MECHANISM OF INHIBITION OF 3-ALPHA,20-BETA-HYDROXYSTEROID DEHYDROGENASE BY A LICORICE-DERIVED STEROIDAL INHIBITOR

Background: Bacterial 3 alpha,20 beta-hydroxysteroid dehydrogenase (3 alpha,20 beta-HSD) reversibly oxidizes the 3 alpha and 20 beta hydroxy l groups of androstanes and pregnanes and uses nicotinamide adenine di nucleotide as a cofactor. 3 alpha,20 beta-HSD belongs to a family of s hort-chain dehydrogenases that has a highly conserved Tyr-X-X-X-Lys se quence. The family includes mammalian enzymes involved in hypertension , digestion, fertility and spermatogenesis. Several members of the enz yme family, including 3 alpha,20 beta-HSD, are competitively inhibited by glycyrrhizic acid, a steroidal compound found in licorice, and its derivative, carbenoxolone, an anti-inflammatory glucocorticoid. Resul ts: The three-dimensional structure of the enzyme-carbenoxolone comple x has been determined and refined at 2.2 Angstrom resolution to a crys tallographic R-factor of 19.4%. The hemisuccinate side chain of carben oxolone makes a hydrogen bond with the hydroxyl group of the conserved residue Tyr152 and occupies the position of the nicotinamide ring of the cofactor. The occupancies of the inhibitor in four independent cat alytic sites refine to 100%, 95%, 54% and 36%. Conclusions: The steroi d binds at the catalytic site in a mode much like the previously propo sed mode of binding of the substrate cortisone. No bound cofactor mole cules were found. The varying occupancy of steroid molecules observed in the four catalytic sites is either due to packing differences or in dicates a cooperative effect among the four sites. The observed bindin g accounts for the inhibition of 3 alpha,20 beta-HSD.