alpha phosphate of ADPR to brake the pyrophosphate bond to produce two products, AMP and ribose-5'-phosphate. The biochemical insight into the in situ observation of ADPRase was discussed.
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Research Products
(8 results)
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[Journal Article] Crystal structure of a homolog of mammalian serine racemase from Schizosaccharomyces pombe2009
Author(s)
Goto,M., Yamauchi,T., Miyahara,I., Kamiya,N., Yoshimura,T., Hihara,H., Kurihara,T., Hirotsu,K., Esaki,N.
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Journal Title
J.Biol. Chem. 284
Pages: 25944-25952
Description
「研究成果報告書概要(和文)」より
Peer Reviewed
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[Journal Article] Crystal structure of a homolog of mammalian serine racemase from Schizosaccharomyces pombe2009
Author(s)
Goto, M., Yamauchi, T., Miyahara, I., Kamiya, N., Yoshimura, T., Hihara, H., Kurihara, T., Hirotsu, K., Esaki, N.
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Journal Title
J.Biol.Chem. 284
Pages: 25944-25952
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Mutational study on aGln90 of Fe-type nitrile hydratase from Rgidiciccys sp. N7712006
Author(s)
Takarada,H., Kawano,Y., Hashimoto,K., Nakayama,H., Ueda,S., Yohda,M., Kamiya,N., Dohnae,N., Maeda,M., Odaka,M.
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Journal Title
Biosci. Biotech. and Biochem. 70
Pages: 881-889
Description
「研究成果報告書概要(和文)」より
Peer Reviewed
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[Journal Article] Mutational study on aGln90 of Fe-type nitrile hydratase from Rgidiciccys sp.N7712006
Author(s)
Takarada, H., Kawano, Y., Hashimoto, K., Nakayama, H., Ueda, S., Yohda, M., Kamiya, N., Dohnae, N., Maeda, M., Odaka, M.
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Journal Title
Biosci.Biotech.and Biochem. 70
Pages: 881-889
Description
「研究成果報告書概要(欧文)」より
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[Presentation] Reaction pathway of ADP-ribose pyrophosphatase, revealed by time-resolved X-ray crystallography2008
Author(s)
Kamiya,N., Kai,K., Nakagawa,N., Kuramitsu,S., Miyahara,I
Organizer
XXI Congress of the International Union of Crystallography
Place of Presentation
Osaka, Japan
Year and Date
2008-08-28
Description
「研究成果報告書概要(和文)」より
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