2003 Fiscal Year Final Research Report Summary
Four-Dimensional Protein Crystallography of Nitrile Hydratase Reaction
| Project/Area Number |
14380321
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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 |
Biophysics
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| Research Institution | RIKEN |
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Principal Investigator |
KAMIYA Nobuo RIKEN, Bio-Crystallography Technology, Senior Scientist, 研究技術開発室・室長(副主任待遇) (60152865)
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| Co-Investigator(Kenkyū-buntansha) |
KAWANO Yoshiaki RIKEN, Bio-Crystallography Technology, Researcher, 研究技術開発室, 研究員 (70321750)
FURUSE Munenori RIKEN, Bio-Crystallography Technology, Post-doc, 研究技術開発室, 協力研究員 (50321807)
NOJIRI Masaki RIKEN, Bio-Crystallography Technology, Post-doc, 研究技術開発室, 基礎科学特別研究員 (20333346)
ODAKA Masafumi RIKEN, Bio-Engineering Lab, Researcher, バイオ工学研究室, 助教授 (20224248)
KOBAYASHI Michihiko University of Tsukuba, Professor, 応用生物化学系, 教授 (70221976)
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| Project Period (FY) |
2002 – 2003
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| Keywords | Nitrile Hydratase / Photo-activation / Four-dimensional Crystallography / SPring-8 / Large-angle Oscillation Technique / Site-directed mutant protein |
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| Research Abstract |
Nitrile hydratase from Rodococcus sp. N-771 is the enzyme that catalyzes the hydration of nitriles to the corresponding amides, and contains a mononuclear non-heme iron as the reaction center (Fe-type NHase). The center is photo-reactive, inactivated by nitrosylation and activated by photo-driven NO release. The photo-activated Fe-type NHase loses the activity within 24 hours under aerobic conditions. Previous studies have revealed that the post-translationally modified cystein sulfenate (aCys114-SO-) of active enzyme is further oxidized under the aerobic conditions to cystein sulfinate (aCys114-SO_2-).
In order to avoid the further oxidation, a crystallization system was constructed under anaerobic conditions of less than 0.1% (v/v) oxygen concentration. The really active structure of intact Fe-type NHase was studied by X-ray crystallography, including complex structures with butyric acid as an inhibitor/stabilizer and with cyclohexyl-isocyanide (ch-NC) as a substrate analogue. We also crystallized the inactive nitrosylated NHase under the anaerobic conditions in the complex form with ch-NC. The dynamic structure changes were traced by using the large-angle oscillation technique (LOT) after photo-activation at a time-resolution of 30min at a RIKEN beamline: BL45XU, SPring-8, the data collection system of which was remodeled for our purpose. Based on the results obtained, the role of aCys 114-SO- in the nitrile hydration mechanism of Fe-type NHase was revealed.
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[Journal Article] Crystal structure of Thermus themophilus HB8 H-protein of the glycine cleavage system, resolved by a six-dimensional molecular-replacement method2003
Author(s)
Nakai, T., Ishijima, J., Masui, R., Kuramitsu, S., Kamiya, N.
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Journal Title
Acta Cryst.Sec.D 59
Pages: 1610-1618
Description
「研究成果報告書概要(和文)」より
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[Journal Article] Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas cholororaphis B23:Sequencing, gene expression, purification and characterization2003
Author(s)
Oinuma, K-I., Hashimoto, Y., Konishi, K., Goda, M., Noguchi, T., Higashibata, H., Kobayashi, M.
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Journal Title
J.Biol.Chem. 278
Pages: 29600-29608
Description
「研究成果報告書概要(和文)」より
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[Journal Article] Crystal structure of Thermus thermophilus HB8 H-protein of the glycine cleavage system, resolved by a six-dimensional molecular-replacement method2003
Author(s)
Nakai, T., Ishijima, J., Masui, R., Kuramitsu, S., Kamiya, N.
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Journal Title
Acta Cryst. Sec. D 50
Pages: 1610-1618
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23: Sequencing, gene expression, purification and characterization2003
Author(s)
Oinuma, K-I., Hashimoto, Y., Konishi, K., Goda, M., Noguchi, T., Higashibata, H., Kobayashi, M.
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Journal Title
J. Biol. Chem. 278
Pages: 29600-29608
Description
「研究成果報告書概要(欧文)」より
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