PUBLICATION
Molecular framework of steroid/retinoid discrimination in 17beta-hydroxysteroid dehydrogenase type 1 and photoreceptor-associated retinol dehydrogenase
- Authors
- Haller, F., Moman, E., Hartmann, R.W., Adamski, J., and Mindnich, R.
- ID
- ZDB-PUB-100420-12
- Date
- 2010
- Source
- Journal of molecular biology 399(2): 255-267 (Journal)
- Registered Authors
- Keywords
- 17β-hydroxysteroid dehydrogenase, photoreceptor associated retinol dehydrogenase, mutagenesis, evolution, protein multifunctionality
- MeSH Terms
-
- 17-Hydroxysteroid Dehydrogenases/genetics
- 17-Hydroxysteroid Dehydrogenases/metabolism*
- Alcohol Oxidoreductases/genetics
- Alcohol Oxidoreductases/metabolism*
- Amino Acid Sequence
- Amino Acid Substitution
- Animals
- Blotting, Western
- Catalytic Domain
- Cell Line
- Gene Expression
- Humans
- Models, Molecular
- Molecular Sequence Data
- Mutagenesis, Site-Directed
- Mutant Proteins/metabolism
- Retinoids/metabolism*
- Sequence Alignment
- Steroids/metabolism*
- Substrate Specificity
- Zebrafish
- PubMed
- 20382160 Full text @ J. Mol. Biol.
Citation
Haller, F., Moman, E., Hartmann, R.W., Adamski, J., and Mindnich, R. (2010) Molecular framework of steroid/retinoid discrimination in 17beta-hydroxysteroid dehydrogenase type 1 and photoreceptor-associated retinol dehydrogenase. Journal of molecular biology. 399(2):255-267.
Abstract
Steroids and retinoids are signaling molecules that influence a variety of physiological processes. 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyzes the reduction of estrone to estradiol, thereby supplying a biologically active estrogen that regulates sex-specific tissue differentiation. Photoreceptor-associated retinol dehydrogenase (prRDH) is evolutionarily closely related to 17beta-HSD1 but reduces all-trans retinal to all-trans retinol, a function important for rhodopsin regeneration in the visual cycle. Multiple sequence alignment revealed a new enzyme-specific amino acid close to the active site that is highly conserved: a methionine (position 144 in human enzyme) in prRDH and a glycine (position145) in 17beta-HSD1. We investigated the role of this conserved residue in substrate discrimination in the human and zebrafish enzymes using mutagenesis, determination of steady state kinetic constants and molecular modeling. Both recombinant enzymes were expressed in HEK 293 cells followed by normalization of expression levels by semi-quantitative western blots.. Exchange of the prRDH specific methionine to glycine resulted in a gain of function: the mutants, in contrast to the wild type (wt) now catalyzed the reduction of estrone in addition to all-trans retinal. We show that both wt (zebrafish and human) 17beta-HSD1 efficiently catalyzed the reduction of all-trans retinal to its alcohol. Exchange of glycine to methionine increased catalytic activity of 17beta-HSD1 towards all-trans retinal in the zebrafish but not 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 consequently methionine insertion benefits all-trans retinal reduction. In contrast, in human 17beta-HSD1 insertion of the bulky methionine causes a structural disruption of the substrate binding site. Our findings illustrate 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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping