title: Aromatic residues in the substrate cleft of RPE65 protein govern retinol isomerization and modulate its progression.
abstract: Previously, we showed that mutating RPE65 residue Phe-103 preferentially produces 13-cis-retinol instead of 11-cis-retinol, supporting a carbocation/radical cation mechanism of retinol isomerization. We asked whether this modulation of specificity can occur with residues other than Phe-103 and what role it plays in substrate binding and isomerization. We modeled the substrate-binding cleft of RPE65 to identify residues lining its surface. Many are phenylalanines and tyrosines, including three Phe residues (Phe-61, Phe-312, and Phe-526) forming an arch-like arrangement astride the cleft and Tyr-338. Also, Phe-418 sits at the neck of the cleft, lending a bend to the volume enclosed by the cleft. All mutations of Phe-61, Phe-312, and Phe-418 result in severely impaired or inactive enzyme. However, mutation of Phe-526 and Tyr-338, like Phe-103, decreases 11-cis-retinol formation, whereas increasing the 13-cis isomer. Significantly, 2 of these 3 residues, Phe-103 and Tyr-338, are located on putatively mobile interstrand loops. We propose that residual densities located in the binding cleft of the RPE65 structure represents a post-cleavage snapshot consistent not only with a fatty acid product, as originally modeled, but also an 11-cis-retinol product. Substrate docking simulations permit 11-cis- or 13-cis-retinyl ester binding in this relatively closed cleft, with the latter favored in F103L, F526A, and Y338A mutant structures, but prohibit binding of all-trans-retinyl ester, suggesting that isomerization occurs early in the temporal sequence, with O-alkyl ester cleavage occurring later. These findings provide insight into the mechanism of isomerization central to the visual cycle.
authors: Laboratory of Retinal Cell and Molecular Biology, NEI, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Preethi
authors: P
authors: Chander
authors: Susan
authors: S
authors: Gentleman
authors: Eugenia
authors: E
authors: Poliakov
authors: T Michael
authors: TM
authors: Redmond
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D051379:Mice; D020125:Mutation, Missense; D010649:Phenylalanine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014801:Vitamin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019745:cis-trans-Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000099178:Retinoid Isomerohydrolase
chemical_list: D010649:Phenylalanine; D014443:Tyrosine; D014801:Vitamin A; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase