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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2014, Vol. 34 Issue (06): 16-22    DOI: 10.13523/j.cb.20140603
    
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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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     
Received: 18 March 2014      Published: 25 June 2014
ZTFLH:  Q789  
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Cite this article:

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.

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


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