Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle

Abstract

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...

RPE65 from Cone-dominant Chicken Is a More Efficient Isomerohydrolase Compared with That from Rod-dominant Species

“…ments (5)(6)(7). In the present study, we have demonstrated that the RPE65 in chicken eyes has significantly higher enzymatic activity, when compared with that in human and bovine eyes.…”

“…1). All of the key amino acid residues required for enzymatic activity, such as the His in the catalytic center (7,19), and the reported palmitoylated Cys residues, Cys-231 and Cys-329 (20), are identical between the chicken and human RPE65 except that the chicken RPE65 lacks the Cys-330, which was reported as a palmitoylation site in bovine RPE65 (20). Cys-330 is conserved in all mammalian RPE65 cloned, including human, bovine, and mouse RPE65, but is replaced by Thr in chicken RPE65 (Fig.…”

Parallel visual cycles in the zebrafish retina

“…The resulting all-trans RAL is first reduced to all-trans ROL by an NADPH-dependent dehydrogenase (Cideciyan et al, 2000;Lion et al, 1975;Rattner et al, 2000;Suzuki et al, 1993), and then transported from the photoreceptor to the second compartment involved in visual pigment recycling, the RPE. Upon arrival in RPE cells, all-trans ROL is esterified into retinylesters (REs) by the lecithin:retinol acyltransferase LRAT (Mondal et al, 2000;Ruiz et al, 1999;Saari and Bredberg, 1989;Saari et al, 1993), and then isomerized to 11-cis ROL by the isomerase RPE65 (or Isomerase I) (Gollapalli and Rando, 2003;Jin et al, 2005;Moiseyev et al, 2005;Redmond et al, 2005). Finally, 11-cis ROL is oxidized to 11-cis RAL by a retinol dehydrogenase (Cideciyan et al, 2000;Driessen et al, 1997;Gamble et al, 2000;Haeseleer et al, 1998;Lion et al, 1975;Simon et al, 1999;Suzuki et al, 1993).…”

“…The 11-cis ROL isomerase RPE65 is a key enzyme in the vertebrate visual cycle (Redmond et al, 2005;Redmond et al, 1998). It is highly expressed in the RPE of all vertebrates and is vital for regenerating the visual pigment 11-cis RAL.…”

“…However due to conflicting data in different studies, the question if an intra-retinal visual cycle exists in rod-dominant species remained controversial until recently (Fan et al, 2003;Feathers et al, 2008;Redmond et al, 2005;Redmond et al, 1998;Seeliger et al, 2001;Wenzel et al, 2007).…”