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Effects of terminal modification on the catalytic efficiency and thermostability of Brucella melitensis 7α-hydroxysteroid dehydrogenase

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Abstract

Brucella melitensis 7α-hydroxysteroid dehydrogenase (Bm7α-HSDH) catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid. In this work, we investigated the effects of terminal modification (His-tags location and terminal truncation) on its catalytic efficiency and thermostability. Compared with C-terminal His-tagged Bm7α-HSDH (C-Bm7α-HSDH), N-Bm7α-HSDH showed a 3.6-fold higher kcat and a 1.3-fold lower Km, resulting in a 7.0-fold higher kcat/Km value toward chenodeoxycholic acid. Circular dichroism spectroscopy indicated that the melting temperature of N-Bm7α-HSDH (46.13 °C) was 3.0 °C lower than that of C-Bm7α-HSDH (49.13 °C). N-Bm7α-HSDH produced 7-oxolithocholic acid in the highest yield of 96.7% in 4 h, whereas the C-Bm7α-HSDH gave 96.4% in 10 h. Moreover, amino acids truncation and His-tag cleave experiments confirmed the C-terminal residues played key roles in catalytic functions. Molecular dynamics simulations further indicated C-terminal His-tagged modification could deform the substrate-binding region to disrupt the enzyme–substrate interactions and catalytic motion. However, the N-terminal His-tag hardly affected the catalytic efficiency due to its location far from the active site of the enzyme. This study provides structural insights into the terminus modifications of hydroxysteroid dehydrogenase on steroid substrate recognition and stabilization, thus affecting its catalytic functions.

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Funding

This project was supported by the National Key research and Development Program of China (2018YFA0900302), the National Science Foundation of China (31970045), 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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Authors and Affiliations

  1. Lab of Brewing Microbiology and Applied Enzymology, School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Zhiyong Liu, Rongzhen Zhang & Yan Xu

  2. Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Wenchi Zhang

  3. School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi City, 214122, China

    Rongzhen Zhang

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  1. Zhiyong Liu
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  4. Yan Xu
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Contributions

All authors have their own contributions to this work. Zhiyong Liu is in charge of the study including writing the first draft. Wenchi Zhang and Yan Xu provides valuable advices and helped with data analysis. Rongzhen Zhang guided the project and revised the manuscript.

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Correspondence to Rongzhen Zhang.

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Liu, Z., Zhang, R., Zhang, W. et al. Effects of terminal modification on the catalytic efficiency and thermostability of Brucella melitensis 7α-hydroxysteroid dehydrogenase. Syst Microbiol and Biomanuf 3, 469–478 (2023). https://doi.org/10.1007/s43393-022-00124-5

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