Resveratrol is an Efficient Uncompetitive Inhibitor of the RPE65 Isomerohydrolase

Abstract

Purpose: : Isomerization and hydrolysis of all-trans retinyl esters to 11-cis retinol is a key reaction in the visual cycle and is catalyzed by RPE65, an iron-dependent isomerohydrolase. The mechanism for the reaction catalyzed by RPE65 remains unknown. Previously, the involvement of free radical formation in the reactions catalyzed by RPE65 has been hypothesized. Resveratrol (3,4',5-trihydrostilbene) is a natural plant phytoalexin which demonstrated an efficient radical scavenging activity. To further determine the role of free radicals in the isomerohydrolase mechanism, we aim to study the effect of resveratrol on the RPE65 isomerohydrolase activity.

Methods: : All-trans-[³H]-retinol was used as a substrate to measure the activities of lecithin:retinol acyltransferase (LRAT) and isomerohydrolase in bovine RPE microsomes. Resveratrol dissolved in dimethylformamide was added in a small volume (less than 1%) from the stock solution into the reaction mixture. The generated retinoids were extracted and analyzed by HPLC with a flow scintillation analyzer. To determine the inhibition mode of the resveratrol, an adenoviral expression vector was used to express chicken RPE65 in 293A cells, and then RPE65 was used in a liposome-based isomerohydrolase assay.

Results: : Resveratrol showed a concentration-dependent inhibition of the isomerohydrolase activity as measured in bovine microsomes, with an IC₅₀ of 250 µM. In the same reaction system, LRAT activity was not affected by the resveratrol at a concentration as high as 2 mM. To analyze the inhibition type, all-trans retinyl palmitate was incorporated in liposomes and used as a substrate for recombinant RPE65. The concentration dependence of RPE65 reaction was measured in the absence and presence of resveratrol. The Lineweaver-Burk graph demonstrated two parallel lines suggesting that resveratrol inhibits the isomerohydrolase reaction in an uncompetitive manner.

Conclusions: : Resveratrol potently and selectively inhibited conversion of all-trans-retinyl ester to 11-cis-retinol catalyzed by RPE65 isomerohydrolase. The uncompetitive mode of the resveratrol inhibition is in agreement with a free radical-mediated mechanism of the RPE65 reaction. This inhibition may be used to slow down the visual cycle and to prevent accumulation of A2E in Stargardt’s disease and age-related macular degeneration.

Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium • age-related macular degeneration