Steroid recognition and engineering of catalysis in mammalian aldo-keto reductases

Of the steroid transforming aldo-keto reductases (AKRs), rat liver 3{dollar}alpha{dollar}-hydroxysteroid dehydrogenase (3{dollar}alpha{dollar}-HSD) is the best studied and provides a model for understanding protein-steroid interactions. Initially the location of the steroid binding site in rat liver 3{dollar}alpha{dollar}-HSD was unknown. Through site-directed mutagenesis of the tryptophans in 3{dollar}alpha{dollar}-HSD and analysis of the mutants by steady-state kinetics and protein fluorescence, we proposed that Trp86 and Trp227 form opposite sides of the binding site. Based on this study, a model for steroid binding in 3{dollar}alpha{dollar}-HSD was proposed and subsequently confirmed when the structure of the 3{dollar}alpha{dollar}-HSD{dollar}cdot{dollar}NADP{dollar}sp+cdot{dollar}testosterone ternary complex was determined. This model may be applicable to other steroid transforming AKRs, including other 3{dollar}alpha{dollar}-, 17{dollar}beta{dollar}-, and 20{dollar}alpha{dollar}-HSDs and the {dollar}Deltasp4{dollar}-3-ketosteroid-5{dollar}beta{dollar}-reductases.; Using both structural and sequence comparisons as a guide, {dollar}Deltasp4{dollar}-3-ketosteroid-5{dollar}beta{dollar}-reductase activity was engineered into rat liver 3{dollar}alpha{dollar}-HSD by mutating His117 of the catalytic tetrad into a glutamic acid. Our results demonstrate that the AKRs use the same general acid (Tyr55) for catalyzing the reduction of both ketones and carbon-carbon double bonds. The identity of amino acid 117 can determine if an AKR functions as an oxidoreductase and/or a double bond reductase. This result has implications for how Nature evolves new enzymatic activities.; In endocrine target tissues, 3{dollar}alpha{dollar}-HSD may function as a molecular switch by interconverting active steroid hormones with their cognate inactive metabolites. We cloned, expressed, and characterized the type 2 3{dollar}alpha{dollar}-HSD isoform from a human prostate cDNA library. The identification of dual 3{dollar}alpha{dollar}-/17{dollar}beta{dollar}-HSD activity in this enzyme demonstrates that the steroid binding site accommodates substrate in two orientations. Also, the bifunctional activity allows this enzyme to remove the potent androgen 5{dollar}alpha{dollar}-DHT from the prostatic androgen pool. However, it cannot catalyze the formation of 5{dollar}alpha{dollar}-DHT and does not act as a molecular switch. This work suggests that different 3{dollar}alpha{dollar}-HSD isoforms may have distinct roles in controlling androgen levels in the prostate.