Mechanism-Based Biodegradability Prediction of Dehalogenase

基于机制的脱卤酶生物降解性预测

• 批准号: 10044197
• 负责人: OHTA Akinori
• 金额: $ 2.62万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10044197/
• 关键词: 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

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.

推导的LinB（1,3,4,6-四氯-1,4-环己二烯卤化水解酶）的氨基酸序列与自养黄杆菌GJIO的卤代烷脱卤酶（DhlA）具有最高水平的相似性，表明LinB属于催化脱卤的卤代烷脱卤酶家族 通过水解机制。 LinB被认为属于α/β-水解酶家族，其在催化反应过程中采用催化三联体，即亲核体-组氨酸-酸。首先，通过定点诱变探测了 LinB 序列中催化三联体的位置。卤代烷脱卤酶LinB 的催化三联体残基被认为是ΔD108、H272 和E132。为了进一步分析，我们确定了LinB 的三维结构。使用悬滴蒸气扩散法对纯化的 LinB 进行结晶。在低温 (100K) 条件下使用同步加速器 X 射线，晶体衍射至 1.58 埃。 LinB 的结构通过用红球菌脱卤素酶 (DhaA) 进行分子替换来解析。

项目成果

Y. Nagata ら: "Complete analysis of genes and enzymes for γ-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26"J. Industrial Microbiology & Biotechnology. 23. 380-390 (1999)

Y. Nagata 等：“少动鞘氨醇单胞菌 UT26 中 γ-六氯环己烷降解的基因和酶的完整分析”J.工业微生物学与生物技术。

Y. Nagata: "Construction and characterisation of histidive-tagged haloalkane dehalogenase(Lin B) of a new substrate class from"Protein Expression and Puritication. 17. 299-304 (1999)

Y. Nagata：“来自《蛋白质表达和纯化》的新底物类别的组氨酸标记卤代烷脱卤酶 (Lin B) 的构建和表征。

Nagata, Y., K. Miyauchi, and M. Takagi: "Complete analysis of genes and enzymes forγ-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26."Industrial Microbiology & Biotechnology. 23. 380-390 (1999)

Nagata, Y.、K. Miyauchi 和 M. Takagi：“少动鞘氨醇单胞菌 UT26 中 γ-六氯环己烷降解的基因和酶的完整分析。”工业微生物学与生物技术 23. 380-390 (1999)。

I. Smatanov: "Crystallization and prelimirary x-ray diffraction analysis of haloalkane deralogenase Lin B from Sphingomouas paucimobilis UT26"Acta Crystallographica Section D. D55. 1231-1233 (1999)

I. Smatanov：“来自 Sphingomouas paucimobilis UT26 的卤代烷脱醛酶 Lin B 的结晶和初步 X 射线衍射分析”《晶体学报》第 D. D55 部分。

Smatanov, I., Y. Nagata, L. A. Svensson, M. Takagi, and J. Marek: "Crystallization and preliminary X-ray diffraction analysis of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26"Acta Crystallographica Section D, Biological Crystallography.

Smatanov, I.、Y. Nagata、L. A. Svensson、M. Takagi 和 J. Marek：“少动鞘氨醇单胞菌 UT26 中卤代烷脱卤酶 LinB 的结晶和初步 X 射线衍射分析”《晶体学报》D 部分，生物晶体学。