NAD(H)-dependent 7 alpha-hydroxysteroid dehydrogenase catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid. Here, we designed mutations of Ile258 adjacent to the catalytic pocket of Brucella melitensis 7 alpha-hydroxysteroid dehydrogenase. The I258M variant gave a 4.7-fold higher k(cat), but 4.5-fold lower K-M, compared with the wild type, resulting in a 21.8-fold higher k(cat)/K-M value for chenodeoxycholic acid oxidation. It presented a 2.0-fold lower K-M value with NAD(+), suggesting stronger binding to the cofactor. I258M produced 7-oxolithocholic acid in the highest yield of 92.3% in 2 h, whereas the wild-type gave 88.4% in 12 h. The I258M mutation increased the half-life from 20.8 to 31.1 h at 30 degrees C. Molecular dynamics simulations indicated increased interactions and a modified tunnel improved the catalytic efficiency, and enhanced rigidity at three regions around the ligand-binding pocket increased the enzyme thermostability. This is the first report about significantly improved catalytic efficiency, cofactor affinity, and enzyme thermostability through single site-mutation of Brucella melitensis 7 alpha-hydroxysteroid dehydrogenase.