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The esterification of alcohols with fatty acids is a universal mechanism to form inert storage forms of sterols, di- and triacylglycerols, and retinoids. In ocular tissues, formation of retinyl esters is an essential step in the enzymatic regeneration of the visual chromophore (11-cis-retinal). Acyl-CoA wax alcohol acyltransferase 2 (AWAT2), also known as multifunctional O-acyltransferase (MFAT), is an integral membrane enzyme with a broad substrate specificity that has been shown to preferentially esterify 11-cis-retinol and thus contribute to formation of a readily available pool of cis retinoids in the eye. However, the mechanism by which this promiscuous enzyme can gain substrate specificity is unknown. Here, we provide evidence for an allosteric modulation of the enzymatic activity by 11-cis retinoids. This regulation is independent from cellular retinaldehyde-binding protein (CRALBP), the major cis-retinoid binding protein. This positive-feedback regulation leads to decreased esterification rates for 9-cis, 13-cis, or all-trans retinols and thus enables preferential synthesis of 11-cis-retinyl esters. Finally, electron microscopy analyses of the purified enzyme indicate that this allosteric effect does not result from formation of functional oligomers. Altogether, these data provide the experimental basis for understanding regulation of AWAT2 substrate specificity.

Retinosomes are intracellular lipid bodies found in the retinal pigment epithelium (RPE). They contain retinyl esters (REs) and are thought to be involved in visual chromophore regeneration during dark adaptation and in case of chromophore depletion. However, key enzymes in chromophore regeneration, retinoid isomerase (RPE65), and lecithin:retinol acyltransferase (LRAT) are located in the endoplasmic reticulum (ER). The mechanism and the enzyme responsible for mobilizing REs from retinosomes remained unknown. Our study demonstrates that patatin-like phospholipase domain containing 2 (PNPLA2) mobilizes all- trans -REs from retinosomes. The absence of PNPLA2 in mouse eyes leads to a significant accumulation of lipid droplets in RPE cells, declined electroretinography (ERG) response, and delayed dark adaptation compared with those of WT control mouse. Our work suggests a function of PNPLA2 as an RE hydrolase in the RPE, mobilizing REs from lipid bodies and functioning as an essential component of the visual cycle. [Display omitted] • PNPLA2 deficiency delays 11- cis -retinal regeneration in the visual cycle • Pnpla2 KO mice accumulate REs in lipid droplets in RPE • PNPLA2 mobilizes REs from lipid droplets via REH activity • PNPLA2 provides the source of substrate for RPE65 in the visual cycle Hara et al. identify the PNPLA2 enzyme that mobilizes vitamin A from the retinosomes in the RPE to serve as the substrate for the isomerase RPE65, an essential enzyme for the regeneration of visual pigment chromophore and maintenance of vision. [ABSTRACT FROM AUTHOR]

It has been proposed that cis-retinol dehydrogenase (cRDH) acts within the body to catalyze the oxidation of 9-cis-retinol, an oxidative step needed for 9-cis-retinoic acid synthesis, the oxidation of 11-cis-retinol [an oxidative step needed for 11-cis-retinal (visual chromophore) synthesis], and 3 α-hydroxysteroid transformations. To assess in vivo the physiological importance of each of these proposed actions of cRDH, we generated cRDH-deficient (cRDH−/−) mice. The cRDH−/− mice reproduce normally and appear to be normal. However, the mutant mice do have a mild visual phenotype of impaired dark adaptation. This phenotype is evidenced by electroretinagram analysis of the mice and by biochemical measures of eye levels of retinoid intermediates during recovery from an intense photobleach. Although it is thought that cRDH is expressed in the eye almost solely in retinal pigment epithelial cells, we detected cRDH expression in other retinal cells, including ganglion cells, amacrine cells, horizontal cells, and the inner segments of the rod photoreceptor cells. Aside from the eye, there are no marked differences in retinoid levels in other tissues throughout the body for cRDH−/− compared with cRDH+/+ mice. Moreover, we did not detect any nonvisual phenotypic changes for cRDH−/− mice, suggesting that these mice do not have problems in metabolizing 3 α-hydroxysteroids. Thus, cRDH may act essentially in the visual cycle but is redundant for catalyzing 9-cis-retinoic acid formation and 3 α-hydroxysteroid metabolism.—Shang, E., K. Lai, A. I. Packer, J. Paik, W. S. Blaner, M. de Morais Vieira, P. Gouras, and D. J. Wolgemuth. Targeted disruption of the mouse cis-retinol dehydrogenase gene: visual and nonvisual functions. J. Lipid Res. 2002. 43: 590–597.

It is known that exogenous 11-cis-retinol inhibits the recovery of photosensitivity of bleached rod outer segments (ROS) and 11-cis-retinol exists in the interphotorecepter matrix. We examined the conversion of 11-cis-retinol with bovine ROS. ROS was incubated with 11-cis-retinol under dim red light. Retinoids were extracted from the reaction mixture with hexane and analyzed by HPLC coupled with a fluorescence spectrophotometer. Isomerization of 11-cis-retinol to all-trans-retinol was observed in the presence of ROS. This isomerization was not suppressed by heat treatment and did not have stereospecificity. In addition, we incubated purified rhodopsin and phospholipids extracted from ROS with 11-cis-retinol. Rhodopsin was found to isomerize 11-cis-retinol to all-trans-retinol as well as ROS, but phospholipids did not. In contrast, the phospholipids inhibited the isomerization of 11-cis-retinol to all-trans-retinol by the purified rhodopsin. Commercially available phospholipids, especially phosphatidylserine, also inhibited the isomerization. Our results suggest that rhodopsin has activity for the isomerization of 11-cis-retinol to all-trans-retinol and may play an important role in the detoxification of 11-cis-retinol in the ROS. [ABSTRACT FROM AUTHOR]

The esterification of alcohols with fatty acids is a universal mechanism to form inert storage forms of sterols, di- and triacylglycerols, and retinoids. In ocular tissues, formation of retinyl esters is an essential step in the enzymatic regeneration of the visual chromophore (11- cis -retinal). Acyl-CoA wax alcohol acyltransferase 2 (AWAT2), also known as multifunctional O -acyltransferase (MFAT), is an integral membrane enzyme with a broad substrate specificity that has been shown to preferentially esterify 11- cis -retinol and thus contribute to formation of a readily available pool of cis retinoids in the eye. However, the mechanism by which this promiscuous enzyme can gain substrate specificity is unknown. Here, we provide evidence for an allosteric modulation of the enzymatic activity by 11- cis retinoids. This regulation is independent from cellular retinaldehyde-binding protein (CRALBP), the major cis -retinoid binding protein. This positive-feedback regulation leads to decreased esterification rates for 9- cis , 13- cis , or all- trans retinols and thus enables preferential synthesis of 11- cis -retinyl esters. Finally, electron microscopy analyses of the purified enzyme indicate that this allosteric effect does not result from formation of functional oligomers. Altogether, these data provide the experimental basis for understanding regulation of AWAT2 substrate specificity., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)