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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2014, Vol. 34 Issue (06): 16-22    DOI: 10.13523/j.cb.20140603
研究报告     
来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶的手性识别过程研究
张滢潭, 杨立荣, 徐刚, 吴坚平
浙江大学化学工程与生物工程学系 杭州 310027
Studying of Chiral Recognition Process of R-2-CPA Dehalogenase from Pseudomonas sp. ZJU26
ZHANG Ying-tan, YANG Li-rong, XU Gang, WU Jian-pin
Zhejiang University, Department of Chemical Engineering and Biological Engineering, Hangzhou 310027, China
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摘要：

立体选择性是2-卤代酸脱卤酶最重要的性质之一，但目前其手性识别过程尚不明确，对其进行研究和解析具有重要意义。以来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶dehDIV-R为模型，研究了R-2-卤代酸脱卤酶的手性识别过程。首先通过测定反应产物的构型，确定dehDIV-R催化底物为SN2反应。通过Discovery Studio 3.0对dehDIV-R进行同源模建及底物分子对接，由对接结果和序列比对确定dehDIV-R立体选择性的关键位点Asn236，预测dehDIV-R的立体选择性与反应时底物到达反应位置的空间位阻密切相关。对dehDIV-R进行虚拟突变，将Asn236位点突变成具有不同空间位阻的残基Ala和Ser，并分别与底物分子进行分子对接，预测突变酶的立体选择性。根据预测结果，对Asn236氨基酸残基进行定点突变，发现在Asn236突变为Ala后的A1酶显示出对RS底物的活力；在Asn236突变为Ser后的S1酶显示出与原始酶相反的立体选择性，实现了立体选择性的反转。与模型的预测结果相符，证明了模型的合理性。
关键词： 2-氯丙酸脱卤酶;  手性识别过程;  分子对接    
Abstract:

Stereoselectivity of 2-haloacid dehalogenase is one of the most important properties in biocatalysis, but the process of chiral recognition is not clear. Therefore resolving this problem becomes a research focus. The process of chiral recognition of dehDIV-R, a R-haloacid dehalogenase from Pseudomonas sp ZJU26 was studied. Firstly, by measuring the configuration of the product, the reaction catalyzed by dehDIV-R was defined as SN2 reaction. Through multiple sequence alignment and homology modeling of dehDIV-R, the key site of stereoselectivity of dehDIV-R was found to be Asn236. After molecular docking studies, the stereoselectivity of dehDIV-R was forecasted to be related to the steric hindrance for substrate to reach the specific reacting site. Asn236 of dehDIV-R was virtually mutated to amino acids residues with different steric hindrance, and the stereoselectivities of the mutant enzymes were predicted. According to the prediction results, the mutations of Asn236/Ala and Asn236/Ser were constructed by site-directing mutagenesis. After measuring their stereoselectivities, A1 with Asn236/Ala showed activity towards S- and R-substrate, and S1 with Asn236/Ser showed inverted activity towards S-substrate instead of R-substrate. These results were consistent with the prediction and the rationality of the model was proved.
Key words: 2-CPA dehalogenase    Process of chiral recognition    Molecular docking
收稿日期: 2014-03-18 出版日期: 2014-06-25
ZTFLH:  Q789  
基金资助:

国家重点基础研究发展计划（2011CB710805）、 国家自然科学基金面上项目（21076187）资助项目
通讯作者: 吴坚平     E-mail: wjp@zju.edu.cn
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引用本文:

张滢潭, 杨立荣, 徐刚, 吴坚平. 来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶的手性识别过程研究[J]. 中国生物工程杂志, 2014, 34(06): 16-22.

ZHANG Ying-tan, YANG Li-rong, XU Gang, WU Jian-pin. Studying of Chiral Recognition Process of R-2-CPA Dehalogenase from Pseudomonas sp. ZJU26. China Biotechnology, 2014, 34(06): 16-22.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140603    或    https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I06/16


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