RPE65, Visual Cycle Retinol Isomerase, Is Not Inherently 11-cis-specific

Abstract

The mechanism of retinol isomerization in the vertebrate retina visual cycle remains controversial. Does the isomerase enzyme RPE65 operate via nucleophilic addition at C 11 of the all-trans substrate, or via a carbocation mechanism? To determine this, we modeled the RPE65 substrate cleft to identify residues interacting with substrate and/or intermediate. We find that wild-type RPE65 in vitro produces 13-cis and 11-cis isomers equally robustly. All Tyr-239 mutations abolish activity. Trp-331 mutations reduce activity (W331Y to ϳ75% of wild type, W331F to ϳ50%, and W331L and W331Q to 0%) establishing a requirement for aromaticity, consistent with cation-carbocation stabilization. Two cleft residues modulate isomerization specificity: Thr-147 is important, because replacement by Ser increases 11-cis relative to 13-cis by 40% compared with wild type. Phe-103 mutations are opposite in action: F103L and F103I dramatically reduce 11-cis synthesis relative to 13-cis synthesis compared with wild type. Thr-147 and Phe-103 thus may be pivotal in controlling RPE65 specificity. Also, mutations affecting RPE65 activity coordinately depress 11-cis and 13-cis isomer production but diverge as 11-cis decreases to zero, whereas 13-cis reaches a plateau consistent with thermal isomerization. Lastly, experiments using labeled retinol showed exchange at 13-cis-retinol C 15 oxygen, thus confirming enzymatic isomerization for both isomers. Thus, RPE65 is not inherently 11-cis-specific and can produce both 11-and 13-cis isomers, supporting a carbocation (or radical cation) mechanism for isomerization. Specific visual cycle selectivity for 11-cis isomers instead resides downstream, attributable to mass action by CRALBP, retinol dehydrogenase 5, and high affinity of opsin apoproteins for 11-cis-retinal.A sequence of metabolic events, termed the visual cycle (1, 2), keeps retinal visual pigments, such as rhodopsin, in a state capable of responding to light. In brief, 11-cis-retinal bound to rhodopsin is photo-isomerized to all-trans-retinal, activating rhodopsin. To regenerate rhodopsin, all-trans-retinal is released, reduced to all-trans-retinol that is transported to the retinal pigment epithelium (RPE), 3 and esterified to all-trans-retinyl esters, the substrate for the retinol isomerase (3). All-transretinyl esters are enzymatically isomerized to yield 11-cis-retinol that is oxidized to 11-cis-retinal and returned to the photoreceptors (3, 4). Recently, the RPE protein RPE65 (5) has been identified as the isomerase central to this cycle (6 -8). The importance of RPE65 in chromophore regeneration had been well established by Rpe65 knock-out mice, which display extreme chromophore starvation (no rhodopsin) in the photoreceptors concurrent with overaccumulation of the all-transretinyl ester substrate of RPE65 in the RPE (9). Consequently, Rpe65 Ϫ/Ϫ mice are extremely insensitive to light. Mutations in the human RPE65 gene cause Leber congenital amaurosis 2, a condition of severe early onset blindness (10 -13), which has been the subject...

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