| Author/Editor | Cassetta, Alberto; Krastanova, Ivet; Kristan, Katja; Brunskole, Mojca; Lamba, Doriano; Lanišnik-Rižner, Tea; Stojan, Jure | |
| Title | Insights into subtle conformational differences in the substrate-binding loop of fungal 17beta-hydroxysteroid dehydrogenase: a combined structural and kinetic approach | |
| Type | članek | |
| Source | Biochem J | |
| Vol. and No. | Letnik 441, št. 1 | |
| Publication year | 2012 | |
| Volume | str. 151-60 | |
| Language | eng | |
| Abstract | 17beta-Hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17beta-HSDcl) is a NADP(H)-dependent enzyme that preferentially catalyzes the interconversion of inactive 17-keto-steroids and their active 17beta-hydroxy counterparts. 17beta-HSDcl belongs to the short-chain dehydrogenase/ reductase superfa mily (SDR). It is currently the only fungal HSD member that has been described and represents one of the model enzymes of the cP1 classical subfamily of NADPH dependent SDR enzymes. A thorough crystallographic analysis has been performed to better understand the structural aspects of this subfamily and provided insights into the evolution of the HSD enzymes. The crystal structures of the 17beta-HSDcl apo, holo and coumestrol-inhibited ternary complex, and the active site Y167F mutant reveal subtle conformational differences in the substrate-binding loop that likely modulate the catalytic activity of 17beta-HSDcl. Coumestrol, a plant-derived non-steroidal compound with estrogenic activity, inhibits 17beta-HSDcl (IC50, 2.8 mM; at 100 muM substrate [4-estrene-3,17-dione]) by occupying the putative steroid-binding site. In addition to an extensive hydrogen-bonding network, coumestrol binding is further stabilized by pi-pi stacking interactions with Tyr-212. A stopped-flow kinetic experiment clearly showed the coenzyme dissociation as the slowest step of the reaction and in addition to the low steroid solubility it prevents the accumulation of enzyme-coenzyme-steroid ternary complex formation. | |
| Descriptors | ASCOMYCETES 17-HYDROXYSTEROID DEHYDROGENASES NADPH DEHYDROGENASE RECOMBINANT PROTEINS BINDING SITES KINETICS ESTROGENS SUBSTRATE SPECIFICITY PROTEIN CONFORMATION COENZYMES CRYSTALLOGRAPHY ESCHERICHIA COLI |