基于分子动力学模拟的高γ-环糊精专一性环糊精糖基转移酶理性改造

Rational engineering of high γ-cyclodextrin specific cyclodextrin glycosyltransferase based on molecular dynamics simulation

摘要:

【背景】环糊精糖基转移酶的分子动力学模拟较传统基因改造而言能有效提高改造效率，减少盲目性。【目的】探究环糊精糖基转移酶的催化专一性机理，为获得产γ-环糊精专一性更高的环糊精糖基转移酶提供高效突变菌株方法。【方法】通过分子对接和分子动力学模拟，获得3种产物类型CGTase与底物的对接模拟结构，并通过定点突变实验进行验证。【结果】分子动力学模拟结果显示α-和β-CGTase与十糖链在酶蛋白S1区域呈现闭合的形态，而γ-CGTase和十糖链在S1区域呈现更易于生成γ-环糊精的张开形态；3种CGTase与十糖链在相同位置存在氢键的氨基酸共有17个相对应位点，其中14个位点的氨基酸种类一致，不一致的3个氨基酸对应α-CGTase位点分别为Y89、D234和Y262。本研究对Y262位点进行定点突变和产物专一性实验，结果显示经过分子动力学预测的Y262L有助于提高产γ-CD专一性，从野生酶的13.7%提高到39.9%，γ-环糊精产物比例提高了3倍。【结论】分子动力学模拟结果对于指导环糊精糖基转移酶的专一性内在机理具有一定的正向指导意义。

关键词:环糊精，环糊精糖基转移酶，分子动力学模拟，产物专一性，催化机理

Abstract:

[Background] Compared with traditional genetic modification, molecular dynamics simulation of cyclodextrin glycosyltransferase can effectively improve the transformation efficiency and reduce blindness. [Objective] To explore the catalytic specificity mechanism of cyclodextrin glycosyltransferase, and to provide an efficient mutation method for obtaining cyclodextrin glycosyltransferase with higher specificity for producing γ-cyclodextrin. [Methods] Through molecular docking and molecular dynamics simulation, the docking simulation structures of three product types of CGTase and substrate were obtained, and verified by site-specific saturation mutation experiment. [Results] The results of molecular dynamics simulation showed that α- and β-CGTase and decarbose chains appeared closed in S1 region, while γ-CGTase and decarbose chains appeared more open in S1 region. There are seventeen corresponding sites in the three CGTase and ten sugar chain amino acids with hydrogen bonds at the same position, of which the amino acid types at fourteen sites are consistent. The corresponding α-CGTase sites of the three inconsistent amino acids were Y89, D234 and Y262, respectively. This study conducted site-directed mutagenesis and product specificity experiments on the Y262 locus. Y262L predicted by molecular dynamics was helpful to increase the specificity of γ-CD production, from 13.7% of the wild enzyme to 39.9%, and the percentage of γ-cyclodextrin products increased by three times. [Conclusion] The results of molecular dynamics simulation have positive significance for guiding the specificity mechanism of cyclodextrin glycosyltransferase.

Key words:Cyclodextrin, Cyclodextrin glucosyltransferase, Molecular dynamics simulation, Product specificity, Catalytic mechanism