Lou Deshuai, Tan Jun, Zhu Liancai, Ji Shunlin, Wang Bochu
Postdoctoral Research Station of Biology, Chongqing University, Chongqing 400030, China.
Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological & Chemical Engineering, Chongqing University of Education, Chongqing 400067, China.
Protein Pept Lett. 2017;24(6):511-516. doi: 10.2174/0929866524666170126150006.
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.
This study was to investigate how to enhance the thermostability of 7α-HSDH from Clostridium absonum (CA 7α-HSDH).
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.
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.
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.
7α-羟基类固醇脱氢酶（7α-HSDHs）能够立体选择性地催化类固醇、芳香族α-酮酯和苯甲醛类似物，在生物转化中发挥关键作用，但其热稳定性较差，这阻碍了它们在生物医学和工业领域的应用。
本研究旨在探究如何提高来自奇异梭菌的7α-羟基类固醇脱氢酶（CA 7α-HSDH）的热稳定性。
基于CA 7α-HSDH的三维结构，使用最近报道的MAESTRO程序计算ΔΔG并预测可提高其热稳定性的单点突变体。对所选突变体进行实验验证。
圆二色光谱结果表明，N89L、N184I和A185I这三个突变体符合三态模型，并且Tm N→I和Tm I→D的值在不同范围内增加。特别是，N184I突变体的Tm N→I最大增加了9.93°C。同时，G189I突变体的变性过程符合二态模型，从变性曲线判断，它比野生型更稳定。然而，酶催化活性分析表明，与野生型CA 7α-HSDH相比，只有N89L突变体具有2.28%的催化效率，其他三个突变体的活性未被检测到。进行分子动力学（MD）模拟以确定突变中发生的结构变化，结果表明无活性的突变体中的β-折叠结构发生了显著变化。
从突变位点的位置判断，β-折叠核心中的残基被认为是增强热稳定性但不保持活性的SDR工程的理想候选位点。
