Deshuai Lou, Jun Tan*, Liancai Zhu, Shunlin Ji and Bochu Wang* Pages 511 - 516 ( 6 )
Background: 7α-Hydroxysteroid dehydrogenases (7α-HSDHs) can stereoselectively catalyze steroids, aromatic α-ketoesters, and benzaldehyde analogues playing a critical role in the biotransformation and poor thermostability that hinders their biomedical and industrial applications.Objective: This study was to investigate how to enhance the thermostability of 7α-HSDH from Clostridium absonum (CA 7α-HSDH). Method: Based on the three-dimensional structure of CA 7α-HSDH, recently reported program MAESTRO was used to compute the ΔΔG and predict the single-point mutants that could enhance its thermostability. The selected mutants were verified experimentally. Results: The results from the circular dichroism spectrum indicated that three of the mutants, N89L, N184I, and A185I, fitted a three-state model and the values for Tm N→I and Tm I→D increased with different ranges. In particular, the Tm N→I for the N184I mutant increased maximally by 9.93°C. Meanwhile, the denaturation process of the G189I mutant fitted the two-state model and it was more stable than the wild type, judging from the denaturation curves. Nevertheless, the enzyme catalytic activity analysis suggested that only the N89L mutant held a 2.28% catalytic efficiency, compared to the wild type, CA 7α-HSDH, and the activities of the other three mutants could not be detected. Molecular dynamics (MD) simulations were performed to determine the structural changes that occurred in the mutations and the results indicated that β-sheet structures in the mutants without detectable activity had changed significantly. Conclusion: Judging from the locations of the mutated sites, residues in the β-sheet core were considered as the favored candidates for SDR engineering to enhance the thermostability but not for activity holding.
Clostridium absonum, hydroxysteroid dehydrogenase, molecular dynamics simulation, β-sheet, short-chain dehydrogenase/ reductase, thermostability.
Postdoctoral Research Station of Biology, Chongqing University, Chongqing 400030, Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological & Chemical Engineering, Chongqing University of Education, Chongqing 400067, Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030, Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030, College of Bioengineering, Chongqing University, No.174, Shapingba Main Street, Chongqing