| Project/Area Number |
10044197
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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 |
応用微生物学・応用生物化学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
OHTA Akinori (1999) Department of Biotechnology, The University of Tokyo, Professor, 大学院・農学生命科学研究科, 教授 (30125885)
高木 正道 (1998) 東京大学, 大学院・農学生命科学研究科, 教授 (50018339)
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| Co-Investigator(Kenkyū-buntansha) |
NAGATA Yuji Department of Biotechnology, The University of Tokyo, Assistant Professor, 大学院・農学生命科学研究科, 助手 (30237531)
TAKAGI Masamichi Department of Bioscience, Tokyo University of Agriculture, Professor, 応用生物科学部・バイオサイエンス学科, 教授 (50018339)
DAMBORSKY Ji Masaryk University, Faculty of Science, 講師
KOCA Jarosla Masaryk University, Faculty of Science, 教授
太田 明徳 東京大学, 大学院・農学生命科学研究科, 助教授 (30125885)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1999: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1998: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Sphingomonas / Environmental pollutant / Chlroinated compound / Biodegradation / Haloalkane dehalogenase / Computer modelling / Hydrolase / ハロアルカンデハロゲナーゼ / dehalogenase / hydrolase / haloalkane / 基質特異性 / ホモロジーモデリング / 部位指定変異導入 / catalytic triad |
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| Research Abstract |
The deduced amino acid sequence of LinB (1, 3, 4, 6-tetrachloro-1, 4-cyclohexadiene halidohydrolase) showed highest level, of similarity to haloalkane dehalogenase (DhlA) from Xanthobacter autotrophicus GJIO, suggesting that LinB belongs to the family of haloalkane dehalogenase enzymes catalyzing the dehalogenation by hydrolytic mechanism. LinB is believed to belong the family of α/β-hydrolases which employ catalytic triad, i.e. nucleophile-histidine-acid, during the catalytic reaction. At first, the position of the catalytic triad within the sequence of LinB was probed by site-directed mutagenesis. The catalytic triad residues of the haloalkane dehalogenase LinB are proposed to be ?D108, H272, and E132 For further analysis, we determined three dimentional structure of LinB. The purified LinB was crystallized using the hanging-drop vapoul-diffusion method. The crystals diffract to 1.58 angstrom using synchrotron X-ray under cryogenic (100K) conditions. The structure of LinB was solved by molecular replacement with dehalogenase (DhaA) from Rhodococcus.
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