Title:The β-Sheet Core is the Favored Candidate of Engineering SDR for Enhancing Thermostability but not for Activity
Volume: 24
Issue: 6
Author(s): Deshuai Lou, Jun Tan*, Liancai Zhu, Shunlin Ji and Bochu Wang*
Affiliation:
- Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological & Chemical Engineering, Chongqing University of Education, Chongqing 400067,China
- College of Bioengineering, Chongqing University, No.174, Shapingba Main Street, Chongqing,China
Keywords:
Clostridium absonum, hydroxysteroid dehydrogenase, molecular dynamics simulation, β-sheet, short-chain dehydrogenase/
reductase, thermostability.
Abstract: 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.
