Met196突变提高Brucella melitensis 7α-羟基类固醇脱氢酶的催化效率和稳定性

Met196 mutation improves catalytic efficiency and stability of Brucella melitensis 7α-hydroxysteroid dehydrogenase

摘要:

【目的】通过计算机辅助设计，理性提高羊布鲁氏菌7α-羟基类固醇脱氢酶的催化效率和稳定性，实现酶的高效稳定催化合成。【方法】通过同源建模、分子对接和蛋白-配体相互作用分析，理性设计关键位点的定向突变。结合酶学性质测定、酶促反应动力学分析和圆二色谱测定等实验，测定突变酶的催化功能和稳定性。【结果】与野生型7α-羟基类固醇脱氢酶相比，Met196Ile和Met196Val突变酶的酶活提高了8.33倍和7.41倍，k_cat/K_m值分别提高了4.93和4.37倍，T_m值分别提高了1.75℃和1.10℃。Met196Ile和Met196Val突变酶催化底物鹅去氧胆酸，合成产物7-氧代-石胆酸所需时间从野生型的8 h缩短为2 h，最高产率约为91%。通过全原子动力学模拟分析了均方根偏差、均方根波动以及蛋白-配体相互作用，阐明了催化性能提高的分子机制。Met196突变诱导的B环(残基Ala145−Pro157)和α7螺旋(残基Val249−Gly265)的刚性增强有利于提高蛋白的稳定性，底物与结合位点或活性位点(Tyr208、Lys212)相互作用力的增强有利于改善催化效率。【结论】本研究采用同源建模和定点突变改造7α-羟基类固醇脱氢酶的催化活力和稳定性，为工业上高效稳定催化合成7-氧代-石胆酸奠定了较坚实的基础，同时为类固醇脱氢酶的理性设计提供了理论指导。

Abstract:

[Objective] Through computer-aided design, we improved the catalytic efficiency and stability of Brucella melitensis 7α-hydroxysteroid dehydrogenase and realized the efficient and stable synthesis of products. [Methods]Directed-mutagenesis at the key sites was rationally designed via homology modeling, molecular docking, and protein-ligand interaction analysis. Enzymatic property determination, enzymatic reaction kinetic analysis, and circular dichroism characterization were carried out to determine the catalytic function and stability of the enzyme. The root-mean-square deviation, root-mean-square fluctuation, and protein-ligand interactions were analyzed through all atom dynamics simulation to clarify the molecular mechanism of Met196 mutations improving catalytic efficiency and stability. [Results]Compared with the wild-type 7α-hydroxysteroid dehydrogenase, Met196Ile and Met196Val increased the specific activity to 8.33 and 7.41 folds, the k_cat/K_m values to 4.93 and 4.37 folds, and the T_m values by 1.75 ℃ and 1.10 ℃, respectively. Furthermore, Met196Ile and Met196Val shortened the duration of the synthesis from chenodeoxycholic acid to 7-oxolithocholic acid from 8 h to 2 h, and the mutants had the highest yield of about 91%. The Met196 mutation-induced rigidity enhancement of loop B (residues Ala145–Pro157) and α7 helix (residues Val249–Gly265) was beneficial to the protein stability. The enhanced interaction between substrate and binding sites or active sites (Tyr208 and Lys212) was conducive to the catalytic efficiency.[Conclusion] This study employed homology modeling and site-directed mutagenesis to modify 7α-hydroxysteroid dehydrogenase and thus improved its catalytic efficiency and stability. The findings laid a solid foundation for the efficient and stable synthesis of 7-oxolithocholic acid in industry and provided theoretical guidance for the rational design of steroid dehydrogenase.