RESUMO
Ring-oxidized retinoids have been synthesized stereoselectively using the Stille cross-coupling reaction. Kinetic constants of mouse class I alcohol dehydrogenase (ADH1) with these retinoids were determined.
Assuntos
Álcool Desidrogenase/metabolismo , Bioquímica/métodos , Retinoides/síntese química , Retinoides/metabolismo , Animais , Diterpenos , Cinética , Camundongos , Oxirredução , Retinaldeído/análogos & derivados , Retinaldeído/síntese química , Retinaldeído/química , Retinaldeído/metabolismo , Retinoides/química , Relação Estrutura-Atividade , Especificidade por Substrato , Vitamina A/análogos & derivados , Vitamina A/síntese química , Vitamina A/química , Vitamina A/metabolismoRESUMO
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.
Assuntos
Álcool Desidrogenase/metabolismo , Polissorbatos/farmacologia , Retinoides/metabolismo , Álcool Desidrogenase/efeitos dos fármacos , Álcool Desidrogenase/genética , Álcool Desidrogenase/isolamento & purificação , Escherichia coli/genética , Humanos , Cinética , Estrutura Molecular , Oxirredução , Reprodutibilidade dos Testes , Retinoides/química , Especificidade por SubstratoRESUMO
Aldo-keto reductases (AKRs) are NAD(P)H-dependent oxidoreductases that catalyse the reduction of a variety of carbonyl compounds, such as carbohydrates, aliphatic and aromatic aldehydes and steroids. We have studied the retinal reductase activity of human aldose reductase (AR), human small-intestine (HSI) AR and pig aldehyde reductase. Human AR and HSI AR were very efficient in the reduction of all- trans -, 9- cis - and 13- cis -retinal ( k (cat)/ K (m)=1100-10300 mM(-1).min(-1)), constituting the first cytosolic NADP(H)-dependent retinal reductases described in humans. Aldehyde reductase showed no activity with these retinal isomers. Glucose was a poor inhibitor ( K (i)=80 mM) of retinal reductase activity of human AR, whereas tolrestat, a classical AKR inhibitor used pharmacologically to treat diabetes, inhibited retinal reduction by human AR and HSI AR. All- trans -retinoic acid failed to inhibit both enzymes. In this paper we present the AKRs as an emergent superfamily of retinal-active enzymes, putatively involved in the regulation of retinoid biological activity through the assimilation of retinoids from beta-carotene and the control of retinal bioavailability.
Assuntos
Oxirredutases do Álcool/metabolismo , Aldeído Redutase/metabolismo , Intestino Delgado/enzimologia , Retinoides/metabolismo , Oxirredutases do Álcool/química , Aldeído Redutase/química , Animais , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Humanos , Cinética , SuínosRESUMO
Influence of vitamin A (retinol) on growth depends on its sequential oxidation to retinal and then to retinoic acid (RA), producing a ligand for RA receptors essential in development of specific tissues. Genetic studies have revealed that aldehyde dehydrogenases function as tissue-specific catalysts for oxidation of retinal to RA. However, enzymes catalyzing the first step of RA synthesis, oxidation of retinol to retinal, remain unclear because none of the present candidate enzymes have expression patterns that fully overlap with those of aldehyde dehydrogenases during development. Here, we provide genetic evidence that alcohol dehydrogenase (ADH) performs this function by demonstrating a role for Adh3, a ubiquitously expressed form. Adh3 null mutant mice exhibit reduced RA generation in vivo, growth deficiency that can be rescued by retinol supplementation, and completely penetrant postnatal lethality during vitamin A deficiency. ADH3 was also shown to have in vitro retinol oxidation activity. Unlike the second step, the first step of RA synthesis is not tissue-restricted because it is catalyzed by ADH3, a ubiquitous enzyme having an ancient origin.
Assuntos
Álcool Desidrogenase/biossíntese , Álcool Desidrogenase/metabolismo , Tretinoína/metabolismo , Álcool Desidrogenase/genética , Oxirredutases do Álcool/biossíntese , Animais , Citosol/enzimologia , Genótipo , Camundongos , Camundongos Transgênicos , Mutação , Oxigênio/metabolismo , Retinaldeído/metabolismo , Fatores de Tempo , Vitamina A/metabolismo , Vitamina A/farmacologia , Deficiência de Vitamina A/metabolismoRESUMO
Studies in knockout mice support the involvement of alcohol dehydrogenases ADH1 and ADH4 in retinoid metabolism, although kinetics with retinoids are not known for the mouse enzymes. Moreover, a role of alcohol dehydrogenase (ADH) in the eye retinoid interconversions cannot be ascertained due to the lack of information on the kinetics with 11-cis-retinoids. We report here the kinetics of human ADH1B1, ADH1B2, ADH4, and mouse ADH1 and ADH4 with all-trans-, 7-cis-, 9-cis-, 11-cis- and 13-cis-isomers of retinol and retinal. These retinoids are substrates for all enzymes tested, except the 13-cis isomers which are not used by ADH1. In general, human and mouse ADH4 exhibit similar activity, higher than that of ADH1, while mouse ADH1 is more efficient than the homologous human enzymes. All tested ADHs use 11-cis-retinoids efficiently. ADH4 shows much higher k(cat)/K(m) values for 11-cis-retinol oxidation than for 11-cis-retinal reduction, a unique property among mammalian ADHs for any alcohol/aldehyde substrate pair. Docking simulations and the kinetic properties of the human ADH4 M141L mutant demonstrated that residue 141, in the middle region of the active site, is essential for such ADH4 specificity. The distinct kinetics of ADH4 with 11-cis-retinol, its wide specificity with retinol isomers and its immunolocalization in several retinal cell layers, including pigment epithelium, support a role of this enzyme in the various retinol oxidations that occur in the retina. Cytosolic ADH4 activity may complement the isomer-specific microsomal enzymes involved in photopigment regeneration and retinoic acid synthesis.