Regulation of patterning and morphogenesis during embryonic development depends on tissue-specific signaling by retinoic acid (RA), the active form of Vitamin A (retinol). The first enzymatic step in RA synthesis, the oxidation of retinol to retinal, is thought to be carried out by the ubiquitous or overlapping activities of redundant alcohol dehydrogenases. The second oxidation step, the conversion of retinal to RA, is performed by retinaldehyde dehydrogenases. Thus, the specific spatiotemporal distribution of retinoid synthesis is believed to be controlled exclusively at the level of the second oxidation reaction. In an N-ethyl-N-nitrosourea (ENU)-induced forward genetic screen we discovered a new midgestation lethal mouse mutant, called trex, which displays craniofacial, limb, and organ abnormalities. The trex phenotype is caused by a mutation in the short-chain dehydrogenase/reductase, RDH10. Using protein modeling, enzymatic assays, and mutant embryos, we determined that RDH10 trex mutant protein lacks the ability to oxidize retinol to retinal, resulting in insufficient RA signaling. Thus, we show that the first oxidative step of Vitamin A metabolism, which is catalyzed in large part by the retinol dehydrogenase RDH10, is critical for the spatiotemporal synthesis of RA. Furthermore, these results identify a new nodal point in RA metabolism during embryogenesis.[Keywords: Retinoic acid; Vitamin A; orofacial cleft; limb] Supplemental material is available at http://www.genesdev.org.

RPE65 is an abundant protein in the retinal pigment epithelium. Mutations in RPE65 are associated with inherited retinal dystrophies. Although it is known that RPE65 is critical for regeneration of 11-cis retinol in the visual cycle, the function of RPE65 is elusive. Here we show that recombinant RPE65, when expressed in QBI-293A and COS-1 cells, has robust enzymatic activity of the previous unidentified isomerohydrolase, an enzyme converting all-trans retinyl ester to 11-cis retinol in the visual cycle. The initial rate for the reaction is 2.9 pmol͞min per mg of RPE65 expressed in 293A cells. The isomerohydrolase activity of RPE65 requires coexpression of lecithin retinol acyltransferase in the same cell to provide its substrate. This enzymatic activity is linearly dependent on the expression levels of RPE65. This study demonstrates that RPE65 is the long-sought isomerohydrolase and fills a major gap in our understanding of the visual cycle. Identification of the function of RPE65 will contribute to the understanding of the pathogenesis for retinal dystrophies associated with RPE65 mutations.isomerase ͉ LRAT ͉ retinal dystrophy ͉ retinyl ester ͉ 11-cis retinal I n vertebrates, vision is initiated in rod and cone photoreceptors.The photosensitive entities in these cells are the visual pigments which consist of an apoprotein, opsin, and a chromophore, 11-cis retinal, which is attached to the opsin by a Schiff's base bond (1). Upon absorption of light by the pigments, 11-cis retinal is isomerized to all-trans retinal, which leads to the conformational changes of opsin and subsequently activates G protein transducin, initiating vision (1, 2). Efficient regeneration of 11-cis retinal, referred to as the visual retinoid cycle (see Fig. 1) is critical for the regeneration of the visual pigments (for reviews, see refs. 3-5) and maintenance of visual function. Disruption of the visual cycle by mutation or dysfunction of one of the numerous enzymes involved in this process leads to a number of blinding disorders (6).The enzyme in the visual cycle that has eluded identification is the isomerohydrolase, which is responsible for the isomerization and hydrolysis of all-trans retinyl ester to 11-cis retinol. Rando and colleagues (7) first proposed that all-trans retinol is first acylated to retinyl esters by lecithin retinol acyltransferase (LRAT). The generated all-trans retinyl ester is then directly isomerized and hydrolized into 11-cis retinol in the retinal pigment epithelium (RPE). This hydrolysis-isomerization process has been proposed to be catalyzed by a single enzyme, referred to as isomerohydrolase, which is associated with the membrane in RPE microsomes (8).Although isomerohydrolase activity had been demonstrated in the RPE almost 20 years ago, identification of the enzyme catalyzing this reaction has been difficult, because this activity is associated with the membrane and is abolished by solubilization in all of the detergents investigated (9). As a result, the isomerohydrolase has not been identified despite in...

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.