title: RPE65, visual cycle retinol isomerase, is not inherently 11-cis-specific: support for a carbocation mechanism of retinol isomerization.
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.
authors.affiliation: Laboratory of Retinal Cell and Molecular Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892, USA. redmond@helix.nih.gov
authors.forename: T Michael
authors.initials: TM
authors.lastname: Redmond
authors.forename: Eugenia
authors.initials: E
authors.lastname: Poliakov
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kuo
authors.forename: Preethi
authors.initials: P
authors.lastname: Chander
authors.forename: Susan
authors.initials: S
authors.lastname: Gentleman
mesh_terms: D000818:Animals; D002244:Carbon / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D012160:Retina / Q000166:cytology / Q000201:enzymology; D013237:Stereoisomerism; D013379:Substrate Specificity; D014443:Tyrosine; D014801:Vitamin A / Q000737:chemistry* / Q000378:metabolism*; D019745:cis-trans-Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099178:Retinoid Isomerohydrolase
chemical_list: D002244:Carbon; D002352:Carrier Proteins; D005136:Eye Proteins; D010100:Oxygen; D014443:Tyrosine; D014801:Vitamin A; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase