Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Abstract

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A 2 treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.In vertebrates, the perception of light is preceded by a quantum event in which a photon of light hits the 11-cis-retinylidene chromophore of rhodopsin in the retina. Photoisomerization of this chromophore to its all-trans isomer results in activation of rhodopsin triggering a cascade of signal transduction events (summarized in Ref. 1). This process simultaneously leads to loss of photoreceptor light sensitivity making continuous regeneration of 11-cis-retinal a requirement to sustain vision. The visual (retinoid) cycle is the fundamental series of reactions responsible for regeneration of the visual chromophore that occurs in photoreceptors and the retinal pigment epithelium (RPE) 4 (summarized in Ref.2). The key component of this enzymatic cycle is the RPE-specific microsomal membrane protein known as RPE65 (3-5). The importance of RPE65 in the retinoid cycle is underscored by the metabolic dysfunction found in Rpe65 Ϫ/Ϫ mice, which is characterized by complete lack of 11-cis-retinoid production accompanied by accumulation of the all-tr...

Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase

“…Also, substrates for CCOs are highly lipophilic and tend to reside in hydrophobic environments, such as plant thylakoid membranes (10) or liposomes in mammals (11)(12)(13). Accordingly, CCOs show varying levels of membrane association, from soluble to integral (14)(15)(16). ACO, VP14, and RPE65 all have surface-exposed hydrophobic patches in analogous regions that may serve as membrane interaction domains.…”

Structure of RPE65 isomerase in a lipidic matrix reveals roles for phospholipids and iron in catalysis

“…Purified RPE65 exhibited retinoid isomerase activity only when bound to all-trans-retinyl ester-incorporated membrane vesicles (10). Moreover, destruction of intact microsomal phospholipids by phospholipase A 2 greatly reduced the production of 11-cis-retinol by native bovine RPE65 (11). Spectroscopic data also indicated that RPE65 adopts functionally and structurally different conformations when bound to lipid bilayers compared with its detergent-solubilized state, although specific changes could not be ascertained (12).…”

“…Historically, the inhibition of RPE retinoid isomerase activity by detergents was the major factor that delayed its link to RPE65, and the mechanism of inhibition remains incompletely understood. In the structure, we observed that residues composing the RPE65 membrane-interaction surface exhibit substantial disorder compared with the rest of the enzyme (11,13). Owing to the roles of these residues in membrane binding as well as substrate recognition and proper positioning of the substrate in the active site, detergent-induced alterations in their native structure, either by stripping the protein of its native phospholipids or by a direct effect, could inactivate the enzyme.…”

Complementation Test ofRpe65Knockout and Tvrm148

“…Recent work concerning the three dimensional structure of B. taurus RPE65 protein [61][62][63][64] provides insight into reasons why the RPE65 F229S mutant protein may be nonfunctional and possibly unstable. The Rpe65 tvrm148 mutation resides in exon 7 of RPE65 1 and results in a substitution of Ser for Phe at amino acid position 229.…”