基于分子模拟技术研究枯草芽孢杆菌Lip-A对映体选择性反转

Study on the Stereoselective Inversion of Bacillus subtilis Lip-A and Its Mutant Lip-AN^(18I) by Molecular Simulation

采用分子动力学及分子对接方法,研究了枯草芽孢杆菌脂肪酶A(Lip-A)及其突变体Lip-AN18I对底物S-1,2-O-异亚丙基-sn-甘油(S-1,2-O-isopropylidene-sn-glycerol,S-IPG)丁酸酯与R-1,2-O-异亚丙基-sn-甘油(R-IPG)丁酸酯立体选择性反转的分子机制。利用SWISS-Model构建突变体Lip-AN18I,通过GROMACS 4.5.4软件对Lip-A与Lip-AN18I分别优化获得合理构象,进而通过分子对接Autodock 4.2软件获得酶与底物复合物的100个构象;通过将立体电子效应的构象约束条件引入到分子对接构象筛选中,筛选到适合于Lip-A及突变体Lip-AN18I活性口袋的反应型构象,对实验数据进行了合理解释;同时,为了减小半柔性对接产生的误差,利用SYBYL 1.3X G_Score打分函数重新评价目标蛋白与配体小分子之间亲和作用力的大小,对实验数据进行了进一步合理解释;基于能量最优与空间互补原则,从分子水平上证实Asn18为调控Lip-A对映体选择性的关键氨基酸之一,为后续的基因工程改造Lip-A获得针对不同手性底物的高效生物催化剂提供了理论指导。

By application of the methods of molecular dynamics and molecular docking, the molecular mechanism in the stereoselective inversion behaviors of Bacillus subtilis lipase A （Lip-A） and its mutant Lip-AN181 were investigated, respectively, towards the S- and R- sterenisomers of 1,2-O-isopropylidene- sn-glycerol （IPG）. The three-dimensional model structures of both Lip-A and Lip-AN181 were firstly constructed by SWISS-Model, and then improved to the reasonable conformations via GROMACS 4.5.4 software. Subsequently, 100 conformations of each enzyme-substrate complex were performed by Autodock 4.2 software. By introducing the stereo-sensing mechanism into the screen of molecular docking conformation, probable binding modes were attained for Lip-A and its mutant Lip-AN181 inside the catalytic cavity, and the experimental data were well explained. In order to reduce the inaccuracy lay in semi-flexible molecular docking, the strength of the affinity force between ligands and target protein was reappraised by the SYBYL G_Score scoring function, and the experimental data were further explained. According to the principle of energy optimization and space complementarity, it was confirmed on the molecular level that Asn 18 was one of the key residues in the process of stereoselective inversion. Therefore, it may provide theoretical guidance for subsequent directed-mutation upon Lip-A to obtain effective biological catalyst towards different chiral compounds.