A novel NAD(H)-dependent 3alpha-HSDH with enhanced activity by magnesium or manganese ions

3α-Hydroxysteroid dehydrogenase (3α-HSDH) plays a crucial role in the metabolism of sex hormones and bile acids. In this study, we heterologously expressed and characterized a novel 3α-HSDH (named Sa 3α-HSDH). Substrate specificity tests showed that Sa 3α-HSDH could catalyze Glycochenodeoxycholic acid (GCDCA) and Glycoursodeoxycholic acid (GUDCA) with catalytic efficiency (kcat/Km) 40.815 and 14.616 s-1 mM-1, respectively. Sa 3α-HSDH is NAD(H) dependent according to the results of coenzyme screening, and one of mesophilic enzymes with optimum temperature 40 °C. Additionally, Sa 3α-HSDH displayed the highest activity at pH 8.5. In this study, effect of metal ions on activity was investigated, and the results showed Mn2+ (10 mM) and Mg2+ (50 mM) could significantly enhance the activity by nearly 140% and 100%, respectively. Fe2+, Cu2+, Fe3+ and K+ could enhance the activity of Sa 3α-HSDH at different levels. Meanwhile, Na+ only displayed activity-declining effect. The three-dimensional structure of Sa 3α-HSDH was predicted and displayed the well-conserved α/β folding patterns (Rossman-fold) with a central β-sheet. These results indicated that Sa 3α-HSDH would contribute to the quantitative determination of serum total bile acids and associated bioconversion.