Enzymes of the short chain and medium chain dehydrogenase/reductase families have been demonstrated to participate in the oxidoreduction of ethanol and retinoids. Mammals and amphibians contain, in the upper digestive tract mucosa, alcohol dehydrogenases of the medium chain dehydrogenase/reductase family, active with ethanol and retinol. In the present work, we searched for a similar enzyme in an avian species (Gallus domesticus). We found that chicken does not contain the homologous enzyme from the medium chain dehydrogenase/reductase family but an oxidoreductase from the aldo-keto reductase family, with retinal reductase and alcohol dehydrogenase activities. The amino acid sequence shows 66 -69% residue identity with the aldose reductase and aldose reductase-like enzymes. Chicken aldo-keto reductase is a monomer of M r 36,000 expressed in eye, tongue, and esophagus. The enzyme can oxidize aliphatic alcohols, such as ethanol, and it is very efficient in all-trans-and 9-cis-retinal reduction (k cat /K m ؍ 5,300 and 32,000 mM ؊1 ⅐min ؊1 , respectively). This finding represents the inclusion of the aldo-keto reductase family, with the (␣/) 8 barrel structure, into the scenario of retinoid metabolism and, therefore, of the regulation of vertebrate development and tissue differentiation.
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...
Glutathione-dependent formaldehyde dehydrogenase (FALDH) is the main enzymatic system for formaldehyde detoxification in all eukaryotic and many prokaryotic organisms. The enzyme of yeasts and some bacteria exhibits about 10-fold higher k cat and K m values than those of the enzyme from animals and plants. Typically Thr-269 and Glu-267 are found in the coenzyme-binding site of yeast FALDH, but Ile-269 and Asp-267 are present in the FALDH of animals. By site-directed mutagenesis we have prepared the T269I and the D267E mutants and the D267E/T269I double mutant of Saccharomyces cerevisiae FALDH with the aim of investigating the role of these residues in the kinetics. The T269I and the D267E mutants have identical kinetic properties as compared with the wild-type enzyme, although T269I is highly unstable. In contrast, the D267E/T269I double mutant is stable and shows low K m (2.5 M) and low k cat (285 min ؊1 ) values with S-hydroxymethylglutathione, similar to those of the human enzyme. Therefore, the simultaneous exchange at both residues is the structural basis of the two distinct FALDH kinetic types. The local structural perturbations imposed by the substitutions are suggested by molecular modeling studies. Finally, we have studied the effect of FALDH deletion and overexpression on the growth of S. cerevisiae. It is concluded that the FALDH gene is not essential but enhances the resistance against formaldehyde (0.3-1 mM). Moreover, the wild-type enzyme (with high k cat and K m ) provides more resistance than the double mutant (with low k cat and K m ).Formaldehyde is a highly reactive compound that is present in the environment as a result of natural processes and human industrial activity. Moreover, formaldehyde is also generated endogenously by several metabolic pathways (1-5). In humans the level of formaldehyde in blood has been estimated as 0.46 -2.81 M (6). The elimination of formaldehyde in eukaryotic cells is mainly carried out by glutathione-dependent formaldehyde dehydrogenase (FALDH) 1 (7). Formaldehyde reacts spontaneously with glutathione (GSH) to form the S-hydroxymethylglutathione (S-HMGSH) adduct which, in the presence of NAD ϩ , is oxidized to S-formylglutathione by FALDH (8). S-Formylglutathione is irreversibly hydrolyzed by S-formylglutathione hydrolase to formate and GSH (8). In addition to the glutathionedependent formaldehyde dehydrogenase activity, FALDH is also active with long chain alcohols, particularly with -hydroxylated fatty acids such as 12-hydroxydodecanoic acid (9 -12). Interestingly FALDH is capable of catalyzing efficiently the NADH-dependent degradation of S-nitrosoglutathione derived from the glutathione nitrosation by nitric oxide (13). FALDH is a dimeric enzyme with subunits of 40 kDa, containing two zinc atoms per subunit, and it appears to be ubiquitous in eukaryotic organisms (14). FALDH is a member of the medium chain alcohol dehydrogenase (ADH) family, and it represents the ADH class (class III) most closely related to the ancestral line that originated by successive g...
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