| 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)
遠藤 勲 宇都宮大学, 農学部, 教授 (00087470)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥13,300,000 (Direct Cost: ¥13,300,000)
Fiscal Year 2003: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2002: ¥9,500,000 (Direct Cost: ¥9,500,000)
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| Keywords | Nitrile Hydratase / Photo-activation / Four-dimensional Crystallography / SPring-8 / Large-angle Oscillation Technique / Site-directed mutant protein / SPning-8 / 小型カッパーゴニオメータ / イソニトリルヒドラターゼ |
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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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