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

In rat ovary, 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD), a membe r of the aldo-keto reductase (AKR) superfamily, converts progesterone into the inactive progestin 20 alpha-hydroxyprogesterone and has been implicated in the termination of pregnancy. Here we report a convenient overexpressio n system that permits the purification of milligram quantities of homogeneo us recombinant 20 alpha-HSD with wild-type enzyme activity. The availabilit y of this enzyme has permitted detailed kinetic, inhibition and fluorescenc e analyses. The enzyme exhibited narrow steroid specificity, catalysing rea ctions only at C-20; it reduced progesterone and 17 alpha-hydroxyprogestero ne and oxidized 20 alpha-hydroxypregnanes. It also turned over common AKR s ubstrates, such as 9,10-phenanthrenequinone and 4-nitrobenzaldehyde, The in trinsic fluorescence spectrum of 20 alpha-HSD was characterized and was que nched on the binding of NADP(H), yielding a K-d(NADP) of 0.36 mu M and a K- d(NADPH) of 0.64 mu M. NADP(H) binding generated an energy transfer band th at could not be quenched by steroids. Inhibition studies conducted with non -steroidal and steroidal anti-inflammatory drugs and synthetic oestrogens i ndicated that even though rat ovarian 20 alpha-HSD and rat liver 3 alpha-hy droxysteroid dehydrogenase (3 alpha-HSD) share more than 67 % amino acid id entity, their inhibition profiles are markedly different. Unlike 3 alpha-HS D, most of these compounds did not inhibit 20 alpha-HSD. Only meclofenamic acid and hexoestrol were potent competitive inhibitors for 20 alpha-HSD, yi elding K-i values of 18.9 and 14.3 mu M respectively. These studies suggest that selective non-steroidal AKR inhibitors could be developed for 20 alph a-HSD that might be useful in maintaining pregnancy and that specific inhib itors might be developed from either N-phenylanthranilates or biphenols.