Mutation of RPE65 can cause severe blindness from birth or early childhood, and RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Here, we show that Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65-/- mice are disorganized compared with those of Rpe65+/+ and Rpe65+/- mice. Rod function, as measured by electroretinography, is abolished in Rpe65-/- mice, although cone function remains. Rpe65-/- mice lack rhodopsin, but not opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65-/- mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/- phenotype, although cone pigment regeneration may be dependent on a separate pathway.

RPE65 is essential for isomerization of vitamin A to the visual chromophore. Mutations in RPE65 cause early-onset blindness, and Rpe65-deficient mice lack 11-cis-retinal but overaccumulate alltrans-retinyl esters in the retinal pigment epithelium (RPE). RPE65 is proposed to be a substrate chaperone but may have an enzymatic role because it is closely related to carotenoid oxygenases. We hypothesize that, by analogy with other carotenoid oxygenases, the predicted iron-coordinating residues of RPE65 are essential for retinoid isomerization. To clarify RPE65's role in isomerization, we reconstituted a robust minimal visual cycle in 293-F cells. Only cells transfected with RPE65 constructs produced 11-cisretinoids, but coexpression with lecithin:retinol acyltransferase was needed for high-level production. Accumulation was significant, amounting to >2 nmol of 11-cis-retinol per culture. Transfection with constructs harboring mutations in residues of RPE65 homologous to those required for interlinked enzymatic activity and iron coordination in related enzymes abolish this isomerization. Iron chelation also abolished isomerization activity. Mutating cysteines implicated in palmitoylation of RPE65 had generally little effect on isomerization activity. Mutations associated with Leber congenital amaurosis͞early-onset blindness cause partial to total loss of isomerization activity in direct relation to their clinical effects. These findings establish a catalytic role, in conjunction with lecithin:retinol acyltransferase, for RPE65 in synthesis of 11-cisretinol, and its identity as the isomerohydrolase.11-cis-retinoids ͉ Leber congenital amaurosis ͉ retinal pigment epithelium R egeneration of 11-cis-retinal, the chromophore of all visual pigments (opsins), occurs by a process in the retinal pigment epithelium (RPE) termed the visual cycle that involves isomerization of all-trans-retinyl esters to 11-cis-retinol. In outline, lecithin:retinol acyltransferase (LRAT) (1, 2) esterifies incoming all-trans-retinol to all-trans-retinyl esters, the substrate for the putative isomerohydrolase (IMH) (3, 4). IMH is postulated to perform a concerted hydrolysis and isomerization yielding 11-cis-retinol, which is trapped by cellular retinaldehyde-binding protein (CRALBP) and then oxidized to 11-cis-retinal by 11-cis-retinol dehydrogenase͞RDH5 (11cisRDH). An alternate mechanism proposes generation of 11-cis-retinol via a retinyl carbocation intermediate (5). Mutations of RDH5, RPE65, LRAT, and CRALBP in the human (6 -12) and cognate disruptions in the mouse (13-16) result in mild to severe blindness due to derangement of RPE retinoid metabolism. CRALBP and RDH5 disruptions do not block the visual cycle but reduce its efficiency (13, 16). Although mutation or loss of LRAT and RPE65 each block the visual cycle, they do so differently. With LRAT loss, no vitamin A accumulates in the RPE, obviating retinoid metabolism (15). With RPE65 loss, all-trans-retinyl ester accumulates to very high levels, but 11-cis-retinoids are not formed (14).Howev...

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