Design of St-2-2 7α-HSDH mutants for altering substrate preference and thermostability

Abstract

Tauroursodeoxycholic acid (TUDCA) is the main active ingredient in bear bile powder. 7α-hydroxysteroid dehydrogenase (7α-HSDH), which selectively catalyzes the oxidation of taurochenodeoxycholic acid (TCDCA) to produce TUDCA is important when using chicken bile powder as a substrate. St-2-2 is an NADP(H)-dependent 7α-HSDH with higher activity on taurocholic acid (TCA) than on TCDCA. Here, we designed mutants of St-2-2 to alter the substrate preference (from TCA to TCDCA). Interestingly, mutant I255G had 3.38-fold increased catalytic activity toward TCDCA compared with St-2-2 and decreased catalytic activity to 4.6% that of St-2-2 toward TCA. In addition, I255T had higher thermostability. For TCDCA, I255G and I255T had lower KM values (0.07 mM, 0.13 mM), higher kcat values (201.2 s−1, 184.8 s−1), and lower ∆∆G values (-3.80 kJ mol−1, -2.20 kJ mol−1) than those of St-2-2. Increased α-helix content, more interactions between surrounding residues, and lower RMSD values indicated increased thermostability of I255T. Both I255G and I255T had newly formed hydrogen bond interactions between the catalytic center and substrates, suggesting stronger binding to TCDCA thus improving catalytic activity. This is the first report about altering substrate preference, catalytic activity, and thermostability through single-site mutation of 7α-HSDH.