Purification, reconstitution, and steady-state kinetics of the trans-membrane 17β-hydroxysteroid dehydrogenase 2

Abstract

Human membrane 17β-hydroxysteroid dehydrogenase 2 is an enzyme essential in the conversion of the highly active 17β-hydroxysteroids into their inactive keto forms in a variety of tissues. 17β-hydroxysteroid dehydrogenase 2 with 6 consecutive histidines at its N terminus was expressed in Sf9 insect cells. This recombinant protein retained its biological activity and facilitated the enzyme purification and provided the most suitable form in our studies. Dodecyl-β-D-maltoside was found to be the best detergent for the solubilization, purification, and reconstitution of this enzyme. The overexpressed integral membrane protein was purified with a high catalytic activity and a purity of more than 90% by nickel-chelated chromatography. For reconstitution, the purified protein was incorporated into dodecyl-β-D-maltoside-destabilized liposomes prepared from L-α-phosphatidylcholine. The detergent was removed by adsorption onto polystyrene beads. The reconstituted enzyme had much higher stability and catalytic activity (2.6 μmol/min/mg of enzyme protein with estradiol) than the detergent-solubilized and purified protein (0.9 μmol/min/mg of enzyme protein with estradiol). The purified and reconstituted protein (with a 2-kDa His tag) was, proved to be a homodimer, and its functional molecular mass was calculated to be 90.4 ± 1.2 kDa based on glycerol gradient analytical ultracentrifugation and chemical cross-linking study. The kinetic studies demonstrated that 17β-hydroxysteroid dehydrogenase 2 was an NAD-preferring dehydrogenase with the Km of NAD being 110 ± 10 μM and that of NADP 9600 ± 100 μM using estradiol as substrate. The kinetic constants using estradiol, testosterone, dihydrotestosterone, and 20α-dihydroprogesterone as substrates were also determined.