title: Differential recognition of the free versus bound retinol by human microsomal retinol/sterol dehydrogenases: characterization of the holo-CRBP dehydrogenase activity of RoDH-4.
abstract: All-trans-retinol is the precursor for all-trans-retinoic acid, the activating ligand for nuclear transcription factors retinoic acid receptors. In the cytosol of various cells, most retinol exists in a bound form, complexed with cellular retinol binding protein type I (holo-CRBP). Whether retinoic acid is produced from the free or bound form of retinol is not yet clear. Here, we present evidence that holo-CRBP is recognized as substrate by human microsomal short-chain dehydrogenase/reductase (SDR) RoDH-4 with the K(m) value close to the liver concentration of holo-CRBP. The ability to utilize holo-CRBP differentiates RoDH-4 from a related enzyme, RoDH-like 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), which is 3-fold more active with free retinol than RoDH-4 but is 15-fold less active toward holo-CRBP. Recognition of the cytosolic holo-CRBP as substrate is consistent with RoDH-4 orientation in the membrane. As established by immunoprecipitation and glycosylation scanning, RoDH-4 faces the cytosolic side of the membrane. Purified RoDH-4, stabilized by reconstitution into proteoliposomes, exhibits the apparent K(m) values for substrates and NAD(+) similar to those of the microsomal enzyme and oxidizes holo-CRBP with the catalytic efficiency (k(cat)/K(m)) of 59 min(-1) mM(-1). Apo-CRBP acts as a strong competitive inhibitor of holo-CRBP oxidation with an apparent K(i) value of 0.2 microM. The results of this study suggest that the human retinol-active SDRs are not functionally equivalent and that, in contrast to RoDH-like 3alpha-HSD, RoDH-4 can access the bound form of retinol for retinoic acid production and is regulated by the apo-/holo-CRBP ratio.
authors.affiliation: Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110, USA.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Lapshina
authors.forename: Olga V
authors.initials: OV
authors.lastname: Belyaeva
authors.forename: Olga V
authors.initials: OV
authors.lastname: Chumakova
authors.forename: Natalia Y
authors.initials: NY
authors.lastname: Kedishvili
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001667:Binding, Competitive; D002384:Catalysis; D006801:Humans; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012177:Retinol-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D054840:Retinol-Binding Proteins, Cellular; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D014801:Vitamin A / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001059:Apoproteins; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; D014801:Vitamin A; D015096:3-Hydroxysteroid Dehydrogenases; D000429:Alcohol Oxidoreductases; C113992:trans-retinol dehydrogenase; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)