RPE65

612119892ENSG00000116745ENSMUSG00000028174Q16518Q91ZQ5NM_000329NM_029987NP_000320NP_084263Retinal pigment epithelium-specific 65 kDa protein, also known as retinoid isomerohydrolase, is an enzyme of the vertebrate visual cycle that is encoded in humans by the RPE65 gene. RPE65 is expressed in the retinal pigment epithelium (RPE, a layer of epithelial cells that nourish the photoreceptor cells) and is responsible for the conversion of all-trans-retinyl esters to 11-cis-retinol during phototransduction. 11-cis-retinol is then used in visual pigment regeneration in photoreceptor cells. RPE65 belongs to the carotenoid oxygenase family of enzymes. Retinal pigment epithelium-specific 65 kDa protein, also known as retinoid isomerohydrolase, is an enzyme of the vertebrate visual cycle that is encoded in humans by the RPE65 gene. RPE65 is expressed in the retinal pigment epithelium (RPE, a layer of epithelial cells that nourish the photoreceptor cells) and is responsible for the conversion of all-trans-retinyl esters to 11-cis-retinol during phototransduction. 11-cis-retinol is then used in visual pigment regeneration in photoreceptor cells. RPE65 belongs to the carotenoid oxygenase family of enzymes. RPE65 is a critical enzyme in the vertebrate visual cycle found the retinal pigmented epithelium. It is also found in rods and cones. The photoisomerization of 11-cis-retinal to all-trans-retinal initiates the phototransduction pathway through which the brain detects light. All-trans-retinol is not photoactive and therefore must be reconverted to 11-cis-retinal before it can recombine with opsin to form an active visual pigment. RPE65 reverses the photoisomerization by converting an all-trans-retinyl ester to 11-cis-retinol. Most commonly, the ester substrate is retinyl palmitate. The other enzymes of the visual cycle complete the reactions necessary to oxidize and esterify all-trans-retinol to a retinyl ester (RPE65's substrate) and to oxidize 11-cis-retinol to 11-cis-retinal (the required photoactive visual pigment component). RPE65 is also referred to as retinol isomerase or retinoid isomerase, owing to past debates about the enzyme's substrate and whether it was involved in ester hydrolysis. RPE65 is a dimer of two symmetrical, enzymatically independent subunits. The active site of each subunit has a seven-bladed beta-propeller structure with four histidines that hold an iron(II) cofactor. This structural motif is common across the studied members of the carotenoid oxygenase family of enzymes. RPE65 is strongly associated with the membrane of the smooth endoplasmic reticulum in RPE cells. The active site of each RPE65 active site contains an Fe(II) cofactor bound by four histidines (His180, His241, His313, and His527), each contributed by a separate blade on the beta-propeller structure. Three of the four histidines are coordinated to nearby glutamic acid residues (Glu148, Glu417, and Glu469), which are thought to help position the histidines to bind the iron cofactor in an octahedral geometry. Phe103, Thr147, and Glu148 surround the active site where they help stabilize the carbocation intermediate and increase the stereoselectivity of RPE65 for 11-cis-retinol over 13-cis-retinol. Reactants and products likely enter and leave the active site through a hydrophobic tunnel which is thought to open into the lipid membrane for direct lipid substrate absorption. A second, smaller tunnel also reaches the active site and may serve as a pathway for water, but is too narrow to transport the retinoid reactants and products. RPE65 is strongly associated with the membrane of the sER. sER is abnormally abundant in RPE cells due to their role in processing lipidic retinoids. Structural studies indicate that RPE65 is partially imbedded in the sER membrane via interactions between its hydrophobic face and the interior of the lipid membrane. This is supported by the need for detergent to solubilize RPE65. A major portion of RPE65’s hydrophobic face, residues 109-126, forms an amphipathic alpha helix that likely contributes to the protein’s membrane affinity. Additionally, Cys112 is palmitoylated in native RPE65, further supporting the theory that the hydrophobic face of RPE65 is imbedded in the membrane. The hydrophobic face contains the entrance to the large tunnel that leads to the enzyme’s active site. The presence of this channel on the hydrophobic face combined with RPE65’s demonstrated ability to absorb substrate direction from the lipid bilayer is consistent with RPE65 being partially embedded in the membrane. RPE65 has been isolated from a wide range of vertebrates including zebra fish, chicken, mice, frogs, and humans. Its structure is highly conserved between species, particularly in the beta-propeller and likely membrane bound regions. The amino acid sequences of human and bovine RPE65 differ by less than 1%. The histidine residues of the beta-propeller structure and the bound iron(II) cofactor are 100% conserved across studied RPE65 orthologs and other members of the carotenoid oxygenase family.