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

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.