X射线衍射晶体法解析脱卤酶DehDIV-<i>R</i>结构的研究 <sup>*</sup>

doi:10.13523/j.cb.20180803

中国生物工程杂志

2018, Vol. 38

Issue (8): 19-25 DOI: 10.13523/j.cb.20180803

研究报告

X射线衍射晶体法解析脱卤酶DehDIV-R结构的研究 ^*

童超迪,吴坚平,杨立荣,徐刚(

)

浙江大学化学工程与生物工程学院杭州 310027

Crystal Structural Analysis of DehDIV-R by X-ray Crystallography

Chao-di TONG,Jian-ping WU,Li-rong YANG,Gang XU(

)

Zhejiang University,Department of Chemical and Biological Engineering,Hangzhou 310027,China

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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(DehDIV-R)为研究对象,采用X射线衍射晶体法进行结构解析。采用ppSUMO载体融合表达DehDIV-R蛋白,依次通过Ni-NTA亲和层析、透析酶切、二次Ni-NTA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 pH 7,12% PEG 6 000,0.2mol/L MgCl₂ ,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DehDIV-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。DehDIV-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。

关键词： R-2-卤代酸脱卤酶; 纯化; 结晶; X射线衍射

Abstract:

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 (DehDIV-R) from Pseudomonas ZJU26 was studied.DehDIV-R was expressed in Escherichia coli BL21(DE3) using ppSUMO 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 pH 7,12% PEG 6000,0.2 mol/L MgCl₂,8 mmol/L CHAPS).The diffraction data of crystals were collected at BL18U1 beamline of Shanghai Synchrotron Radiation Facility(SSRF).The crystal structure of DehDIV-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 DehDIV-R have laid a foundation for further understanding the relationship between structure and function.

Key words: R-2-haloacid dehalogenase Purification Crystallization X-ray diffraction

收稿日期: 2018-03-09 出版日期: 2018-09-11

ZTFLH:

Q814

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

通讯作者: 徐刚 E-mail: xugang_1030@zju.edu.cn

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引用本文:

童超迪,吴坚平,杨立荣,徐刚. X射线衍射晶体法解析脱卤酶DehDIV-R结构的研究 ^*[J]. 中国生物工程杂志, 2018, 38(8): 19-25.

Chao-di TONG,Jian-ping WU,Li-rong YANG,Gang XU. Crystal Structural Analysis of DehDIV-R by X-ray Crystallography. China Biotechnology, 2018, 38(8): 19-25.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180803 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I8/19

图1 第一次Ni-NTA亲和层析纯化DehDIV-R的SDS-PAGE分析图

图2 经ULP1酶切后第二次Ni-NTA亲和层析纯化DehDIV-R的SDS-PAGE分析图

图3 DehDIV-R凝胶过滤层析图谱

图4 凝胶过滤层析纯化DehDIV-R的SDS-PAGE分析图

图5 初筛得到的DehDIV-R晶体

图6 最优化条件下的DehDIV-R晶体

表1 脱卤酶DehDIV-R晶体衍射数据统计结果

图7 DehDIV-R X射线衍射画面

表2 脱卤酶DehDIV-R晶体结构精修结果

图8 脱卤酶DehDIV-R晶体结构Ramachandran图

图9 脱卤酶DehDIV-R整体结构示意图(a)与活性口袋(b)

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