RPE65 operates in the vertebrate visual cycle by stereospecifically binding all-trans-retinyl esters

RPE65 is a major protein of unknown function found associated with the retinyl pigment epithelial (RPE) membranes [Hamel, C. P., Tsilou, E., Pfeffer, B. A., Hooks, J. J., Detrick, B., and Redmond, T. M. (1993) J. Biol. Chem. 268, 15751-15757; Bavik, C. O., Levy, F., Hellman, U., Wernstedt, C., and Eriksson, U. (1993) J. Biol. Chem. 268, 20540-20546]. RPE65 knockouts fail to synthesize 11-cis-retinal, the chromophore of rhodopsin, and accumulate all-trans-retinyl esters in the RPE. Previous studies have also shown that RPE65 is specifically labeled with all-trans-retinyl ester based affinity labeling agents, suggesting a retinyl ester binding role for the protein. In the present work, we show that purified RPE65 binds all-trans-retinyl palmitate (tRP) with a K(D) = 20 pM. These quantitative experiments are performed by measuring the quenching of RPE65 fluorescence by added tRP. The binding for tRP is highly specific because 11-cis-retinyl palmitate binds with a K(D) = 14 nM, 11-cis-retinol binds with a K(D) = 3.8 nM, and all-trans-retinol (vitamin A) binds with a K(D) = 10.8 nM. This stereospecificity for tRP is to be compared to the binding of retinoids to BSA, where virtually no discrimination is found in the binding of the same retinoids. This work provides further evidence that RPE65 functions by binding to and mobilizing the highly hydrophobic all-trans-retinyl esters, allowing them to enter the visual cycle.     

A palmitoylation switch mechanism in the regulation of the visual cycle

RPE65 is essential for the biosynthesis of 11-cis-retinal, the chromophore of rhodopsin. Here, we show that the membrane-associated form (mRPE65) is triply palmitoylated and is a chaperone for all-trans-retinyl esters, allowing their entry into the visual cycle for processing into 11-cis-retinal. The soluble form of RPE65 (sRPE65) is not palmitoylated and is a chaperone for vitamin A, rather than all-trans-retinyl esters. Thus, the palmitoylation of RPE65 controls its ligand binding selectivity. The two chaperones are interconverted by lecithin retinol acyl transferase (LRAT) acting as a molecular switch. Here mRPE65 is a palmitoyl donor, revealing a new acyl carrier protein role for palmitoylated proteins. When chromophore synthesis is not required, mRPE65 is converted into sRPE65 by LRAT, and further chromophore synthesis is blocked. The studies reveal new roles for palmitoylated proteins as molecular switches and LRAT as a palmitoyl transferase whose role is to catalyze the mRPE65 to sRPE65 conversion.     

Palmitoyl transferase activity of lecithin retinol acyl transferase

Lecithin retinol acyl transferase (LRAT) has the essential role of catalyzing the transfer of an acyl group from the sn-1 position of lecithin to vitamin A to generate all-trans-retinyl esters (tREs). In vitro studies had shown previously that LRAT also can exchange palmitoyl groups between RPE65, a tRE binding protein essential for vision, and tREs. This exchange is likely to be of regulatory significance in the operation of the visual cycle. In the current study, the substrate specificity of LRAT is explored with palmitoylated amino acids and dipeptides as RPE65 surrogates. Both O- and S-substituted palmitoylated analogues are excellent substrates for tLRAT, a readily expressed and readily purified form of LRAT. Using vitamin A as the palmitoyl acceptor, tREs are readily formed. The cognate of these reactions occurs in crude retinal pigment epithelial (RPE) membranes as well. RPE membranes containing LRAT transfer palmitoyl groups from radiolabeled [1-(14)C]-l-alpha-dipalmitoyl diphosphatidylcholine (DPPC) to RPE65. Palmitoyl transfer is abolished by preincubation with a specific LRAT antagonist both in membranes and with purified tLRAT. These experiments are consistent with an expanded role for LRAT function as a protein palmitoyl transferase.     

Rpe65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of beta-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.     

Computational studies for the structure and function of mRPE65

The mRPE65 protein is one form of the RPE65 protein and plays a very important role in the visual cycle. However, its 3D structure and detailed mechanism of function are still unclear because of difficulties with isolation and crystallization. This computational study reports a model for the mRPE65 protein structure derived from a model for sRPE65. The natural substrate for RPE65 has been shown to be a retinyl ester and, by utilizing the Autodock and the Ligplot programs, the interactions between the ester and the protein as well as the effects of several mutations on these interactions are studied. Finally, the position of the binding site is proposed based on an iterative process and the effects of the mutations on the binding site are also discussed.     

Preferential release of 11-cis-retinol from retinal pigment epithelial cells in the presence of cellular retinaldehyde-binding protein

In photoreceptor cells of the retina, photoisomerization of 11-cis-retinal to all-trans-retinal triggers phototransduction. Regeneration of 11-cis-retinal proceeds via a complex set of reactions in photoreceptors and in adjacent retinal pigment epithelial cells where all-trans-retinol is isomerized to 11-cis-retinol. Our results show that isomerization in vitro only occurs in the presence of apo-cellular retinaldehyde-binding protein. This retinoid-binding protein may drive the reaction by mass action, overcoming the thermodynamically unfavorable isomerization. Furthermore, this 11-cis-retinol/11-cis-retinal-specific binding protein potently stimulates hydrolysis of endogenous 11-cis-retinyl esters but has no effect on hydrolysis of all-trans-retinyl esters. Apo-cellular retinaldehyde-binding protein probably exerts its effect by trapping the 11-cis-retinol product. When retinoid-depleted retinal pigment epithelial microsomes were preincubated with different amounts of all-trans-retinol to form all-trans-retinyl esters and then [3H]all-trans-retinol was added, as predicted, the specific radioactivity of [3H]all-trans-retinyl esters increased during subsequent reaction. However, the specific radioactivity of newly formed 11-cis-retinol stayed constant during the course of the reaction, and it was largely unaffected by expansion of the all-trans-retinyl ester pool during the preincubation. The absence of dilution establishes that most of the ester pool does not participate in isomerization, which in turn suggests that a retinoid intermediate other than all-trans-retinyl ester is on the isomerization reaction pathway.     

Retinal pigment epithelium-retinal G protein receptor-opsin mediates light-dependent translocation of all-trans-retinyl esters for synthesis of visual chromophore in retinal pigment epithelial cells

Visual perception begins with the absorption of a photon by an opsin pigment, inducing isomerization of its 11-cis-retinaldehyde chromophore. After a brief period of activation, the resulting all-trans-retinaldehyde dissociates from the opsin apoprotein rendering it insensitive to light. Restoring light sensitivity to apo-opsin requires thermal re-isomerization of all-trans-retinaldehyde to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle in retinal pigment epithelial (RPE) cells. Vertebrates can see over a 10(8)-fold range of background illumination. This implies that the visual cycle can regenerate a visual chromophore over a similarly broad range. However, nothing is known about how the visual cycle is regulated. Here we show that RPE cells, functionally or physically separated from photoreceptors, respond to light by mobilizing all-trans-retinyl esters. These retinyl esters are substrates for the retinoid isomerase and hence critical for regenerating visual chromophore. We show in knock-out mice and by RNA interference in human RPE cells that this mobilization is mediated by a protein called "RPE-retinal G protein receptor" (RGR) opsin. These data establish that RPE cells are intrinsically sensitive to light. Finally, we show that in the dark, RGR-opsin inhibits lecithin:retinol acyltransferase and all-trans-retinyl ester hydrolase in vitro and that this inhibition is released upon exposure to light. The results of this study suggest that RGR-opsin mediates light-dependent translocation of all-trans-retinyl esters from a storage pool in lipid droplets to an "isomerase pool" in membranes of the endoplasmic reticulum. This translocation permits insoluble all-trans-retinyl esters to be utilized as substrate for the synthesis of a new visual chromophore.     

Quantitation of biological retinoids by high-pressure liquid chromatography: primary internal standardization using tritiated retinoids

A single method is described for quantitation of 14 retinoids found in biological material. The method consists of reversed-phase HPLC, internal standardization, and carrier extraction procedures with three synthetic retinoids. Primary standardization of HPLC uv detector is achieved using tritiated all-trans-retinoic acid, all-trans-retinol, all-trans-retinyl palmitate, and all-trans-retinyl acetate. Extraction methods are standardized by correlating the uv absorbance of retinoids at 340 nm with radioactivity of tritiated retinoids of known specific activity. Quantitation of 10 pg of tritiated or 5 ng of nonradioactive retinoid per 0.1 g sample in a polarity range from 4-oxo-retinoic acid to retinyl stearate can be achieved in a single, 50-min chromatographic run. A single HPLC pump, a C18 reversed-phase analytical column, a multistep three-solvent gradient, and inexpensive solvents based on methanol, water, and chloroform comprise this cost-effective chromatographic system. Our primary standardization method allows investigators employing different procedures to compare results between laboratories by standardizing the HPLC uv detector with commercially available tritiated retinoids. With this method we were able to quantitate nanomolar amounts of endogenous retinoic acids and retinyl esters, that "HPLC uv only" conditions usually would not detect in the circulation and liver of rats under physiological conditions.     

Hydrolysis of 11-cis- and all-trans-retinyl palmitate by homogenates of human retinal epithelial cells

The retinal epithelium plays an important role in the storage and metabolism of retinoids in the eye. Studies were conducted to examine the enzymatic hydrolysis of retinyl esters by human retinal epithelial cells. Homogenates prepared from these cells were found to hydrolyze both the 11-cis- and all-trans-isomers of retinyl palmitate. Retinyl ester hydrolysis was time-, protein-, and pH-dependent. The 11-cis isomer was hydrolyzed at a rate which was approximately 20 times greater than that of the all-trans isomer. The 11-cis-retinyl palmitate hydrolase activity did not require detergents, unlike the all-trans-retinyl palmitate hydrolase activity, which required detergents for activity. The 11-cis-retinyl palmitate hydrolase activity was maximally active with the addition of 1.0% sodium taurocholate at about pH 8.5, was abolished by incubation at 50 degrees C for 10 min, and was quantitatively recovered in the pellet after centrifugation at 100,000 X g for 1 h. The rate of hydrolysis of 11-cis-retinyl palmitate became saturated with increasing concentrations of 11-cis-retinyl palmitate; under the assay conditions employed, the hydrolase activity had an apparent Km of 19 microM toward 11-cis-retinyl palmitate. All-trans-retinol and 11-cis-retinyl did not affect the rate of hydrolysis of 11-cis-retinyl palmitate, and addition of all-trans-retinyl palmitate only weakly inhibited the 11-cis-retinyl palmitate hydrolytic activities. These data indicate that the human retinal epithelium possesses distinct activities for the hydrolysis of 11-cis- and all-trans-retinyl esters and raise the possibility that these activities may provide a means of distinguishing the stereoisomers of retinol in this tissue.     

Aberrant metabolites in mouse models of congenital blinding diseases: formation and storage of retinyl esters

Regeneration of the visual chromophore, 11-cis-retinal, is a critical step in restoring photoreceptors to their dark-adapted conditions. This regeneration process, called the retinoid cycle, takes place in the photoreceptor outer segments and the retinal pigment epithelium (RPE). Disabling mutations in nearly all of the retinoid cycle genes are linked to human conditions that cause congenital or progressive defects in vision. Several mouse models with disrupted genes related to this cycle contain abnormal fatty acid retinyl ester levels in the RPE. To investigate the mechanisms of retinyl ester accumulation, we generated single or double knockout mice lacking retinoid cycle genes. All-trans-retinyl esters accumulated in mice lacking RPE65, but they are reduced in double knockout mice also lacking opsin, suggesting a connection between visual pigment regeneration and the retinoid cycle. Only Rdh5-deficient mice accumulate cis-retinyl esters, regardless of the simultaneous disruption of RPE65, opsin, and prRDH. 13-cis-Retinoids are produced at higher levels when the flow of retinoid through the cycle was increased, and these esters are stored in specific structures called retinosomes. Most importantly, retinylamine, a specific and effective inhibitor of the 11-cis-retinol formation, also inhibits the production of 13-cis-retinyl esters. The data presented here support the idea that 13-cis-retinyl esters are formed through an aberrant enzymatic isomerization process.     

Role of LRAT on the retinoid isomerase activity and membrane association of Rpe65

Absorption of a photon by a vertebrate opsin pigment induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical re-isomerization of the chromophore via an enzyme pathway called the visual cycle. The retinoid isomerase in this pathway is Rpe65, a membrane-associated protein in the retinal pigment epithelium (RPE) with no predicted membrane-spanning segments. It has been suggested that Rpe65 is S-palmitoylated by lecithin:retinol acyl transferase (LRAT) on Cys(231), Cys(329), and Cys(330), and that this palmitoylation is required for isomerase activity and the association of Rpe65 with membranes. Here we show that the affinity of Rpe65 for membranes is similar in wild-type and lrat(-/-) mice. The isomerase activity of Rpe65 is also similar in both strains when all-trans-retinyl palmitate is used as substrate. With all-trans-retinol substrate, isomerase activity is present in wild-type but undetectable in RPE homogenates from lrat(-/-) mice. Substitution of Cys(231), Cys(329), and Cys(330) with Ser or Ala did not affect the affinity of Rpe65 for membranes. Further, these Cys residues are not palmitoylated in Rpe65 by mass spectrometric analysis. Global inhibition of protein palmitoylation by 2-bromopalmitate did not affect the solubility or isomerase activity of Rpe65. Finally, we show that soluble and membrane-associated Rpe65 possesses similar isomerase specific activities. These results indicate that LRAT is not required for isomerase activity beyond synthesis of retinyl-ester substrate, and that the association of Rpe65 with membranes is neither dependent upon LRAT nor the result of S-palmitoylation. The affinity of Rpe65 for membranes is probably an intrinsic feature of this protein.     

Identification of the key residues determining the product specificity of isomerohydrolase

The efficient recycling of the chromophore of visual pigments, 11-cis-retinal, through the retinoid visual cycle is an essential process for maintaining normal vision. RPE65 is the isomerohydrolase in retinal pigment epithelium and generates predominantly 11-cis-retinol (11cROL) and a minor amount of 13-cis-retinol (13cROL), from all-trans-retinyl ester (atRE). We recently identified and characterized novel homologues of RPE65, RPE65c, and 13-cis-isomerohydrolase (13cIMH), which are expressed in the zebrafish inner retina and brain, respectively. Although these two homologues have 97% identical amino acid sequences, they exhibit distinct product specificities. Under the same assay conditions, RPE65c generated predominantly 11cROL, similar to RPE65, while 13cIMH generated exclusively 13cROL from atRE substrate. To study the impacts of the key residues determining the isomerization product specificity of RPE65, we replaced candidate residues by site-directed mutagenesis in RPE65c and 13cIMH. Point mutations at residues Tyr58, Phe103, and Leu133 in RPE65c resulted in significantly altered isomerization product specificities. In particular, our results showed that residue 58 is a primary determinant of isomerization specificity, because the Y58N mutation in RPE65c and its reciprocal N58Y mutation in 13cIMH completely reversed the respective enzyme isomerization product specificities. These findings will contribute to the elucidation of molecular mechanisms underlying the isomerization reaction catalyzed by RPE65.     

Mole quantity of RPE65 and its productivity in the generation of 11-cis-retinal from retinyl esters in the living mouse eye

RPE65, a protein expressed in cells of the retinal pigment epithelium of the eye, is essential for the synthesis by isomerohydrolase of 11-cis-retinal, the chromophore of rod and cone opsins. Recent work has established that RPE65 is a retinyl ester binding protein, and as all-trans-retinyl esters are the substrate for isomerohydrolase activity, the hypothesis has emerged that RPE65 serves to deliver substrate to this enzyme or complex. We bred mice with five distinct combinations of the RPE65 Leu450/Met450 variants (Leu/Leu, Met/Met, Leu/Met, Leu/-, and Met/-), measured in mice of each genotype the mole quantity of RPE65 per eye, and measured the initial rate of rhodopsin regeneration after a nearly complete bleach of rhodopsin to estimate the maximum rate of 11-cis-retinal synthesis in vivo. The quantity of RPE65 per eye ranged from 5.7 pmol (Balb/c) to 0.32 pmol (C57BL/6N x Rpe65(-)(/)(-)); the initial rate of rhodopsin regeneration was a Michaelis function of RPE65, where V(max) = 18 pmol/min per eye and K(m) = 1.7 pmol, and not dependent on the Leu450/Met450 variant. At RPE65 levels well below the K(m), the rate of production of 11-cis-retinal per RPE65 molecule was approximately 10 min(-)(1). Thus, the results imply that as a chaperone each RPE65 molecule can deliver retinyl ester to the isomerohydrolase at a rate of 10 molecules/min; should RPE65 itself be identified as the isomerase, each copy must be able to produce at least 10 molecules of 11-cis-retinal per minute.     

All-trans-retinyl esters are the substrates for isomerization in the vertebrate visual cycle

The identification of the critical enzyme(s) that carries out the trans to cis isomerization producing 11-cis-retinol during the operation of the visual cycle remains elusive. Confusion exists in the literature as to the exact nature of the isomerization substrate. At issue is whether it is an all-trans-retinyl ester or all-trans-retinol (vitamin A). As both putative substrates interconvert rapidly in retinal pigment epithelial membranes, the choice of substrate can be ambiguous. The two enzymes that effect interconversion of all-trans-retinol and all-trans-retinyl esters are lecithin retinol acyl transferase (LRAT) and retinyl ester hydrolase (REH). The retinyl ester or all-trans-retinol pools are radioactively labeled separately in the presence of inhibitors of LRAT and REH, effectively preventing their interconversion. Pulse-chase experiments unambiguously demonstrate that all-trans-retinyl esters, and not all-trans-retinol, are the precursors of 11-cis-retinol. When the all-trans-retinyl ester pool is radioactively labeled, the resulting 11-cis-retinol is labeled with the same specific activity as the precursor ester. The converse is true with vitamin A. These data unambiguously establish all-trans-retinyl esters as the precursors of 11-cis-retinol.     

Retinyl ester homeostasis in the adipose differentiation-related protein-deficient retina

The retinal pigmented epithelium (RPE) plays an essential role in vision, including storing and converting retinyl esters of the visual chromophore, 11-cis-retinal. Retinyl ester storage structures (RESTs), specialized lipid droplets within the RPE, take up retinyl esters synthesized in the endoplasmic reticulum. Here we report studies of mice lacking exons 2 and 3 of the gene encoding adipose differentiation-related protein (Adfp), a structural component of RESTs. We found that dark adaptation was slower in Adfp(Delta2-3/Delta2-3) than in Adfp(+/+) mice and that Adfp(Delta2-3/Delta2-3) mice had consistently delayed clearances of all-trans-retinal and all-trans-retinol from rod photoreceptor cells. Two-photon microscopy revealed aberrant trafficking of all-trans-retinyl esters in the RPE of Adfp(Delta2-3/Delta2-3) mice, a problem caused by abnormal maintenance of RESTs in the dark-adapted state. Retinyl ester accumulation was also reduced in Adfp(Delta2-3/Delta2-3) as compared with Adfp(+/+) mice. These observations suggest that Adfp plays a unique role in vision by maintaining proper storage and trafficking of retinoids within the eye.     

Membrane phospholipids as an energy source in the operation of the visual cycle

Biology depends on the coupling of the free energy of hydrolysis of phosphate esters, such as ATP, to drive processes which would otherwise be thermodynamically unfavorable. Carboxyl esters are like phosphate esters in their ability to hydrolyze with substantial negative free energies, enabling them to participate in group transfer processes as well. In particular, membrane phospholipids constitute an enormous store of potential energy that could be used to fuel energetically unfavorable processes. One such process involves the biosynthesis of 11-cis-retinal, the chromophore of rhodopsin, from all-trans-retinol (vitamin A). The difference in free energy between an all-trans retinoid and its corresponding 11-cis retinoid is approximately 4 kcal/mol. This energy is provided for in a minimally two-step process involving membrane phospholipids as the energy source. First, all-trans-retinol is esterified in the retinal pigment epithelium by lecithin retinol acyl transferase (LRAT) to produce an all-trans-retinyl ester. Second, this ester is transformed into 11-cis-retinol by an isomerohydrolase in a process that couples the negative free energy of hydrolysis of the acyl ester to the formation of the strained 11-cis-retinol.     

Inhibition of RPE65 Retinol Isomerase Activity by Inhibitors of Lipid Metabolism

RPE65 is the isomerase catalyzing conversion of all-trans-retinyl ester (atRE) into 11-cis-retinol in the retinal visual cycle. Crystal structures of RPE65 and site-directed mutagenesis reveal aspects of its catalytic mechanism, especially retinyl moiety isomerization, but other aspects remain to be determined. To investigate potential interactions between RPE65 and lipid metabolism enzymes, HEK293-F cells were transfected with expression vectors for visual cycle proteins and co-transfected with either fatty acyl:CoA ligases (ACSLs) 1, 3, or 6 or the SLC27A family fatty acyl-CoA synthase FATP2/SLCA27A2 to test their effect on isomerase activity. These experiments showed that RPE65 activity was reduced by co-expression of ACSLs or FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently inhibited RPE65 isomerase activity in cellulo. We found triacsin C to be a competitive inhibitor of RPE65 (IC₅₀ = 500 nm). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0–1 μm atRE. Other inhibitors of ACSLs had modest inhibitory effects compared with triascin C. In conclusion, we have identified an inhibitor of ACSLs as a potent inhibitor of RPE65 that competes with the atRE substrate of RPE65 for binding. Triacsin C, with an alkenyl chain resembling but not identical to either acyl or retinyl chains, may compete with binding of the acyl moiety of atRE via the alkenyl moiety. Its inhibitory effect, however, may reside in its nitrosohydrazone/triazene moiety.     

Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye

Visual sensation in vertebrates is triggered when light strikes retinal photoreceptor cells causing photoisomerization of the rhodopsin chromophore 11-cis-retinal to all-trans-retinal. The regeneration of preillumination conditions of the photoreceptor cells requires formation of 11-cis-retinal in the adjacent retinal pigment epithelium (RPE). Using the intrinsic fluorescence of all-trans-retinyl esters, noninvasive two-photon microscopy revealed previously uncharacterized structures (6.9 +/- 1.1 microm in length and 0.8 +/- 0.2 microm in diameter) distinct from other cellular organelles, termed the retinyl ester storage particles (RESTs), or retinosomes. These structures form autonomous all-trans-retinyl ester-rich intracellular compartments distinct from other organelles and colocalize with adipose differentiation-related protein. As demonstrated by in vivo experiments using wild-type mice, the RESTs participate in 11-cis-retinal formation. RESTs accumulate in Rpe65-/- mice incapable of carrying out the enzymatic isomerization, and correspondingly, are absent in the eyes of Lrat-/- mice deficient in retinyl ester synthesis. These results indicate that RESTs located close to the RPE plasma membrane are essential components in 11-cis-retinal production.     

Molecular logic of 11-cis-retinoid biosynthesis in a cone-dominated species

The biochemical pathway to visual chromophore biosynthesis in rod-dominated animals involves minimally a two component system in which all-trans-retinyl esters, generated by the action of lecithin retinol acyltransferase (LRAT) on vitamin A, are processed into 11-cis-retinol by isomerohydrolase. Possible differences in retinoid metabolism in cone-dominated animals have been noted in the literature, so it was of interest to explore whether these differences are tangential or fundamental. Central to this issue is whether cone-dominated animals use an isomerohydrolase (IMH)-based mechanism in the predominant pathway to 11-cis-retinoids. Here, it is shown that all-trans-retinyl esters (tREs) are the direct precursors of 11-cis-retinol formation in chicken retinyl pigment epithelium/retina preparations. This conclusion is based on at least three avenues of evidence. First, reagents that block tRE synthesis from vitamin A also block 11-cis-retinol synthesis. Second, pulse-chase experiments also establish that tREs are the precursors to 11-cis-retinol. Finally, 11-cis-retinyl-bromoacetate, a known affinity-labeling agent of isomerohydrolase, also blocks chromophore biosynthesis in the cone system.     

Substrate specificities and mechanism in the enzymatic processing of vitamin A into 11-cis-retinol

The biosynthesis of 11-cis-retinol in the retinal pigment epithelium requires two consecutive enzymatic reactions. The first involves the esterification of all-trans-retinol by lecithin retinol acyltransferase (LRAT). The second reaction involves the direct conversion of an all-trans-retinyl ester into 11-cis-retinol by an isomerase-like enzyme. This latter reaction couples the free energy of hydrolysis of an ester to the thermodynamically uphill trans to cis conversion, thus providing the energy to drive the latter process. In this paper both enzymes are studied with respect to their substrate specificities to provide information on mechanism. The isomerase is shown to be highly specific with respect to the ionylidene ring system and substitution at C15, whereas sterically bulkier substituents at C9 and C11 are permitted. C5 and C13 demethyl retinoids are isomerized, removing from consideration isomerization mechanisms involving C-H abstraction at the C5 or C13 methyl groups of the retinoid. On the other hand, C9 demethyl retinoids are not isomerized. A C-H abstraction mechanism is unlikely at the C9 methyl group as well, because no kinetic deuterium isotope effect is found with all-trans-19,19,19-trideuterioretinoids and isomerization of unlabeled retinoids occurs without the incorporation of deuterium when the isomerization is performed in D2O. LRAT proved to be broadly specific for retinols but was relatively inert with other hydrophobic alcohols including cholesterol. The enzyme is also highly specific for phosphatidylcholine analogues versus other potential membranous acyl donors such as phosphatidylethanolamine and phosphatidylserine.