Molecular framework of steroid/retinoid discrimination in 17β-hydroxysteroid dehydrogenase type 1 and photoreceptor-associated retinol dehydrogenase.

Steroids and retinoids are signaling molecules that control a variety of physiological processes. 17beta-Hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyzes the reduction of estrone to estradiol, supplying biologically active estrogen-regulating sex-specific differentiation. Photoreceptor-associated retinol dehydrogenase (prRDH) is evolutionarily closely related to 17beta-HSD1 but reduces all-trans retinal to all-trans retinol, contributing to rhodopsin regeneration in the visual cycle. Sequence alignment revealed a new enzyme-specific conserved amino acid close to the active site: methionine (position 144 in human enzyme) in prRDH and glycine (position 145) in 17beta-HSD1. We investigated the role of this residue in substrate discrimination in human and zebrafish enzymes. Both recombinant enzymes were expressed in HEK 293 cells followed by normalization of expression by semiquantitative Western blots. Changing of the prRDH-specific methionine to glycine resulted in a gain of function: the mutants now catalyzed the reduction of estrone and all-trans retinal. Human and zebrafish wild-type 17beta-HSD1s efficiently catalyzed the reduction of all-trans retinal to its alcohol. Exchange of glycine for methionine increased the catalytic activity of 17beta-HSD1 toward all-trans retinal in zebrafish but not in the human enzyme, in which the opposite effect was observed. Molecular modeling showed that the zebrafish 17beta-HSD1 substrate-binding pocket is similar to that of prRDH and methionine insertion benefits all-trans retinal reduction. In contrast, in human 17beta-HSD1, the insertion of the bulky methionine causes a disruption of substrate-binding site. We demonstrate for the first time the role of a single amino acid in the evolution of these functionally diverse enzymes and suggest new physiological functions for 17beta-HSD1 in retinoid metabolism. This has implications for the validation of inhibitors of 17beta-HSD1 developed for cancer treatment