X射线衍射晶体法解析脱卤酶DehDIV-R结构的研究  

Crystal Structural Analysis of Deh DIV-R by X-ray Crystallography

作　　者：童超迪 吴坚平[1] 杨立荣[1] 徐刚[1] TONG Chao-di;WU Jian-ping;YANG Li-rong;XU Gang(Zhejiang University,Department of Chemical and Biological Engineering,Hangzhou 310027,China)

机构地区：[1]浙江大学化学工程与生物工程学院

出　　处：《中国生物工程杂志》2018年第8期19-25,共7页China Biotechnology

基　　金：国家自然科学基金面上项目(21076187);国家973计划(2011CB710800)资助项目

摘　　要：R-2-卤代酸脱卤酶能立体选择性水解R-2-卤代酸。解析酶的单晶结构对提高酶的选择性和活性提供了直接的结构指导,是目前酶结构领域研究的前沿。以实验室前期得到的来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶（Deh DIV-R）为研究对象,采用X射线衍射晶体法进行结构解析。采用pp SUMO载体融合表达Deh DIV-R蛋白,依次通过Ni-NTA亲和层析、透析酶切、二次NiNTA亲和层析以及凝胶过滤层析纯化得到单一条带,且均一性好的蛋白。接着对结晶条件进行初筛与优化,得到的最佳结晶条件为0.1mol/L HEPES p H 7,12%PEG 6 000,0.2mol/L Mg Cl2,8mmol/L CHAPS。晶体在上海同步辐射光源BL18U1线站上收集衍射数据,采用分子置换法成功解析获得了分辨率为2.35的Deh DIV-R的晶体结构。Ramachandran图表明98.02%的氨基酸位于最适区,证明了该结构的合理性。Deh DIV-R的纯化、结晶以及结构解析为进一步深入了解其结构和功能奠定了基础。R-2-haloacid dehalogenase can selectively hydrolyze R-2-haloacid and have important applications in the synthesis of chiral compounds. The analysis of the crystal structure provides a direct structural guide to improve the selectivity and activity of the enzyme,which is the frontier in the field of enzymatic structure research. The crystal structure of R-2-chlorpropionic acid dehalogenase（ Deh DIV-R） from Pseudomonas ZJU26 was studied. Deh DIV-R was expressed in Escherichia coli BL21（ DE3） using pp SUMO as vectors,and purified by Ni-NTA affinity chromatography,ULPI digestion,second Ni-NTA affinity chromatography and gel filtration chromatography. High-quality crystals were obtained in optimal conditions（ 0. 1 mol/L HEPES p H 7,12% PEG6000,0. 2 mol/L Mg Cl2,8 mmol/L CHAPS）. The diffraction data of crystals were collected at BL18 U1 beamline of Shanghai Synchrotron Radiation Facility（ SSRF）. The crystal structure of Deh DIV-R with a resolution of 2. 35was successfully resolved by Molecular Replacement（ MR）. The Ramachandran plot shows that 98. 02% of the amino acids are in the optimum region,indicating the rationality of the structure. The purification,crystallization and structural analysis of the Deh DIV-R have laid a foundation for further understanding the relationship between structure and function.

关 键 词：R-2-卤代酸脱卤酶 纯化 结晶 X射线衍射 

分 类 号：Q814[生物学—生物工程]