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 approximately 75% of wild type, W331F to approximately 50%, and W331L and W331Q to 0%) establishing a requirement for aromaticity, consistent with cation-pi 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.
Highlights
The mechanism of retinol isomerization in the vertebrate retina visual cycle remains controversial
Does the isomerase enzyme RPE65 operate via nucleophilic addition at C11 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
In this report we show that RPE65 is a leaky retinol isomerase and that this feature is consistent with a carbocation mechanism of action in RPE65 retinol isomerase activity
Summary
Site-directed Mutagenesis of RPE65—QuikChange XL sitedirected mutagenesis kit (Stratagene, La Jolla, CA) was used for mutagenesis of the RPE65 open reading frame cloned in pVitro (Invivogen, San Diego, CA). 24 h after transfection, all-trans-retinol was added to a final concentration of 2.5 M, and the cells were cultured for a further 7 h and harvested for analysis. The reaction was extracted with 2 ϫ 1 ml of hexane, and the extracts were pooled, dried, redissolved in hexane, and fractionated by normal phase HPLC as described above with n-octanol replaced by chloroform (85.4% n-hexane, 11.2% ethyl acetate, 2% 1,4-dioxane, 1.4% chloroform), and the all-trans-retinol peak was collected and quantified. The labeled all-trans-retinol was dried under vacuum and redissolved in 10 l of 2,5-dihydroxybenzoic acid in acetone (0.5 M) and analyzed by a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) method (Voyager-DE STR, Applied Biosystems, Foster City, CA) in positive ion reflective mode, modified from Wingerath et al [37]. Orientation of the retinyl moiety in the substrate cleft was based on the experimental findings of a requirement for aromaticity at aa 331 commensurate with cation- stabilization of the retinoid intermediate
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