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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Research Article

Expression and Functional Characterization of a Novel NAD(H)- dependent 3α-HSDH

Author(s): Deshuai Lou*, Zixin Zhou, Xiaoli Zhang, Yangyang Cao, Qian Long, Cunhong Luo, Qiang Li, Xi Liu and Jun Tan*

Volume 29, Issue 11, 2022

Published on: 06 October, 2022

Page: [946 - 953] Pages: 8

DOI: 10.2174/0929866529666220822102311

Price: $65

Abstract

Background: 3α-Hydroxysteroid dehydrogenase (3α-HSDH) reversibly catalyzes the oxidation of the C3-hydroxyl group of steroids, and has been used in clinical applications to detect serum total bile acid (TBA). In this study, A novel 3α-HSDH (called Sb 3α-HSDH) was expressed and characterized.

Methods: Plasmid pGEX-6p-1 was used for the expression of Sb 3α-HSDH in Escherichia coli (BL21), and activities were determined by recording the change in absorbance at 340 nm with/without adding of ions. A prediction of its three-dimensional structure was performed with AlphaFold.

Results: The substrate specificity test indicated that Sb 3α-HSDH is NAD(H)-dependent and has no activity with NADP(H). We also showed that Sb 3α-HSDH can catalyze the oxidation reaction of GCDCA and GUDCA with catalytic efficiencies (kcat/Km) of 29.060 and 45.839 s-1mM-1, respectively. The temperature dependence of catalysis suggests that Sb 3α-HSDH is a member of the mesophilic enzymes with its best activity at about 45 °C. The optimum pH of Sb 3α-HSDH was found to be between pH 8.0 and 9.0. The effect of ions, including K+, Mg2+, Na+, Cu2+, Mn2+, Fe2+, and Fe3+ on enzyme activity was evaluated and K+ and Mg2+ were found to enhance the activity of Sb 3α-HSDH by about 20% at concentrations of 200 mM and 50 mM, respectively. The well-conserved GIG motif, the active sites, and the Rossmann fold in the threedimensional structure indicate that Sb 3α-HSDH belongs to the “classical” type of SDR superfamily.

Conclusion: We expressed and characterized a novel NAD(H)-dependent 3α-HSDH with typical threedimensional characteristics of the SDRs that exhibited substrate specificity to GCDCA and GUDCA.

Keywords: 3α-Hydroxysteroid dehydrogenase, Heterologous expression, Biochemical property, Catalytic activity, Short-chain dehydrogenase/reductase, Metal ions

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