Molecular determinants of steroid recognition and catalysis in aldo-keto reductases. Lessons from 3 alpha-hydroxysteroid dehydrogenase

Hydroxysteroid Dehydrogenases (HSDs) regulate the occupancy of steroid horm one receptors by converting active steroid hormones into their cognate inac tive metabolites. HSDs belong to either the Short-chain Dehydrogenase/Reduc tases (SDRs) or the Aldo-Keto Reductases (AKRs). The AKRs include virtually all mammalian 3 alpha-HSDs, Type 5 17 beta-HSD, ovarian 20 alpha-HSDs as w ell as the steroid 5 beta-reductases. Selective inhibitors of 3 alpha-HSD i soforms could control occupancy of the androgen and GABAA receptors, while broader based AKR inhibitors targeting 3 alpha-HSD, 20 alpha-HSD and prosta glandin F-2 alpha synthase could maintain pregnancy. We have determined thr ee X-ray crystal structures of rat liver 3 alpha-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 bei ng 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) part icipates in a 'proton-relay' in which Tyr55 acts as general acid/base catal yst. Its bifunctionality relies on contributions from His117 and Lys84 whic h alter the pK(b) and pK(a), respectively of this residue. Point mutation o f the tetrad results in different enzymatic activities. H117E mutants displ ay 5 beta-reductase activity while Y55F and Y55S mutants retain quinone red uctase activity. Our results suggest that different transition states are i nvolved in these reaction mechanisms. The ternary complex structure shows t hat the mature steroid binding pocket is comprised of ten residues recruite d from five loops, and that there is significant movement of a C-terminal l oop on binding ligand. Mutagenesis of pocket tryptophans shows that steroid substrates and classes of nonsteroidal inhibitors exhibit different bindin g modes which may reflect ligand-induced loop movement. Exploitation of the se findings using steroidal and nonsteroidal mechanism based inactivators m ay lead to selective and broad based AKR inhibitors. (C) 1999 Elsevier Scie nce Ltd. All rights reserved.