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 (k/K) 40.815 and 14.616 s mM, 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 Mn (10 mM) and Mg (50 mM) could significantly enhance the activity by nearly 140% and 100%, respectively. Fe, Cu, Fe 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.