Biochemical Properties of Purified Human Retinol Dehydrogenase 12 (RDH12):  Catalytic Efficiency toward Retinoids and C₉ Aldehydes and Effects of Cellular Retinol-Binding Protein Type I (CRBPI) and Cellular Retinaldehyde-Binding Protein (CRALBP) on the Oxidation and Reduction of Retinoids

Abstract

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.

Kinetic Analysis of Human Enzyme RDH10 Defines the Characteristics of a Physiologically Relevant Retinol Dehydrogenase

“…This PCR product was subcloned between the XbaI and HindIII sites of pET28a vector (Novagen, Madison, WI) as an intermediate step before transferring the RDH10 cDNA into the SmaI-NotI sites of the pVL1393 vector modified as described previously (18,19). The final construct of RDH10 in pVL1393-encoded RDH10 fused with the His 6 tag on the C terminus.…”

“…Determination of kinetic constants of RDH10 was performed as described previously in detail for RDH12 and RDH11 (18,19). All-trans-retinol, all-trans-retinaldehyde, and 9-cis-retinaldehyde were obtained from Sigma and purified by highpressure liquid chromatography (HPLC).…”

“…The amount of protein varied between 3 and 20 g, so that the amount of the product did not exceed 10% of the substrate. Reaction rates were determined based on the percent substrate conversion as described previously (18,19). Kinetic constants were calculated using GraFit (Erithacus Software Ltd., UK) and expressed as the mean Ϯ S.D.…”

“…ApoCRBPI and holoCRBPI were prepared as described previously (19). The A 350 /A 280 ratio of the holo-CRBPI was 1.75.…”

Identification of Key Residues Determining Isomerohydrolase Activity of Human RPE65

“…) (24) in comparison with other retinoid-processing enzymes (25,26). This hypothesis is supported by the observation that endogenous RPE65 exists at high levels in the RPE (11 g/eye in bovine), probably to compensate for its relatively low activity (27).…”