Abstract
NAD(H)-dependent 7α-hydroxysteroid dehydrogenase catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid. Here, we designed mutations of Ile258 adjacent to the catalytic pocket of Brucella melitensis 7α-hydroxysteroid dehydrogenase. The I258M variant gave a 4.7-fold higher kcat, but 4.5-fold lower KM, compared with the wild type, resulting in a 21.8-fold higher kcat/KM value for chenodeoxycholic acid oxidation. It presented a 2.0-fold lower KM value with NAD+, suggesting stronger binding to the cofactor. I258M produced 7-oxolithocholic acid in the highest yield of 92.3% in 2 h, whereas the wild-type gave 88.4% in 12 h. The I258M mutation increased the half-life from 20.8 to 31.1 h at 30 °C. Molecular dynamics simulations indicated increased interactions and a modified tunnel improved the catalytic efficiency, and enhanced rigidity at three regions around the ligand-binding pocket increased the enzyme thermostability. This is the first report about significantly improved catalytic efficiency, cofactor affinity, and enzyme thermostability through single site-mutation of Brucella melitensis 7α-hydroxysteroid dehydrogenase.
Key points
• Sequence and structure analysis guided the site mutation design.
• Thermostability, catalytic efficiency and 7-oxo-LCA production were determined.
• MD simulation was performed to indicate the improvement by I258M mutation.
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Acknowledgements
We are grateful to the Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, Jiangnan University in encouraging us for the studies.
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This project was supported by the National Key research and Development Program of China (2018YFA0900302), the National Science Foundation of China (31970045), Postgraduate Research & Practice Innovation Program of Jiangsu Province (1012050205205974), the National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-12), the Program of Introducing Talents of Discipline to Universities (111-2-06), and Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
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All authors have their own contributions to this work. The first author, ZL, was in charge of the study including writing the manuscript. Dr. WZ and Dr. YX have been providing valuable advices. Dr. RZ guided the project and revised the manuscript.
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Liu, Z., Zhang, R., Zhang, W. et al. Ile258Met mutation of Brucella melitensis 7α-hydroxysteroid dehydrogenase significantly enhances catalytic efficiency, cofactor affinity, and thermostability. Appl Microbiol Biotechnol 105, 3573–3586 (2021). https://doi.org/10.1007/s00253-021-11299-7
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DOI: https://doi.org/10.1007/s00253-021-11299-7