Human Hep27 was originally isolated from growth-arrested HepG2 cells and identified as a member of the superfamily of short-chain dehydrogenases/reductases (SDR). Its substrate specificity has not been determined, but a cross-species comparison suggests that it occurs in widely divergent species, such as human, Cenorhabditis elegans, Drosophila and Arabidopsis thaliana. In this study, Hep27 was expressed as a His(6) fusion protein, and subjected to a substrate screen, using a compound library of SDR substrates, comprising steroids, retinoids, sugars and carbonyl compounds. Whereas no steroid dehydrogenase or retinoid activity was detected, it was found that Hep27 catalyzed the NADPH-dependent reduction of dicarbonyl compounds, like 3,4-hexanedione and 1-phenyl-1,2-propanedione with similar turnover numbers as DCXR (a mitochondrial dicarbonyl reductase/xylulose reductase). In contrast, Hep27 does not convert sugar substrates like xylulose or threose. Based on its substrate specificity and expression in endothelial tissues, it is suggested that Hep27 functions as a dicarbonyl reductase in enzymatic inactivation of reactive carbonyls, involved in covalent modification of cellular components.

Mutations in the gene encoding 11-cis-retinol dehydrogenase (RDH5; EC 1.1.1.105) are associated with fundus albipunctatus, an autosomal recessive eye disease characterized by stationary night blindness and accumulation of white spots in the retina. In addition, some mutated alleles are associated with development of cone dystrophy, especially in elderly patients. The numbers of identified RDH5 mutations linked to fundus albipunctatus have increased considerably during recent years. In this work, we have characterized the biochemical and cell biological properties of 11 mutants of RDH5 to understand the molecular pathology of the disease. All RDH5 mutants showed decreased protein stability and subcellular mislocalization and, in most cases, loss of enzymatic activity in vitro and in vivo. Surprisingly, mutant A294P displays significant enzymatic activity. Cross-linking studies and molecular modeling showed that RDH5 is dimeric, and co-expression analyses of wild-type and mutated alleles showed that the mutated enzymes, in a transdominant-negative manner, influenced the in vivo enzymatic properties of functional variants of the enzyme, particularly the A294P mutant. Thus, under certain conditions, nonfunctional alleles act in a dominantnegative way on functional but relatively unstable mutated alleles. However, in heterozygous individuals carrying one wild-type allele, the disease is recessive, probably due to the stability of the wild-type enzyme.

No abstract

Retinoic acid is generated by a two-step mechanism. First, retinol is converted into retinal by a retinol dehydrogenase, and, subsequently, retinoic acid is formed by a retinal dehydrogenase. In vitro, several enzymes are suggested to act in this metabolic pathway. However, little is known regarding their capacity to contribute to retinoic acid biosynthesis in vivo. We have developed a versatile cell reporter system to analyze the role of several of these enzymes in 9-cis-retinoic acid biosynthesis in vivo. Using a Gal4-retinoid X receptor fusion proteinbased luciferase reporter assay, the formation of 9-cisretinoic acid from 9-cis-retinol was measured in cells transfected with expression plasmids encoding different combinations of retinol and retinal dehydrogenases. The results suggested that efficient formation of 9-cisretinoic acid required co-expression of retinol and retinal dehydrogenases. Interestingly, the cytosolic alcohol dehydrogenase 4 failed to efficiently catalyze 9-cis-retinol oxidation. A structure-activity analysis showed that mutants of two retinol dehydrogenases, devoid of the carboxyl-terminal cytoplasmic tails, displayed greatly reduced enzymatic activities in vivo, but were active in vitro. The cytoplasmic tails mediate efficient endoplasmic reticulum localization of the enzymes, suggesting that the unique milieu in the endoplasmic reticulum compartment is necessary for in vivo activity of microsomal retinol dehydrogenases.Retinol (vitamin A) and its derivatives are essential dietary compounds needed in a variety of physiological processes, e.g. embryonic development, reproduction, cell differentiation, postnatal growth, maintenance of the immune system, and vision (1-3). The metabolically active retinoids are 9-cis-retinoic acid (9cRA) 1 and all-trans-retinoic acid (atRA) in extraocular tissues, acting as ligands for two classes of nuclear retinoid receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) (4). In ocular tissues, 11-cis-retinal serves as the chromophore of the visual pigments (2).Most cells obtain all-trans-retinol as the main source of retinoids, and it is known that activation of retinol into the active metabolites occur via tissue-specific isomerization and oxidation reactions. First, 9-cis-, 11-cis-, or all-trans-retinols are oxidized by retinol dehydrogenases (RDHs) into the corresponding retinals. For generation of the two isomers of RA, 9-cis-and all-trans-retinal are then further oxidized by a class of retinal dehydrogenases (Raldhs) (for reviews, see Refs. 5 and 6). It has been suggested that oxidation of the two isomers of retinol are the rate-limiting factors in the pathways generating 9cRA and atRA.Two classes of RDHs have been implicated in oxidation of the different stereo isomers of retinol, i.e. microsomal members of the short chain alcohol dehydrogenase/reductases, and the cytosolic medium chain alcohol dehydrogenases (ADHs). Several members of both classes of enzymes can oxidize different stereo isomers of retinol in vitro. However,...

No abstract