Retinal Reductase Research Articles

Selective biosynthesis of retinol in S. cerevisiae

The vitamin A component retinol has become an increasingly sought-after cosmetic ingredient. In previous efforts for microbial biosynthesis of vitamin A, a mixture of retinoids was produced. In order to efficiently produce retinol at high purity, the precursor and NADPH supply was first enhanced to improve retinoids accumulation in the S. cerevisiae strain constructed from a β-carotene producer by introducing β-carotene 15,15ʹ-dioxygenase, following by screening of heterologous and endogenous oxidoreductases for retinal reduction. Env9 was found as an endogenous retinal reductase and its activity was verified in vitro. By co-expressing Env9 with the E. coli ybbO, as much as 443.43 mg/L of retinol was produced at 98.76% purity in bi-phasic shake-flask culture when the antioxidant butylated hydroxytoluene was added to prevent retinoids degradation. The retinol titer reached 2479.34 mg/L in fed-batch fermentation. The success in selective biosynthesis of retinol would lay a solid foundation for its biotechnological production.Graphical

Development of a Gene Therapy Vector for RDH12-Associated Retinal Dystrophy.

Early-onset severe retinal dystrophy (EOSRD) is a genetically heterogeneous group of diseases resulting in serious visual disability in children. A significant number of EOSRD cases, often diagnosed as Leber congenital amaurosis (LCA13), are associated with mutations in the gene encoding retinol dehydrogenase 12 (RDH12). RDH12 is a member of the enzyme family of short-chain dehydrogenases/reductases. In the retina, RDH12 plays a critical role in reducing toxic retinaldehydes generated by visual cycle activity that is required for the light response of the photoreceptor cells. Individuals with RDH12 deficiency exhibit widespread retinal degeneration impacting both rods and cones. Although Rdh12-deficient (Rdh12-/-) mice do not exhibit retinal degeneration, functional deficits relevant to visual cycle function can be demonstrated. In the present study, we describe the development and preclinical testing of a recombinant adeno-associated viral (rAAV) vector that has the potential for use in treating EOSRD due to RDH12 mutations. Wild-type and Rdh12-/- mice that received a subretinal injection of rAAV2/5 carrying a human RDH12 cDNA driven by a human rhodopsin-kinase promoter exhibited transgene expression that was stable, correctly localized, and did not cause retinal toxicity. In addition, administration of the vector reconstituted retinal reductase activity in the retinas of Rdh12-/- mice and decreased susceptibility to light damage associated with Rdh12 deficiency, thus demonstrating potential therapeutic efficacy in an animal model that does not exhibit a retinal degeneration phenotype. These findings support further efforts to develop gene replacement therapy for individuals with RDH12 mutations.

Exome Sequencing Identified a Recessive RDH12 Mutation in a Family with Severe Early-Onset Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is the most important hereditary retinal disease caused by progressive degeneration of the photoreceptor cells. This study is to identify gene mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in a Chinese family using next-generation sequencing technology. A Chinese family with 7 members including two individuals affected with severe early-onset RP was studied. All patients underwent a complete ophthalmic examination. Exome sequencing was performed on a single RP patient (the proband of this family) and direct Sanger sequencing on other family members and normal controls was followed to confirm the causal mutations. A homozygous mutation c.437T<A (p.V146D) in the retinol dehydrogenase 12 (RDH12) gene, which encodes an NADPH-dependent retinal reductase, was identified as being related to the phenotype of this arRP family. This homozygous mutation was detected in the two affected patients, but not present in other family members and 600 normal controls. Another three normal members in the family were found to carry this heterozygous missense mutation. Our results emphasize the importance of c.437T<A (p.V146D) substitution in RDH12 and provide further support for the causative role of this mutation in the pathogenesis and clinical diagnosis of RP.

DHRS3, a retinal reductase, is differentially regulated by retinoic acid and lipopolysaccharide-induced inflammation in THP-1 cells and rat liver

Both retinoid status and inflammation have been shown to control the level of expression of retinoid homeostatic genes. In the present study, DHRS3, previously shown to possess retinal reductase activity, was identified by microarray analysis of THP-1 monocytes as a possible gene target of all-trans-retinoic acid (RA). In these cells, DHRS3 mRNA increased 30- to 40-fold after treatment with ≤20 nM RA for 24 h, while DHRS3 protein also increased. Of several synthetic retinoids tested, only Am580, a RA receptor-α-selective retinoid, increased DHRS3 mRNA expression. The full-length DHRS3 cDNA was cloned from rat liver and subjected to in vitro transcription-translation. Two major ∼30- and 35-kDa proteins were detected. In adult rat tissues, DHRS3 mRNA was most abundant in the adrenal gland, liver, and ovary. In the liver, DHRS3 is expressed in hepatocytes and possibly in all liver cells. To evaluate whether DHRS3 is regulated in the liver by RA and/or inflammatory stimuli, we treated rats for 6 h with RA or LPS or both. DHRS3 mRNA was doubled by RA but reduced by >90% after treatment with LPS in the absence and presence of RA. On the basis of our results, DHRS3 mRNA expression is regulated by RA in a tissue- or cell-type specific manner; the RA-induced increase in DHRS3 may contribute to retinoid storage; and a reduction of DHRS3 expression in the liver during inflammation may contribute to the perturbation of whole body vitamin A metabolism that has previously been shown to occur in conditions of inflammatory stress.

Influence of dietary spices on thein vivoabsorption of ingested β-carotene in experimental rats

Animal studies were conducted to evaluate the influence of dietary spice compounds, piperine, capsaicin and ginger, on the absorption of orally administered β-carotene and its conversion to vitamin A. In rats maintained on these spice-containing diets for 8 weeks, concentrations of β-carotene and retinol were determined in the serum, liver and intestine 4 h after a single oral administration of β-carotene. β-Carotene concentration was significantly increased in the serum, liver and intestine of piperine- and ginger-fed rats, suggesting improved absorption of β-carotene. However, retinol concentration was not significantly changed in these animals, suggesting that the bioconversion of β-carotene to vitamin A was not similarly influenced. Between the two enzymes involved in the bioconversion of β-carotene to vitamin A, the activity of intestinal and hepatic β-carotene 15,15'-dioxygenase was either unaffected or lowered by these spice treatments. The activity of intestinal and hepatic retinal reductase was unaffected by the dietary spices. Activities of these two enzymes involved in the bioconversion of β-carotene to retinal were inhibited by the test spices in vitro, thus corroborating with the in vivo observation. Although the bioconversion of β-carotene was not promoted, increased absorption and tissue levels of β-carotene by the dietary spices may contribute to a higher antioxidant protection.

Oxidation of PAH trans-Dihydrodiols by Human Aldo-Keto Reductase AKR1B10

AKR1B10 has been identified as a potential biomarker for human nonsmall cell lung carcinoma and as a tobacco exposure and response gene. AKR1B10 functions as an efficient retinal reductase in vitro and may regulate retinoic acid homeostasis. However, the possibility that this enzyme is able to activate polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols to form reactive and redox-active o-quinones has not been investigated to date. AKR1B10 was found to oxidize a wide range of PAH trans-dihydrodiol substrates in vitro to yield PAH o-quinones. Reactions of AKR1B10 proceeded with improper stereochemistry, since it was specific for the minor (+)-benzo[a]pyrene-7S,8S-dihydrodiol diastereomer formed in vivo. However, AKR1B10 displayed reasonable activity in the oxidation of both the (-)-R,R and (+)-S,S stereoisomers of benzo[g]chrysene-11,12-dihydrodiol and oxidized the potentially relevant, albeit minor, (+)-benz[a]anthracene-3S,4S-dihydrodiol metabolite. We find that AKR1B10 is therefore likely to play a contributing role in the activation of PAH trans-dihydrodiols in human lung. AKR1B10 retinal reductase activity was confirmed in vitro and found to be 5- to 150-fold greater than the oxidation of PAH trans-dihydrodiols examined. AKR1B10 was highly expressed at the mRNA and protein levels in human lung adenocarcinoma A549 cells, and robust retinal reductase activity was measured in lysates of these cells. The much greater catalytic efficiency of retinal reduction compared to PAH trans-dihydrodiol metabolism suggests AKR1B10 may play a greater role in lung carcinogenesis through dysregulation of retinoic acid homeostasis than through oxidation of PAH trans-dihydrodiols.

A 27.368 kDa retinal reductase in New Zealand white rabbit liver cytosol encoded by the peroxisomal retinol dehydrogenase–reductase cDNA: purification and characterization of the enzymeThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

We obtained a full-length cDNA based on a sequence deposited in GenBank (accession No. AB045133), annotated as rabbit peroxisomal NADP(H)-dependent retinol dehydrogenase-reductase (NDRD). The rabbit NDRD gene, like its mouse and human homologs, harbors 2 initiation sites, one of which theoretically encodes a 29.6 kDa protein with 279 amino acids, and the other encodes a 27.4 kDa protein with 260 amino acids. The purification of a rabbit cytosolic retinol oxidoreductase with a subunit molecular mass of 34 kDa and an N terminus that is not completely identical to that of NDRD, has been reported. An enzyme responsible for the all-trans retinal reductase activity in the liver cytosol of New Zealand white rabbit was purified to homogeneity using differential centrifugation and successive chromatographic analyses. The subunit molecular mass of the purified enzyme, revealed by SDS-PAGE, was approximately 27 kDa. The intact molecular mass, measured by MALDI-TOF mass spectrometry, was 27.368 kDa. The 60 kDa relative mobility observed in size-exclusion chromatography indicates that the native protein probably exists as a dimer. The purified enzyme was positively confirmed to be the product of NDRD by peptide mass fingerprinting, tandem mass spectrometry, and N-terminal sequencing. Taken together, the results suggested that the native protein is truncated at the N terminus.

DHRS3, a retinal reductase, is regulated by retinoic acid and lipopolysaccharide in THP1 cells and rat liver in vivo

DHRS3, a retinal reductase, is regulated by retinoic acid and lipopolysaccharide in THP1 cells and rat liver in vivo

Characterization of an Oligomeric Carbonyl Reductase of Dog Liver: Its Identity with Peroxisomal Tetrameric Carbonyl Reductase

Dog liver contains an oligomeric NADPH-dependent carbonyl reductase (CR) with substrate specificity for alkyl phenyl ketones, but its endogenous substrate and primary structure remain unknown. In this study, we examined the molecular weight and substrate specificity of the enzyme purified from dog liver. The enzyme is a ca. 100-kDa tetramer composing of 27-kDa subunit, and reduces all-trans-retinal and alpha-dicarbonyl compounds including isatin, which are substrates for pig peroxisomal tetrameric carbonyl reductase (PTCR). In addition, the dog enzyme resembles pig PTCR in inhibitor sensitivity to flavonoids, myristic acid, lithocholic acid, bromosulfophthalein and flufenamic acid. Furthermore, the amino acid sequence of dog CR determined by protein sequencing and cDNA cloning was 84% identical to that of pig PTCR and had a C-terminal peroxisomal targeting signal type 1, Ser-His-Leu. The immunoprecipitation using the anti-pig PTCR antibody shows that the dog enzyme is a major form of soluble NADPH-dependent all-trans-retinal reductase in dog liver. Thus, dog oligomeric CR is PTCR, and may play a role in retinoid metabolism as a retinal reductase.

Effects of Low-Carbohydrate Diet and Pycnogenol®Treatment on Retinal Antioxidant Enzymes in Normal and Diabetic Rats

Because chronic hyperglycemia of uncontrolled diabetes mellitus may lead to increased reactive oxygen species and decreased enzymatic antioxidant defenses responsible for pathological processes in diabetic retinopathy, this study examined the hypothesis that a low-carbohydrate, high-fat diet, either alone or in combination with Pinus maritima can reduce hyperglycemia, restoring a more balanced, oxidative condition. Normal and streptozotocininduced diabetic rats were fed either a regular or low-carbohydrate diet for 30 or 90 d. In addition, normal and diabetic rats on the chronic (90-d) low-carbohydrate diet were treated with daily intraperitoneal Pinus maritima doses (10 mg/kg) for 14 consecutive days. Retinas were fractionated to assay activities of glutathione peroxidase, glutathione reductase, and gamma-glutamyl transferase. After 30 d, the low-carbohydrate diet reduced glycemic parameters and normalized aspartate aminotransferase activity in diabetic animals, suggesting less organ damage. No differences were observed between males and females in any measured glycemic parameters. Whereas all diabetic control animals developed cataracts bilaterally, no treated diabetic animals developed cataracts. There were no deleterious effects on retinal antioxidant defenses with either a 30-d or chronic low-carbohydrate diet. When diet was combined with Pinus maritima treatment, both retinal glutathione peroxidase and glutathione reductase activities increased, suggesting that a low-carbohydrate diet plus Pinus maritima may be an effective antioxidant and antihyperglycemic therapy, reducing the risk of diabetic retinopathy and cataract formation.

Properties of short-chain dehydrogenase/reductase RalR1: characterization of purified enzyme, its orientation in the microsomal membrane, and distribution in human tissues and cell lines.

Recently, we reported the first biochemical characterization of a novel member of the short-chain dehydrogenase/reductase superfamily, retinal reductase 1 (RalR1) (Kedishvili et al. (2002) J. Biol. Chem. 277, 28909-28915). In the present study, we purified the recombinant enzyme from the microsomal membranes of insect Sf9 cells, determined its catalytic efficiency for the reduction of retinal and the oxidation of retinol, established its transmembrane topology, and examined the distribution of RalR1 in human tissues and cell lines. Purified RalR1-His(6) exhibited the apparent K(m) values for all-trans-retinal and all-trans-retinol of 0.12 and 0.6 microM, respectively. The catalytic efficiency (k(cat)/K(m)) for the reduction of all-trans-retinal (150,000 min(-1) mM(-1)) was 8-fold higher than that for the oxidation of all-trans-retinol (18,000 min(-1) mM(-1)). Protease protection assays and site-directed mutagenesis suggested that the enzyme is anchored in the membrane by the N-terminal signal-anchor domain, with the majority of the polypeptide chain located on the cytosolic side of the membrane. An important feature that prevented the translocation of RalR1 across the membrane was the positively charged R(25)K motif flanking the N-terminal signal-anchor. The cytosolic orientation of RalR1 suggested that, in intact cells, the enzyme would function predominantly as a reductase. Western blot analysis revealed that RalR1 is expressed in a wide variety of normal human tissues and cancer cell lines. The expression pattern and the high catalytic efficiency of RalR1 are consistent with the hypothesis that RalR1 contributes to the reduction of retinal in various human tissues.

Human aldose reductase and human small intestine aldose reductase are efficient retinal reductases: consequences for retinoid metabolism.

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.

Characterization of Mouse Short-chain Aldehyde Reductase (SCALD), an Enzyme Regulated by Sterol Regulatory Element-binding Proteins

Sterol regulatory element-binding proteins (SREBPs) enhance transcription of genes encoding all of the proteins required for the cellular synthesis and uptake of cholesterol and unsaturated fatty acids. Here, we use suppression subtractive hybridization to identify a previously unrecognized SREBP-enhanced gene in mice. The gene encodes a membrane-bound enzyme that we designate SCALD, for short-chain aldehyde reductase. We expressed SCALD in bacteria, purified it extensively, and studied its catalytic properties in detergent solution. The enzyme specifically uses NADPH to reduce a variety of short-chain aldehydes, including nonanal and 4-hydroxy-2-nonenal. The enzyme also reduces retinaldehydes, showing equal activity for all-trans-retinal and 9-cis-retinal. Northern blot analysis indicates that SCALD is expressed most abundantly in mouse liver and testis. In the liver of mice, SCALD is suppressed by fasting and induced by refeeding, consistent with regulation by SREBPs. In testis, SCALD expression is restricted to pachytene spermatocytes, as revealed by visualization of mRNA and protein. SCALD is also expressed in four layers of the retina, including the outer segment of rods and cones, as revealed by immunohistochemistry. SCALD appears to be the mouse ortholog of the human protein that has been designated variously as prostate short-chain dehydrogenase/reductase 1, retinal reductase 1, and retinol dehydrogenase 11. In view of its ability to reduce short-chain aldehydes in addition to retinals, we propose that SCALD may be induced by SREBP in liver and other tissues to prevent toxicity from fatty aldehydes that are generated from oxidation of unsaturated fatty acids that are synthesized as a result of SREBP activity.

Crystal Structure of the Vertebrate NADP(H)-dependent Alcohol Dehydrogenase (ADH8)

The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8–NADP + have been now determined and refined to resolutions of 2.2 Å and 1.8 Å, respectively. The coenzyme and substrate specificity of ADH8, that has 50–65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K m value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.

Reduction of all-trans-retinal in the mouse liver peroxisome fraction by the short-chain dehydrogenase/reductase RRD: induction by the PPAR alpha ligand clofibrate.

The mouse liver 16,000 g fraction, which contains peroxisomes, reduces all-trans-retinal, but has limited ability to dehydrogenate retinol enzymatically. Feeding mice for 2 weeks with a diet containing clofibrate (0.5%, w/w), a PPAR alpha ligand and peroxisome proliferator, increased the 16,000 g fraction approximately 2-fold in protein, approximately 2-fold in specific activity of retinal reduction, and approximately 4-fold in retinal reductase units compared to controls, and caused a 50% decrease in liver retinol. An increase in both reductase specific activity and units indicates that clofibrate/PPAR alpha induced expression of retinal-reducing enzymes(s), in addition to increasing reductase(s) content. We expressed a cDNA from the NCBI data bank that encodes a peroxisome short-chain dehydrogenase/reductase. The enzyme, mouse retinal reductase (RRD, also known as human 2,4-dienoyl-CoA reductase), reduces all-trans-retinal [V(m) = 40 nmol min(-1) (mg of protein)(-1); K(0.5) = 2.3 microM] and has 4- and 60-fold less activity with 13-cis-retinal and 9-cis-retinal, respectively. Recombinant RRD functions with both unbound and CRBP(I) (cellular retinol-binding protein)-bound retinal, but apo-CRBP(I) inhibits the reductase. RRD mRNA expression was initiated on embryo day 7. Most adult tissues assayed expressed the mRNA. Liver, kidney, and heart had the most intense expression, with much less intense expression in brain, spleen, and lung. Clofibrate feeding increased the amount of RRD protein in the 16,000 g fraction of liver, consistent with the clofibrate-induced increase in reductase activity. These data relate retinoid metabolism, PPAR alpha, peroxisomes, and RRD, and are consistent with a further function of CRBP(I) in retinoid metabolism.

Low zinc intake decreases the lymphatic output of retinol in rats infused intraduodenally with β-carotene

Previously, we have shown that the lymphatic absorption of retinol is significantly decreased in rats fed a low zinc diet. This study was conducted to determine whether the absorption of β-carotene also is altered in zinc-deficient male rats. The absorption of β-carotene was estimated by determining the amount of retinol appearing in the mesenteric lymph during intraduodenal infusion of β-carotene. One group of rats was fed the AIN-93G diet but low in zinc (LZ; 3 mg/kg) and the other was fed the same diet adequate in zinc (AZ; 30 mg/kg). The LZ and AZ rats were trained to meal feed equal amounts of the diets twice daily. At 6 weeks, each rat with lymph cannula was infused via an intraduodenal catheter at 3 ml/h for 8 h with a lipid emulsion containing 65.0 nmol β-carotene, 565.1 μmol triolein, 27.8 kBq 14C-triolein ( 14C-OA), 72 mg albumin, and 396 μmol Na-taurocholate in 24 ml PBS (pH, 6.7). The lymphatic output of retinol over the 8-h period was significantly lower in LZ rats than in AZ rats. The absorption of 14C-OA also was significantly lower in LZ rats. No significant differences were observed between groups in intestinal β-carotene 15,15′-dioxygenase, retinal reductase, and retinal oxidase activities. The findings demonstrate that low zinc intake or marginal zinc deficiency significantly lowers the absorption of β-carotene as estimated by lymphatic retinol output. The results also indicate that the decrease in retinol output in LZ rats is not linked to defects in β-carotene cleavage and subsequent conversion of retinal to retinol in the intestinal mucosa. This study suggests that zinc status is an important factor determining the intestinal absorption of β-carotene and hence the nutritional status of vitamin A.

Carotenoderma--a review of the current literature.

Carotenoderma--a review of the current literature.

Human pancreas protein 2 (PAN2) has a retinal reductase activity and is ubiquitously expressed in human tissues

Human gene for pancreas protein 2 (PAN2) is a novel member of the short-chain dehydrogenase/reductase gene superfamily. The properties of PAN2 protein have not yet been characterized. We present the first evidence that human PAN2 is a ubiquitously expressed microsomal enzyme that recognizes retinoids but not steroids as substrates with the apparent K m values between 0.08 μM and 0.4 μM. PAN2 is ∼4-fold more efficient in the reductive than in the oxidative direction. The apparent K m values for NADP + and NADPH are 0.65 μM and 0.32 μM versus 1200 μM and 1060 μM for NAD + and NADH, respectively. Kinetic constants and expression pattern of PAN2 suggest that it is likely to function as a reductase in vivo and might contribute to the reduction of retinaldehyde to retinol in most human tissues.

Evidence That the Human Gene for Prostate Short-chain Dehydrogenase/Reductase (PSDR1) Encodes a Novel Retinal Reductase (RalR1)

All-trans-retinoic acid is a metabolite of vitamin A (all-trans-retinol) that functions as an activating ligand for a family of nuclear retinoic acid receptors. The intracellular levels of retinoic acid in tissues are tightly regulated, although the mechanisms underlying the control of retinoid metabolism at the level of specific enzymes are not completely understood. In this report we present the first characterization of the retinoid substrate specificity of a novel short-chain dehydrogenase/reductase (SDR) encoded by RalR1/PSDR1, a cDNA recently isolated from the human prostate (Lin, B., White, J. T., Ferguson, C., Wang, S., Vessella, R., Bumgarner, R., True, L. D., Hood, L., and Nelson, P. S. (2001) Cancer Res. 61, 1611-1618). We demonstrate that RalR1 exhibits an oxidoreductive catalytic activity toward retinoids, but not steroids, with at least an 800-fold lower apparent K(m) values for NADP+ and NADPH versus NAD+ and NADH as cofactors. The enzyme is approximately 50-fold more efficient for the reduction of all-trans-retinal than for the oxidation of all-trans-retinol. Importantly, RalR1 reduces all-trans-retinal in the presence of a 10-fold molar excess of cellular retinol-binding protein type I, which is believed to sequester all-trans-retinal from nonspecific enzymes. As shown by immunostaining of human prostate and LNCaP cells with monoclonal anti-RalR1 antibodies, the enzyme is highly expressed in the epithelial cell layer of human prostate and localizes to the endoplasmic reticulum. The enzymatic properties and expression pattern of RalR1 in prostate epithelium suggest that it might play a role in the regulation of retinoid homeostasis in human prostate.

A Vertebrate Aldo-keto Reductase Active with Retinoids and Ethanol

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).min(-1), respectively). This finding represents the inclusion of the aldo-keto reductase family, with the (alpha/beta)(8) barrel structure, into the scenario of retinoid metabolism and, therefore, of the regulation of vertebrate development and tissue differentiation.