cDNA cloning and characterization of a new human microsomal NAD+-dependent dehydrogenase that oxidizes all-trans-retinol and 3alpha-hydroxysteroids.
We report the cDNA sequence and catalytic properties of a new member of the short chain dehydrogenase/reductase superfamily. The 1134-base pair cDNA isolated from the human liver cDNA library encodes a 317-amino acid protein, retinol dehydrogenase 4 (RoDH-4), which exhibits the strongest similarity with rat all-trans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3, and mouse cis-retinol/androgen dehydrogenase (</=73% identity). The mRNA for RoDH-4 is abundant in adult liver, where it is translated into RoDH-4 protein, which is associated with microsomal membranes, as evidenced by Western blot analysis. Significant amounts of RoDH-4 message are detected in fetal liver and lung. Recombinant RoDH-4, expressed in microsomes of Sf9 insect cells using BacoluGold Baculovirus system, oxidizes all-trans-retinol and 13-cis-retinol to corresponding aldehydes and oxidizes the 3alpha-hydroxysteroids androstane-diol and androsterone to dihydrotestosterone and androstanedione, respectively. NAD+ and NADH are the preferred cofactors, with apparent Km values 250-1500 times lower than those for NADP+ and NADPH. All-trans-retinol and 13-cis-retinol inhibit RoDH-4 catalyzed oxidation of androsterone with apparent Ki values of 5.8 and 3.5 microM, respectively. All-trans-retinol bound to cellular retinol-binding protein (type I) exhibits a similar Ki value of 3.6 microM. Unliganded cellular retinol-binding protein has no effect on RoDH activity. Citral and acyclic isoprenoids also act as inhibitors of RoDH-4 activity. Ethanol is not inhibitory. Thus, we have identified and characterized a sterol/retinol-oxidizing short chain dehydrogenase/reductase that prefers NAD+ and recognizes all-trans-retinol as substrate. RoDH-4 can potentially contribute to the biosynthesis of two powerful modulators of gene expression: retinoic acid from retinol and dihydrotestosterone from 3alpha-androstane-diol.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
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Kedishvili
D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D012172:Retinaldehyde / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013256:Steroids / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D013256:Steroids; D009243:NAD; D000429:Alcohol Oxidoreductases; C113992:trans-retinol dehydrogenase; D012172:Retinaldehyde