Cloning of a cDNA for Liver Microsomal Retinol Dehydrogenase

Abstract

Retinoic acid, a hormone biosynthesized from retinol, controls numerous biological systems by regulating eukaryotic gene expression from conception through death. This work reports the cloning and expression of a liver cDNA encoding a microsomal retinol dehydrogenase (RoDH), which catalyzes the primary and rate-limiting step in retinoic acid synthesis. The predicted amino acid sequence and biochemical data obtained from the recombinant enzyme verify it as a short-chain alcohol dehydrogenase. Like microsomal RoDH, the recombinant enzyme recognized as substrate retinol bound to cellular retinol-binding protein, had higher activity with NADP rather than NAD, was stimulated by ethanol or phosphatidylcholine, was not inhibited by 4-methylpyrazole, was inhibited by phenylarsine oxide and carbenoxolone and localized to microsomes. RoDH recognized the physiological form of retinol, holocellular retinol-binding protein, with a Km of 0.9 microM, a value lower than the approximately 5 microM concentration of holocellular retinol binding protein in liver. Northern and Western blot analyses revealed RoDH expression only in rat liver, despite enzymatic activity in liver, brain, kidney, lung, and testes. These data suggest that tissue-specific isozyme(s) of short chain alcohol dehydrogenases catalyze the first step in retinoic acid biogenesis and further strengthen the evidence that the "cassette" of retinol bound to cellular retinol-binding protein serves as a physiological substrate.

cDNA Cloning and Characterization of a New Human Microsomal NAD+-dependent Dehydrogenase that Oxidizes All-trans-retinol and 3α-Hydroxysteroids

“…2). A very strong signal was observed in human liver, similar to the expression patterns of rat all-trans-retinol dehydrogenases (3,4,12). In addition, relatively high levels of hybridizing message were detected in fetal liver and lung (Fig.…”

“…32 P]dATP-labeled coding region of rat RoDH-1 prepared by PCR amplification of the rat liver mRNA with gene-specific primers designed according to a published sequence (3). The hybridization conditions were as follows: 25% formamide, 5ϫ Denhardt's solution (0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400), 5ϫ saline-sodium-phosphate-EDTA, 0.1 mg/ml salmon sperm DNA, and 0.1% SDS at 42°C overnight.…”

“…Recently, this family of enzymes has expanded to include the retinol-oxidizing dehydrogenases (2)(3)(4)(5)(6). Retinol dehydrogenases are involved in the biosynthesis of all-trans-retinoic acid, the activating ligand for a family of nuclear receptors (7).…”

Identification of the reactive cysteine residue (Cys227) in human carbonyl reductase

“…Members of the mammalian branch of the evolutionary tree include, in addition to carbonyl reductase, various dehydrogenases and reductases involved in the metabolism of prostaglandins, steroids, pterins and retinol ( [8] and references therein [24]). Typically, residue identities between the different enzymes are 20±30%, indicating early deviation from a common origin.…”

Retinoid activation of retinoic acid receptor but not retinoid X receptor is sufficient to rescue lethal defect in retinoic acid synthesis

“…Biochemical studies indicate that both isomers of RA can be generated in vitro from vitamin A (retinol) by cytosolic alcohol dehydrogenases (21) and microsomal short-chain dehydrogenases (22,23), which each catalyze oxidation of all-trans-retinol and 9-cis-retinol to the corresponding retinaldehyde derivatives, followed by cytosolic retinaldehyde dehydrogenase (Raldh), which catalyzes further oxidation of both all-trans-retinaldehyde and 9-cis-retinaldehyde to produce all-trans-RA or 9-cis-RA (24,25). Mouse genetic studies support a role in RA synthesis for alcohol dehydrogenase genes Adh1, Adh3, and Adh4 (26,27) as well as Raldh genes Raldh2 (Aldh1a2) (28,29) and Raldh1 (Aldh1a1) (30).…”