R-2-卤代酸脱卤酶的同源模建及底物对映体选择性研究

Homology Modeling and Substrate Enantioselectivity for R-2-haloacid Dehalogenase

对来自假单胞菌ZJU26中的R-2-卤代酸脱卤酶(DehI-R)进行同源模建,分析其与底物的相互作用,为解析酶的底物对映体选择性提供理论依据。采用Sybyl中的APM模块首次构建并优化了R-2-卤代酸脱卤酶的三维结构,并用Procheck验证结构模型的合理性。使用Surflex-Docking模块将结构模型与底物分别进行对接,分析相互之间的作用。序列比对结果显示,R-2-卤代酸脱卤酶与恶臭假单胞菌PP3中DehI的相似性达23.71%。Deh-R模建后的结构与模板很好的吻合。模型比对分析DehI-R中参与催化的残基,除Asn203外大部分都比较保守。分子对接结果表明,R-2-氯丙酸和S-2-氯丙酸都可以结合到活性位点上,决定其选择性的是位点Asn203,在RS-2-卤代酸脱卤酶所对应位点的残基为Ala,相比之下,Asn具备较大的空间位阻,从而阻止了S-2-氯丙酸的反应。利用Sybyl中的Biopolymer模块对R-2-卤代酸脱卤酶中的Asn203突变成具有不同空间位阻的Ala、Gly和Gln。突变酶与底物对接结果进一步证实了Asn203位点对R-2-卤代酸脱卤酶的底物对映体选择性作用。

A model of R-2-haloacid dehalogenase（DehI-R）from Pseudomonas sp.ZJU26,built by homology modeling,was docked with substrates to analyze the interaction between them,for the purpose of providing the theoretical basis of the enzyme＇s enantioselectivity.The three dimensional structure of the DehI-R was constructed by APM in Sybyl.The reliability of the model was assessed by Procheck.The interaction of the enzyme and substrate was analyzed by Surflex-Docking.Sequence alignment indicated that the dehI-R from Pseudomonas sp.ZJU26 had 23.71% identity with the DehI from Pseudomonas putida PP3.The constructed 3D model of the R-2-haloacid dehalogenase adopted a similar folding pattern to the template and the two matched well.Structure alignment displayed that most residues for catalytic site or substrate binding site were conserve except Asn203.Molecular docking results indicated that both of the substrates could bind to the enzyme.The residue dictating the enzyme＇s selectivity was Asn203 in R-2-haloacid dehalogenases,while it was Ala in RS-2-haloacid dehalogenases.Compared with the Ala in RS-2-haloacid dehalogenases.Comparatively,the Asn203 in the DehI-R,with huger steric hindrance,inhibited the dehalogenation of the S-2-chloropropionate（S-2-CPA）.The Asn203 was mutated to residues with various steric hindrance,such as Ala,Gly and Gln,by Biopolymer in Sybyl.The docking results of the mutants and the substrates proved the function of Asn203 in the substrate enantioselectivity for R-2-haloacid dehalogenases.The model can be used as a starting point for the direct evolution for the enzyme in future.