中文摘要：

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。

英文摘要：

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 stereoselectivity of dehDIV-R was forecasted to be related be Asn236. After molecular docking studies, the 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-directlng 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.