Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.
Retinol dehydrogenase 12 (RDH12) is a novel member of the short-chain dehydrogenase/reductase superfamily of proteins that was recently linked to Leber's congenital amaurosis 3 (LCA). We report the first biochemical characterization of purified human RDH12 and analysis of its expression in human tissues. RDH12 exhibits ~2000-fold lower K m values for NADP + and NADPH than for NAD + and NADH and recognizes both retinoids and lipid peroxidation products (C 9 aldehydes) as substrates. The k cat values of RDH12 for retinaldehydes and C 9 aldehydes are similar, but the K m values are, in general, lower for retinoids. The enzyme exhibits the highest catalytic efficiency for all-trans-retinal (k cat /K m ~900 min −1 μM −1 ), followed by 11-cis-retinal (450 min −1 mM −1 ) and 9-cis-retinal (100 min −1 mM −1 ). Analysis of RDH12 activity toward retinoids in the presence of cellular retinol-binding protein (CRBP) type I or cellular retinaldehyde-binding protein (CRALBP) suggests that RDH12 utilizes the unbound forms of all-trans-and 11-cis-retinoids. As a result, the widely expressed CRBPI, which binds all-trans-retinol with much higher affinity than all-transretinaldehyde, restricts the oxidation of all-trans-retinol by RDH12, but has little effect on the reduction of all-trans-retinaldehyde, and CRALBP inhibits the reduction of 11-cis-retinal stronger than the oxidation of 11-cis-retinol, in accord with its higher affinity for 11-cis-retinal. Together, the tissue distribution of RDH12 and its catalytic properties suggest that, in most tissues, RDH12 primarily contributes to the reduction of all-trans-retinaldehyde; however, at saturating concentrations of peroxidic aldehydes in the cells undergoing oxidative stress, for example, photoreceptors, RDH12 might also play a role in detoxification of lipid peroxidation products.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.