cDNA cloning and characterization of a new human microsomal NAD+-dependent dehydrogenase that oxidizes all-trans-retinol and 3α-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 (less than or equal to 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 3 alpha-hydroxysteroids androstane-diol and androsterone to dihydrotestosterone and androstanedione, respectively. NAD(+) and NADH are the preferred cofactors, with apparent K-m values 250-1500 times lower than those for NADP(+) and NADPH. AU-trans-retinol and 13-cis-retinol inhibit RoDH-4 catalyzed oxidation of androsterone with apparent K-i values of 5.8 and 3.5 mu M, respectively. All-trans-retinol bound to cellular retinol-binding protein (type I) exhibits a similar K-i value of 3.6 mu M, 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 3 alpha-androstane-diol.