Publications

• Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids.

  Gallego O., Belyaeva O.V., Porte S., Ruiz F.X., Stetsenko A.V., Shabrova E.V., Kostereva N.V., Farres J., Pares X., Kedishvili N.Y.

  Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto ... >> More

  Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo. << Less

  Biochem. J. 399:101-109(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Further characterization of human microsomal 3alpha-hydroxysteroid dehydrogenase.

  Chetyrkin S.V., Hu J., Gough W.H., Dumaual N., Kedishvili N.Y.

  This manuscript reports further characterization of the recently discovered human short-chain alcohol dehydrogenase, proposed to oxidize 3alpha-androstanediol to dihydrotestosterone in testis and prostate (M. G. Biswas and D. W. Russell, 1997, J. Biol. Chem. 272, 15959-15966). Enzyme expressed usi ... >> More

  This manuscript reports further characterization of the recently discovered human short-chain alcohol dehydrogenase, proposed to oxidize 3alpha-androstanediol to dihydrotestosterone in testis and prostate (M. G. Biswas and D. W. Russell, 1997, J. Biol. Chem. 272, 15959-15966). Enzyme expressed using the Baculovirus System localized in the microsomal fraction and catalyzed oxidation and reduction of the functional groups on steroids at carbons 3 and 17. Autoradiography assays revealed that the enzyme was most efficient as a 3alpha-hydroxysteroid oxidoreductase. High affinity of the enzyme for NADH (Km of 0.18 microM), lack of stereospecificity in the reductive direction, and poor efficiency for 3beta-versus 3alpha-hydroxyl oxidation could account for the observed transient accumulation of 3beta-stereoisomers in the oxidative reaction. Consistent with the 65% sequence identity with RoDH dehydrogenases, the enzyme oxidized all-trans-retinol with the Km value of 3.2 microM and Vmax value of 1.2 nmol/min per milligram microsomes. 13-cis-Retinol and all-trans-retinol bound to the cellular retinol-binding protein were not substrates. Neurosteroid allopregnanolone was a better substrate than all-trans-retinol with the Km and Vmax values of 0.24 microM and 14.7 nmol/min per milligram microsomes. Northern blot analysis revealed that the corresponding mRNA was present in adult human brain (caudate nucleus, amygdala, hippocampus, substantia nigra, thalamus) and spinal cord in addition to other tissues. The message was also detected in fetal lung, liver, and brain. Antibodies against the enzyme recognized a protein of approximately 35 kDa in the particulate fraction of human tissues. This study presents new information about enzymatic properties, substrate specificity, and tissue distribution of this enzyme, and provides a better insight into its possible physiological function(s). << Less

  Arch. Biochem. Biophys. 386:1-10(2001) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Retinoids, omega-hydroxyfatty acids and cytotoxic aldehydes as physiological substrates, and H2-receptor antagonists as pharmacological inhibitors, of human class IV alcohol dehydrogenase.

  Allali-Hassani A., Peralba J.M., Martras S., Farres J., Pares X.

  Kinetic constants of human class IV alcohol dehydrogenase (sigmasigma-ADH) support a role of the enzyme in retinoid metabolism, fatty acid omega-oxidation, and elimination of cytotoxic aldehydes produced by lipid peroxidation. Class IV is the human ADH form most efficient in the reduction of 4-hyd ... >> More

  Kinetic constants of human class IV alcohol dehydrogenase (sigmasigma-ADH) support a role of the enzyme in retinoid metabolism, fatty acid omega-oxidation, and elimination of cytotoxic aldehydes produced by lipid peroxidation. Class IV is the human ADH form most efficient in the reduction of 4-hydroxynonenal (k(cat)/Km: 39,500 mM(-1) min(-1)). Class IV shows high activity with all-trans-retinol and 9-cis-retinol, while 13-cis-retinol is not a substrate but an inhibitor. Both all-trans-retinoic and 13-cis-retinoic acids are potent competitive inhibitors of retinol oxidation (Ki: 3-10 microM) which can be a basis for the regulation of the retinoic acid generation and of the pharmacological actions of the 13-cis-isomer. The inhibition of class IV retinol oxidation by ethanol (Ki: 6-10 mM) may be the origin of toxic and teratogenic effects of ethanol. H2-receptor antagonists are poor inhibitors of human and rat classes I and IV (Ki > 0.3 mM) suggesting a small interference in ethanol metabolism at the pharmacological doses of these common drugs. << Less

  FEBS Lett. 426:362-366(1998) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• cDNA cloning and characterization of a new human microsomal NAD+-dependent dehydrogenase that oxidizes all-trans-retinol and 3alpha-hydroxysteroids.

  Gough W.H., VanOoteghem S., Sint T., Kedishvili N.Y.

  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 similarit ... >> More

  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. << Less

  J. Biol. Chem. 273:19778-19785(1998) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Kinetics of human alcohol dehydrogenase with ring-oxidized retinoids: effect of Tween 80.

  Martras S., Alvarez R., Gallego O., Dominguez M., de Lera A.R., Farres J., Pares X.

  Human alcohol dehydrogenases (ADH1 and ADH4) actively use retinoids oxidized at the cyclohexenyl ring (4-oxo-, 4-hydroxy-, and 3,4-didehydro-retinoids), which are functional compounds in several cells and tissues (i.e., in human skin). Remarkably, activities with 4-oxo-retinal and 4-hydroxy-retino ... >> More

  Human alcohol dehydrogenases (ADH1 and ADH4) actively use retinoids oxidized at the cyclohexenyl ring (4-oxo-, 4-hydroxy-, and 3,4-didehydro-retinoids), which are functional compounds in several cells and tissues (i.e., in human skin). Remarkably, activities with 4-oxo-retinal and 4-hydroxy-retinol (kcat = 2050 min(-1) for ADH4) are the highest among retinoids, similar to those of the best aliphatic alcohols. Thus, ADH1 and ADH4 provide a metabolic pathway for the synthesis of the corresponding retinoic acids. Tween 80, a widely used detergent in the retinoid activity assay, behaves as a competitive inhibitor. The Km values for all-trans-retinol (2-3 microM), estimated in the absence of detergent, are 10-fold lower than those obtained at the usual 0.02% Tween 80. This suggests a contribution of ADH to retinoid metabolism more relevant than previously expected. However, Tween 80 stabilizes retinoids in water solution and provides a reliable and reproducible assay, suitable for comparing different ADHs and different retinoid substrates. << Less

  Arch. Biochem. Biophys. 430:210-217(2004) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Alcohol dehydrogenase 2 is a major hepatic enzyme for human retinol metabolism.

  Hellgren M., Stroemberg P., Gallego O., Martras S., Farres J., Persson B., Pares X., Hoeoeg J.O.

  The metabolism of all-trans- and 9-cis-retinol/ retinaldehyde has been investigated with focus on the activities of human, mouse and rat alcohol dehydrogenase 2 (ADH2), an intriguing enzyme with apparently different functions in human and rodents. Kinetic constants were determined with an HPLC met ... >> More

  The metabolism of all-trans- and 9-cis-retinol/ retinaldehyde has been investigated with focus on the activities of human, mouse and rat alcohol dehydrogenase 2 (ADH2), an intriguing enzyme with apparently different functions in human and rodents. Kinetic constants were determined with an HPLC method and a structural approach was implemented by in silico substrate dockings. For human ADH2, the determined K(m) values ranged from 0.05 to 0.3 microM and k(cat) values from 2.3 to 17.6 min(-1), while the catalytic efficiency for 9-cis-retinol showed the highest value for any substrate. In contrast, poor activities were detected for the rodent enzymes. A mouse ADH2 mutant (ADH2Pro47His) was studied that resembles the human ADH2 setup. This mutation increased the retinoid activity up to 100-fold. The K(m) values of human ADH2 are the lowest among all known human retinol dehydrogenases, which clearly support a role in hepatic retinol oxidation at physiological concentrations. << Less

  Cell. Mol. Life Sci. 64:498-505(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.