| Project/Area Number |
15H03832
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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 |
Bio-related chemistry
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| Research Institution | Akita University |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
野口 恵一 東京農工大学, 学内共同利用施設等, 准教授 (00251588)
松村 洋寿 秋田大学, 工学(系)研究科(研究院), 講師 (60741824)
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| Co-Investigator(Renkei-kenkyūsha) |
KUROKI RYOTA 独立行政法人日本原子力研究開発機構, 量子ビーム応用研究部門, ユニット長 (30391246)
YOHDA MASAFUMI 東京農工大学, 大学院工学研究院, 教授 (50250105)
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| Project Period (FY) |
2015-04-01 – 2018-03-31
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| Project Status |
Completed (Fiscal Year 2017)
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| Budget Amount *help |
¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000)
Fiscal Year 2017: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2016: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2015: ¥9,100,000 (Direct Cost: ¥7,000,000、Indirect Cost: ¥2,100,000)
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| Keywords | 触媒反応機構 / 反応中間体 / 翻訳後修飾 / 時間分割結晶構造解析 / 顕微分光法 / SFX / XFEL / 時間分割構造解析 / 中性子構造解析 / 質量分析 / FTIR |
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| Outline of Final Research Achievements |
In the hydration mechanism of nitrile hydratase (NHase), the substrate nitrile molecule, coordinated to the non-heme iron is attacked nucleophilically by the O-atom of the cysteine-sulfenate ligand, to form a novel cyclic intermediate. To uncover the reaction mechanism followed by the cyclic intermediate by serial femtosecond crystallography, we performed the following researches. βTyr37, which was suggested to be involved in the proton-transfer to the N-atom of the cyclic intermediate based on the hybrid quantum mechanics/molecular mechanics (QM/MM) method, was substituted by Phe, Ile or Ala. All mutant NHases showed significant catalytic activity. And, the crystal structure of the βY37K mutant was basically unchanged except for the substituted residue. We identified the reaction intermediate species of NHase by using time-resolved microspectroscopy. We determined the crystallization condition which is enough small for SFX analyses.
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