The specificity of alcohol dehydrogenase with cis‐retinoids

Abstract

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-cisretinoids. We report here the kinetics of human ADH1B1, ADH1B2, ADH4, and mouse ADH1 and ADH4 with alltrans-, 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-cisretinol, 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.Keywords: alcohol dehydrogenase; enzyme kinetics; retina; retinoid metabolism; retinol dehydrogenase.Retinoids are essential in several physiological processes such as development, growth and cellular maintenance [1,2]. The active forms of retinol are its oxidized derivatives alltrans-and 9-cis-retinoic acid which perform their function through the binding to specific nuclear receptors [3,4]. Retinoic acids are synthesized by two enzymatic reactions which include retinol oxidation to retinal, and oxidation of retinal to retinoic acid. Two types of enzymes have been implicated in the first reaction: the alcohol dehydrogenases (ADH) of the medium-chain dehydrogensase/reductase family and the retinol dehydrogenases of the short-chain dehydrogenase/reductase (SDR) family [5]. In mammals, ADH is a cytosolic NAD + -dependent enzyme formed by two subunits of 40 kDa, with two zinc atoms per subunit [6].Genomic studies indicate that five ADH classes (ADH1-ADH5) exist in mammals [7]. It is well established that ADH1 and ADH4 [5,8], and to a lesser extent ADH2 [9], are involved in retinoid metabolism. Recently, it has been proposed that ADH3, the ubiquitous enzyme responsible for formaldehyde elimination, could also have a role in retinoic acid generation in vivo [10]. Nevertheless, the high activity toward retinoids and the spatiotemporal colocalization of ADH1 and ADH4 with retinoic acid during embryogenesis and in adult tis...

Retinol Dehydrogenase (RDH12) Protects Photoreceptors from Light-induced Degeneration in Mice

“…RDHs constitute two groups of enzymes that belong to microsomal and soluble short-chain dehydrogenase/reductase (9,10,(27)(28)(29) and alcohol dehydrogenase families that possess RDH activity (30). In addition to biochemical assays confirming the specificity of various dehydrogenases (9,31,32), mouse genetic studies promise to provide a powerful approach to elucidate their function (14,(32)(33)(34).…”

The Role of Retinoid X Receptor α in Regulating Alcohol Metabolism

“…ADH isozyme activities in the liver cytosol were measured by detecting the reduction of NAD ϩ at 340 nm. ADH1 activity was measured with 0.3 mM NAD ϩ and 10 mM ethanol in 0.1 M glycine-NaOH buffer, pH 10.5, as described previously (Martras et al, 2004). The ADH2 activity was determined with 5 mM benzyl alcohol and 2.4 mM NAD ϩ in 0.1 M glycine-NaOH buffer, pH, 10, as reported previously (Stromberg et al, 2004).…”

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

“…In contrast to most alcohol/aldehyde pairs, the human and rat ADH2 enzymes showed higher activities for retinols than for retinaldehydes. Similarly, human and mouse ADH4 showed higher turnover for the alcoholic forms of most tested retinoids as compared to the corresponding aldehydes [8,12,22]. At the same time human ADH2 has been suggested to participate in the first-pass metabolism of ethanol in the liver, an action that has been shown to competitively inhibit retinol conversion [29].…”

“…The k cat values of human ADH2 for retinol oxidation are comparable to those for aliphatic alcohols at pH 7.5 [16], thus suggesting a common rate-limiting step, probably the dissociation of cofac- tor. This differs from other ADHs, which show lower k cat values with retinol isomers than with aliphatic substrates [22]. Since ADH2 is mainly expressed in the liver [27], the kinetic constants strongly suggest that the human enzyme is a major component of the hepatic retinol oxidation.…”

“…The experiments with Tween 80 were performed to relate results to earlier published values [14,19]. Retinoid concentration was measured using the specific molar absorption coefficients [8,14,19,22,23]. Protein concentrations were determined with the Bio-Rad protein assay, standardized with BSA, complemented with amino acid analysis on an Amersham Pharmacia Biotech Alpha Plus Analyzer.…”