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

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.