Mechanism of inhibition of 3α,20β-hydroxysteroid dehydrogenaseby a licorice-derived steroidal inhibitor

Background: Bacterial 3α, 20β-hydroxysteroid dehydrogenase (3α, 20β-HSD) reversibly oxidizes the 3α and 20β hydroxyl groups of androstanes and pregnanes and uses nicotinamide adenine dinucleotide as a cofactor. 3α, 20β-HSD belongs to a family of short-chain dehydrogenases that has a highly conserved Tyr-X-X-X-Lys sequence. The family includes mammalian enzymes involved in hypertension, digestion, fertility and sperm atogenesis. Several members of the enzyme family, including 3α, 20β-HSD, are competitively inhibited by glycyrrhizic acid, a steroidal compound found in licorice, and its derivative, carbenoxolone, ananti-inflammatory glucocorticoid. Results The three-dimensional structure of the enzyme-carbenoxolone complex has been determined and refined at 2.2 å resolution to a crystallographic R-factor of 19.4%. The hemisuccinate side chain of carbenoxolone 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 catalytic sites refine to 100%, 95%, 54% and 36%. Conclusion The steroid binds at the catalytic site in a mode much like the previously proposed mode of binding of the substrate cortisone. No bound cofactor molecules were found. The varying occupancy of steroid molecules observed in the four catalytic sites is either due to packing differences or indicates a cooperative effect among the four sites. The observed binding accounts for the inhibition of 3α,20β-HSD.