Characterization of homogeneous recombinant rat ovarian 20α-hydroxysteroid dehydrogenase: fluorescent properties and inhibition profile

Abstract

In rat ovary, 20α-hydroxysteroid dehydrogenase (20α-HSD), a member of the aldo-keto reductase (AKR) superfamily, converts progesterone into the inactive progestin 20α-hydroxyprogesterone and has been implicated in the termination of pregnancy. Here we report a convenient overexpression system that permits the purification of milligram quantities of homogeneous recombinant 20α-HSD with wild-type enzyme activity. The availability of this enzyme has permitted detailed kinetic, inhibition and fluorescence analyses. The enzyme exhibited narrow steroid specificity, catalysing reactions only at C-20; it reduced progesterone and 17α-hydroxyprogesterone and oxidized 20α-hydroxypregnanes. It also turned over common AKR substrates, such as 9,10-phenanthrenequinone and 4-nitrobenzaldehyde. The intrinsic fluorescence spectrum of 20α-HSD was characterized and was quenched on the binding of NADP(H), yielding a KdNADP of 0.36 μM and a KdNADP of 0.64 μM. NADP(H) binding generated an energy transfer band that could not be quenched by steroids. Inhibition studies conducted with non-steroidal and steroidal anti-inflammatory drugs and synthetic oestrogens indicated that even though rat ovarian 20α-HSD and rat liver 3α-hydroxysteroid dehydrogenase (3α-HSD) share more than 67% amino acid identity, their inhibition profiles are markedly different. Unlike 3α-HSD, most of these compounds did not inhibit 20α-HSD. Only meclofenamic acid and hexoestrol were potent competitive inhibitors for 20α-HSD, yielding Ki values of 18.9 and 14.3 μM respectively. These studies suggest that selective non-steroidal AKR inhibitors could be developed for 20α-HSD that might be useful in maintaining pregnancy and that specific inhibitors might be developed from either N-phenylanthranilates or biphenols.