Molecular determinants of steroid recognition and catalysis in aldo-keto reductases. Lessons from 3alpha-hydroxysteroid dehydrogenase.
Hydroxysteroid Dehydrogenases (HSDs) regulate the occupancy of steroid hormone receptors by converting active steroid hormones into their cognate inactive metabolites. HSDs belong to either the Short-chain Dehydrogenase/Reductases (SDRs) or the Aldo-Keto Reductases (AKRs). The AKRs include virtually all mammalian 3alpha-HSDs, Type 5 17beta-HSD, ovarian 20alpha-HSDs as well as the steroid 5beta-reductases. Selective inhibitors of 3alpha-HSD isoforms could control occupancy of the androgen and GABA(A) receptors, while broader based AKR inhibitors targeting 3alpha-HSD, 20alpha-HSD and prostaglandin F2alpha synthase could maintain pregnancy. We have determined three X-ray crystal structures of rat liver 3alpha-HSD, a representative AKR. These structures are of the apoenzyme (E), the binary-complex (E.NADP-), and the ternary complex (E.NADP+.testosterone). These structures are being used with site-directed mutagenesis to define the molecular determinants of steroid recognition and catalysis as a first step in rational inhibitor design. A conserved catalytic tetrad (Tyr55, Lys84, His117 and Asp50) participates in a 'proton-relay' in which Tyr55 acts as general acid/base catalyst. Its bifunctionality relies on contributions from His117 and Lys84 which alter the pKb and pKa, respectively of this residue. Point mutation of the tetrad results in different enzymatic activities. H117E mutants display 5beta-reductase activity while Y55F and Y55S mutants retain quinone reductase activity. Our results suggest that different transition states are involved in these reaction mechanisms. The ternary complex structure shows that the mature steroid binding pocket is comprised of ten residues recruited from five loops, and that there is significant movement of a C-terminal loop on binding ligand. Mutagenesis of pocket tryptophans shows that steroid substrates and classes of nonsteroidal inhibitors exhibit different binding modes which may reflect ligand-induced loop movement. Exploitation of these findings using steroidal and nonsteroidal mechanism based inactivators may lead to selective and broad based AKR inhibitors.