Purification and properties of three kinds of α-hydroxysteriod dehydrogenases from Brevibacterium fuscum DC33

Abstract

The cell-free extract of Brevibacterium fuscum DC33 contained three kinds of hydroxysteriod dehydrogenase (3a-, 7a-, and 12a-hydroxysteriod dehydrogenases). 7a-Hydroxysteroid dehydrogenase (EC 1.1.1.59) was purified to electrophoretical homogeneity by ion exchange chromatography, affinity chromatography, and preparative electrophoresis. Its molecular weight was 104, 000 and the enzyme was composed of four identical subunits. The enzyme had an optimum pH of 5.3 for dehydrocholic acid reduction, and around 10 for cholic acid oxidation. It was stable in a pH range of 5.7 to 10.5 at 5°C overnight. The enzyme was most active at 25° to 30°C. The activity was not affected by incubation at 30°C for 30 min, but it was lost at 40°C for 30 min. Withe the assumption of two-substrate kinetics, we calculated various kinetic constants for dehydrocholic acid, 7, 12-diketolithocholic acid, 12-ketochenodeoxycholic acid, and 3, 12-diketolithocholic acid (for the structure of bile acids, see Table 2) together with NAD⁺ or NADH. The enzyme was active only toward hydroxysteroids with a 7a-hydroxyl group. The production of 7-ketochenodeoxycholic acid from cholic acid and of 3, 12-diketolithocholic acid from dehydrocholic acid by the purified 7a-hydroxysteroid dehydrogenase was confirmed by thin-layer chromatography.

12a-Hydroxysteroid dehydrogenase was purified by a similar method. It was active toward hydroxysteroids with a 12a-hydroxyl group.

3a-Hydroxysteroid dehydrogenase was purified by preparative electrophoresis. It was active toward hydroxysteroids with a 3a-hydroxyl group.