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cDNA cloning, tissue distribution, and substrate characteristics of a cis-Retinol/3alpha-hydroxysterol short-chain
1Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214, USA.
Abstract:
We report here a mouse cDNA that encodes a 316-amino acid short-chain dehydrogenase that prefers NAD+ as its cofactor and recognizes as substrates androgens and retinols, i.e. has steroid 3alpha- and 17beta-dehydrogenase and cis/trans-retinol catalytic activities. This cis-retinol/androgen dehydrogenase type 2 (CRAD2) shares close amino acid similarity with mouse retinol dehydrogenase isozyme types 1 and 2 and CRAD1 (86, 84, and 87%, respectively). CRAD2 exhibits cooperative kinetics with 3alpha-adiol (3alpha-hydroxysteroid dehydrogenase activity) and testosterone (17beta-hydroxysteroid dehydrogenase activity), but Michaelis-Menten kinetics with androsterone (3alpha-hydroxysteroid dehydrogenase activity), 11-cis-retinol, all-trans-retinol, and 9-cis-retinol, with V/K0.5 values of 1.6, 0.2, 0.1, 0.04, 0.005, and not saturated, respectively. Carbenoxolone (IC50 = 2 microM) and 4-methylpyrazole (IC50 = 5 mM) inhibited CRAD2, but neither ethanol nor phosphatidylcholine had marked effects on its activity. Liver expressed CRAD2 mRNA intensely, with expression in lung, eye, kidney, and brain (2.9, 2, 1.6, and 0.6% of liver mRNA, respectively). CRAD2 represents the fifth isozyme in a group of short-chain dehydrogenase/reductase isozymes (retinol dehydrogenases 1-3 and CRAD1), closely related in primary amino acid sequence (approximately 85%), that are expressed in different quantities in various tissues, have different substrate specificities, and may serve different physiological functions. CRAD2 may alter the amounts of active and inactive androgens and/or convert retinols into retinals. These data expand insight into the multifunctional nature of short-chain dehydrogenases/reductases and into the enzymology of steroid and retinoid metabolism.
Insights
A newly identified enzyme, cis-retinol/androgen dehydrogenase type 2 (CRAD2), demonstrates dual activity in metabolizing androgens and retinols. This discovery enhances understanding of short-chain dehydrogenases/reductases and their roles in steroid and retinoid pathways.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Short-chain dehydrogenases/reductases (SDRs) are a diverse enzyme family involved in various metabolic processes.
- Steroid and retinoid metabolism are crucial for numerous physiological functions, including hormone regulation and vision.
Purpose of the Study:
- To identify and characterize a novel mouse cDNA encoding a short-chain dehydrogenase with dual substrate specificity.
- To elucidate the enzymatic activities, kinetic properties, and tissue expression patterns of the newly identified enzyme, designated cis-retinol/androgen dehydrogenase type 2 (CRAD2).
Main Methods:
- cDNA cloning and sequencing to identify the gene encoding CRAD2.
- Enzyme activity assays to determine substrate specificity (androgens and retinols) and cofactor preference (NAD+).
- Kinetic analysis (cooperative and Michaelis-Menten kinetics) and inhibitor studies (carbenoxolone, 4-methylpyrazole).
- Quantitative analysis of CRAD2 mRNA expression in various mouse tissues using RT-PCR.
Main Results:
- A mouse cDNA encoding a 316-amino acid SDR, CRAD2, was identified.
- CRAD2 exhibits both steroid 3alpha- and 17beta-dehydrogenase activities, along with cis/trans-retinol dehydrogenase activities.
- CRAD2 shares high amino acid similarity (84-87%) with known mouse retinol dehydrogenase isozymes and CRAD1.
- The enzyme displayed cooperative kinetics with 3alpha-adiol and testosterone, and Michaelis-Menten kinetics with other substrates.
- CRAD2 expression was highest in the liver, with detectable levels in lung, eye, kidney, and brain.
- Carbenoxolone and 4-methylpyrazole inhibited CRAD2 activity.
Conclusions:
- CRAD2 is a novel multifunctional enzyme with significant roles in both androgen and retinol metabolism.
- Its distinct kinetic properties and tissue-specific expression suggest specialized physiological functions within the SDR family.
- CRAD2 contributes to the understanding of the complex enzymology of steroid and retinoid metabolism and the diverse roles of SDRs.