Aromatic Residues in the Substrate Cleft of RPE65 Protein Govern Retinol Isomerization and Modulate Its Progression

Previously, we showed that mutating RPE65 residue Phe-103 preferentially produces 13-cis-retinol instead of 11-cis-retinol, supporting a carbocation/radical cation mechanism of retinol isomerization. We asked whether this modulation of specificity can occur with residues other than Phe-103 and what role it plays in substrate binding and isomerization. We modeled the substrate-binding cleft of RPE65 to identify residues lining its surface. Many are phenylalanines and tyrosines, including three Phe residues (Phe-61, Phe-312, and Phe-526) forming an arch-like arrangement astride the cleft and Tyr-338. Also, Phe-418 sits at the neck of the cleft, lending a bend to the volume enclosed by the cleft. All mutations of Phe-61, Phe-312, and Phe-418 result in severely impaired or inactive enzyme. However, mutation of Phe-526 and Tyr-338, like Phe-103, decreases 11-cis-retinol formation, whereas increasing the 13-cis isomer. Significantly, 2 of these 3 residues, Phe-103 and Tyr-338, are located on putatively mobile interstrand loops. We propose that residual densities located in the binding cleft of the RPE65 structure represents a post-cleavage snapshot consistent not only with a fatty acid product, as originally modeled, but also an 11-cis-retinol product. Substrate docking simulations permit 11-cis- or 13-cis-retinyl ester binding in this relatively closed cleft, with the latter favored in F103L, F526A, and Y338A mutant structures, but prohibit binding of all-trans-retinyl ester, suggesting that isomerization occurs early in the temporal sequence, with O-alkyl ester cleavage occurring later. These findings provide insight into the mechanism of isomerization central to the visual cycle.     

FATP1 Inhibits 11-cis Retinol Formation via Interaction with the Visual Cycle Retinoid Isomerase RPE65 and Lecithin:Retinol Acyltransferase

The isomerization of all-trans retinol (vitamin A) to 11-cis retinol in the retinal pigment epithelium (RPE) is a key step in the visual process for the regeneration of the visual pigment chromophore, 11-cis retinal. LRAT and RPE65 are recognized as the minimal isomerase catalytic components. However, regulators of this rate-limiting step are not fully identified and could account for the phenotypic variability associated with inherited retinal degeneration (RD) caused by mutations in the RPE65 gene. To identify new RPE65 partners, we screened a porcine RPE mRNA library using a yeast two-hybrid assay with full-length human RPE65. One identified clone (here named FATP1c), containing the cytosolic C-terminal sequence from the fatty acid transport protein 1 (FATP1 or SLC27A1, solute carrier family 27 member 1), was demonstrated to interact dose-dependently with the native RPE65 and with LRAT. Furthermore, these interacting proteins colocalize in the RPE. Cellular reconstitution of human interacting proteins shows that FATP1 markedly inhibits 11-cis retinol production by acting on the production of all-trans retinyl esters and the isomerase activity of RPE65. The identification of this new visual cycle inhibitory component in RPE may contribute to further understanding of retinal pathogenesis.     

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

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 A² 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.     

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.     

Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13-cis, 9-cis, all-trans, all-trans-4-oxo, 9-cis-4-oxo, 13-cis-4-oxo), three retinal isomers (13-cis-, 9-cis-, and all-trans) and two retinol isomers (13-cis- and all-trans). Human serum samples were subjected to this HPLC analysis and at least, all-trans retinol, 13-cis retinol, and all-trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all-trans retinoic acid in human sera.     

Conditional Ablation of Retinol Dehydrogenase 10 in the Retinal Pigmented Epithelium Causes Delayed Dark Adaption in Mice

Regeneration of the visual chromophore, 11-cis-retinal, is a crucial step in the visual cycle required to sustain vision. This cycle consists of sequential biochemical reactions that occur in photoreceptor cells and the retinal pigmented epithelium (RPE). Oxidation of 11-cis-retinol to 11-cis-retinal is accomplished by a family of enzymes termed 11-cis-retinol dehydrogenases, including RDH5 and RDH11. Double deletion of Rdh5 and Rdh11 does not limit the production of 11-cis-retinal in mice. Here we describe a third retinol dehydrogenase in the RPE, RDH10, which can produce 11-cis-retinal. Mice with a conditional knock-out of Rdh10 in RPE cells (Rdh10 cKO) displayed delayed 11-cis-retinal regeneration and dark adaption after bright light illumination. Retinal function measured by electroretinogram after light exposure was also delayed in Rdh10 cKO mice as compared with controls. Double deletion of Rdh5 and Rdh10 (cDKO) in mice caused elevated 11/13-cis-retinyl ester content also seen in Rdh5^−/−Rdh11^−/− mice as compared with Rdh5^−/− mice. Normal retinal morphology was observed in 6-month-old Rdh10 cKO and cDKO mice, suggesting that loss of Rdh10 in the RPE does not negatively affect the health of the retina. Compensatory expression of other retinol dehydrogenases was observed in both Rdh5^−/− and Rdh10 cKO mice. These results indicate that RDH10 acts in cooperation with other RDH isoforms to produce the 11-cis-retinal chromophore needed for vision.     

Retinyl esters are the substrate for isomerohydrolase

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.     

RDH13L,an Enzyme Responsible for the Aldehyde-Alcohol Redox Coupling Reaction (AL-OL Coupling Reaction) to Supply 11-cis Retinal in the Carp Cone Retinoid Cycle

Cone photoreceptors require effective pigment regeneration mechanisms to maintain their sensitivity in the light. Our previous studies in carp cones suggested the presence of an unconventional and very effective mechanism to produce 11-cis retinal, the necessary component in pigment regeneration. In this reaction (aldehyde-alcohol redox coupling reaction, AL-OL coupling reaction), formation of 11-cis retinal, i.e. oxidation of 11-cis retinol is coupled to reduction of an aldehyde at a 1:1 molar ratio without exogenous NADP(H) which is usually required in this kind of reaction. Here, we identified carp retinol dehydrogenase 13-like (RDH13L) as an enzyme catalyzing the AL-OL coupling reaction. RDH13L was partially purified from purified carp cones, identified as a candidate protein, and its AL-OL coupling activity was confirmed using recombinant RDH13L. We further examined the substrate specificity, subcellular localization, and expression level of RDH13L. Based on these results, we concluded that RDH13L contributes to a significant part, but not all, of the AL-OL coupling activity in carp cones. RDH13L contained tightly bound NADP⁺ which presumably functions as a cofactor in the reaction. Mouse RDH14, a mouse homolog of carp RDH13L, also showed the AL-OL coupling activity. Interestingly, although carp cone membranes, carp RDH13L and mouse RDH14 all showed the coupling activity at 15–37 °C, they also showed a conventional NADP⁺-dependent 11-cis retinol oxidation activity above 25 °C without addition of aldehydes. This dual mechanism of 11-cis retinal synthesis attained by carp RDH13L and mouse RDH14 probably contribute to effective pigment regeneration in cones that function in the light.     

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.     

Biotransformation of Unsaturated Long-Chain Fatty Acids by Eubacterium lentum

Eubacterium lentum (33 strains) isomerized the 12-cis double bond of C₁₈ fatty acids with cis double bonds at C-9 and C-12 into an 11-trans double bond before reduction of the 9-cis double bond. The 14-cis double bond of homo-γ-linolenic acid was isomerized by 29 strains into a 13-trans double bond. The same strains isomerized the 14-cis double bond of arachidonic acid into a 13-trans double bond and then isomerized the 8-cis double bond into a 7-trans double bond; the 13-cis double bond of 10-cis, 13-cis-nonadecadienoic acid was isomerized into a 12-trans double bond. None of these isomerization products was further reduced. Studies with resting cells showed optimal isomerization velocity at a linoleic acid concentration of 37.5 μM; higher concentrations were inhibitory. The pH optimum for isomerization was 7.5 to 8.5. The isomerase was inhibited by the sulfhydryl reagents iodoacetamide, bromoacetate, and N-ethylmaleimide and by the chelators EDTA and 1,10-phenanthroline.     

Negative charge of the glutamic acid 417 residue is crucial for isomerohydrolase activity of RPE65

RPE65 is the isomerohydrolase essential for regeneration of 11-cis retinal, the chromophore of visual pigments. Here we compared the impacts of two mutations in RPE65, E417Q identified in patients with Leber congenital amaurosis (LCA), and E417D on isomerohydrolase activity. Although both mutations decreased the stability of RPE65 and altered its sub-cellular localization, E417Q abolished isomerohydrolase activity whereas the E417D mutant retained partial enzymatic activity suggesting that the negative charge of E417 is important for RPE65 catalytic activity. Loss of charge at this position may represent a mechanism by which the E417Q mutation causes blindness in LCA patients.     

Simultaneous analysis of retinol,all-trans- and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in plasma by liquid chromatography using on-column concentration after single-phase fluid extraction

A reversed-phase high-performance liquid chromatographic method for the simultaneous analysis of retinol, all-trans-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma and cell culture medium is described. Sample preparation involves precipitation of proteins and extraction of retinoids with 60% acetonitrile. After centrifugation, the acetonitrile content of the supernatant is reduced to 45%, allowing on-column concentration of analytes. Injection volumes up to 2.0 ml (equivalent to 0.525 ml of sample) can be used without compromising chromatographic resolution of all-trans-retinoic acid and 13-cis-retinoic acid. Retinoids were stable in this extract and showed no isomerization when stored in the dark in a cooled autosampler, allowing automated analysis of large series of samples. Recoveries from spiked plasma samples were between 95 and 103%. Although no internal standard was used, the inter-assay precision for all retinoids was better than 6% and 4% at concentrations of 30 nM and 100 nM, respectively. The method is a valuable tool for the study of cellular metabolism of all-trans-retinoic acid, as polar metabolites of this compound can be detected with high sensitivity in cell culture media.     

Identification of a Novel Palmitylation Site Essential for Membrane Association and Isomerohydrolase Activity of RPE65

RPE65 is a membrane-associated protein abundantly expressed in the retinal pigment epithelium, which converts all-trans-retinyl ester to 11-cis-retinol, a key step in the retinoid visual cycle. Although three cysteine residues (Cys-231, Cys-329, and Cys-330) were identified to be palmitylated in RPE65, recent studies showed that a triple mutant, with all three Cys replaced by an alanine residue, was still palmitylated and remained membrane-associated, suggesting that there are other yet to be identified palmitylated Cys residues in RPE65. Here we mapped the entire RPE65 using mass spectrometry analysis and demonstrated that a trypsin-digested RPE65 fragment (residues 98-118), which contains two Cys residues (Cys-106 and Cys-112), was singly palmitylated in both native bovine and recombinant human RPE65. To determine whether Cys-106 or Cys-112 is the palmitylation site, these Cys were separately replaced by alanine. Mass spectrometry analysis of purified wild-type RPE65 and C106A and C112A mutants showed that mutation of Cys-106 did not affect the palmitylation status of the fragment 98-118, whereas mutation of Cys-112 abolished palmitylation in this fragment. Subcellular fractionation and immunocytochemistry analyses both showed that mutation of Cys-112 dissociated RPE65 from the membrane, whereas the C106A mutant remained associated with the membrane. In vitro isomerohydrolase activity assay showed that C106A has an intact enzymatic activity similar to that of wtRPE65, whereas C112A lost its enzymatic activity. These results indicate that the newly identified Cys-112 palmitylation site is essential for the membrane association and activity of RPE65.Both rod and cone visual pigments in vertebrates require 11-cis-retinal as the chromophore. Isomerization of 11-cis-retinal to all-trans-retinal by a photon triggers the phototransduction cascade and initiates vision (1, 2). Recycling of 11-cis-retinal through the retinoid visual cycle is an essential process for the regeneration of visual pigments and for normal vision (3, 4). The key step in the visual cycle is to isomerize all-trans-retinyl ester to 11-cis-retinol in retinal pigment epithelium (RPE)² (5, 6). This isomerization process is known to be catalyzed by an isomerohydrolase in the RPE. Several recent lines of evidence suggest that RPE65 is the isomerohydrolase in the visual cycle (7-9).RPE65 is a microsomal protein, abundantly expressed in the RPE (10-12). RPE65 knock-out (Rpe65^-/-) mice showed a lack of 11-cis-retinoids, overaccumulation of all-trans-retinyl ester, impaired visual function, and early degeneration of cone photoreceptors (7-9). RPE65 is an iron(II)-dependent enzyme, in which an iron is coordinated by four conserved histidine (His) residues (His-180, -241, -313, and -527) based on molecular modeling using a crystal structure of apocarotenoid monooxygenase as a template (8, 13-15). RPE65 lacks any predicted transmembrane helix and is associated with the microsomal membrane (11). Previous studies have shown that membrane association of RPE65 is essential for its isomerohydrolase activity (7). However, the structural basis for its membrane association has not been revealed. An earlier study showed that three Cys residues (Cys-231, 329 and 330) in RPE65 were palmitylated, which were suggested to be responsible for its membrane association (16). However, triple mutations of all the three Cys residues did not completely dissociate RPE65 from the membrane (17, 18). Moreover, the triple Cys mutant remains palmitylated (17). These results suggested that either the site of palmitylation responsible for the membrane association of RPE65 had not yet been identified or other mechanisms, such as hydrophobic interactions, anchor the protein to cellular membranes (17, 19).In this study, we used the combination of mass spectrometric analysis and site-directed mutagenesis to identify the palmitylated site in RPE65. Moreover, we determined the role of this site in the membrane association and enzymatic activity of RPE65.     

Evolution and the origin of the visual retinoid cycle in vertebrates

Absorption of a photon by visual pigments induces isomerization of 11-cis-retinaldehyde (RAL) chromophore to all-trans-RAL. Since the opsins lacking 11-cis-RAL lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called ‘visual cycle’. Protostomes and vertebrates use essentially different machinery of visual pigment regeneration, and the origin and early evolution of the vertebrate visual cycle is an unsolved mystery. Here we compare visual retinoid cycles between different photoreceptors of vertebrates, including rods, cones and non-visual photoreceptors, as well as between vertebrates and invertebrates. The visual cycle systems in ascidians, the closest living relatives of vertebrates, show an intermediate state between vertebrates and non-chordate invertebrates. The ascidian larva may use retinochrome-like opsin as the major isomerase. The entire process of the visual cycle can occur inside the photoreceptor cells with distinct subcellular compartmentalization, although the visual cycle components are also present in surrounding non-photoreceptor cells. The adult ascidian probably uses RPE65 isomerase, and trans-to-cis isomerization may occur in distinct cellular compartments, which is similar to the vertebrate situation. The complete transition to the sophisticated retinoid cycle of vertebrates may have required acquisition of new genes, such as interphotoreceptor retinoid-binding protein, and functional evolution of the visual cycle genes.     

Diacylglycerol O-Acyltransferase Type-1 Synthesizes Retinyl Esters in the Retina and Retinal Pigment Epithelium

Retinyl esters represent an insoluble storage form of vitamin A and are substrates for the retinoid isomerase (Rpe65) in cells of the retinal pigment epithelium (RPE). The major retinyl-ester synthase in RPE cells is lecithin:retinol acyl-transferase (LRAT). A second palmitoyl coenzyme A-dependent retinyl-ester synthase activity has been observed in RPE homogenates but the protein responsible has not been identified. Here we show that diacylglycerol O-acyltransferase-1 (DGAT1) is expressed in multiple cells of the retina including RPE and Müller glial cells. DGAT1 catalyzes the synthesis of retinyl esters from multiple retinol isomers with similar catalytic efficiencies. Loss of DGAT1 in dgat1 ^-/- mice has no effect on retinal anatomy or the ultrastructure of photoreceptor outer-segments (OS) and RPE cells. Levels of visual chromophore in dgat1 ^-/- mice were also normal. However, the normal build-up of all-trans-retinyl esters (all-trans-RE’s) in the RPE during the first hour after a deep photobleach of visual pigments in the retina was not seen in dgat1 ^-/- mice. Further, total retinyl-ester synthase activity was reduced in both dgat1 ^-/- retina and RPE.     

Binding affinities of CRBPI and CRBPII for 9-cis-retinoids

Background

Cellular retinol binding-protein I (CRBPI) and cellular retinol binding-protein II (CRBPII) serve as intracellular retinoid chaperones that bind retinol and retinal with high affinity and facilitate substrate delivery to select enzymes that catalyze retinoic acid (RA) and retinyl ester biosynthesis. Recently, 9-cis-RA has been identified in vivo in the pancreas, where it contributes to regulating glucose-stimulated insulin secretion. In vitro, 9-cis-RA activates RXR (retinoid × receptors), which serve as therapeutic targets for treating cancer and metabolic diseases. Binding affinities and structure–function relationships have been well characterized for CRBPI and CRBPII with all-trans-retinoids, but not for 9-cis-retinoids. This study extended current knowledge by establishing binding affinities for CRBPI and CRBPII with 9-cis-retinoids.

Methods

We have determined apparent dissociation constants, K′_d, through monitoring binding of 9-cis-retinol, 9-cis-retinal, and 9-cis-RA with CRBPI and CRBPII by fluorescence spectroscopy, and analyzing the data with non-linear regression. We compared these data to the data we obtained for all-trans- and 13-cis-retinoids under identical conditions.

Results

CRBPI and CRBPII, respectively, bind 9-cis-retinol (K′_d, 11 nM and 68 nM) and 9-cis-retinal (K′_d, 8 nM and 5 nM) with high affinity. No significant 9-cis-RA binding was observed with CRBPI or CRBPII.

Conclusions

CRBPI and CRBPII bind 9-cis-retinol and 9-cis-retinal with high affinities, albeit with affinities somewhat lower than for all-trans-retinol and all-trans-retinal.

General significance

These data provide further insight into structure–binding relationships of cellular retinol binding-proteins and are consistent with a model of 9-cis-RA biosynthesis that involves chaperoned delivery of 9-cis-retinoids to enzymes that recognize retinoid binding-proteins.     

Configuration of the carotenoid in the reaction centers of photosynthetic bacteria. Comparison of the resonance Raman spectrum of the reaction center of Rhodopseudomonas sphaeroides G1C with those of cis-trans isomers of β-carotene

The resonance Raman spectrum of the reaction center of Rhodopseudomonas sphaeroides G1C as well as those of the cis-trans isomers of β-carotene (all-trans, 9-cis, 13-cis, 15-cis and 9-cis, 13-cis- (or 9-cis, 13′-cis)) have been recorded at liquid N₂ temperature by use of the 457.9, 488.0 and 514.5 nm excitation lines. Comparison of the spectra indicated that the carotenoid in the reaction center takes the 15-cis configuration.     

Substrate Specificity and Subcellular Localization of the Aldehyde-Alcohol Redox-coupling Reaction in Carp Cones

Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.     

Aromatic lipophilic spin traps effectively inhibit RPE65 isomerohydrolase activity

We previously showed that RPE65 does not specifically produce 11-cis-retinol only but also 13-cis-retinol, supporting a carbocation or radical cation mechanism of isomerization. The intrinsic properties of conjugated polyene chains result in facile formation of radical cations in oxidative conditions. We hypothesized that such radical intermediates, if involved in the mechanism of RPE65, could be stabilized by spin traps. We tested a variety of hydrophilic and lipophilic spin traps for their ability to inhibit RPE65 isomerohydrolase activity. We found that the aromatic lipophilic spin traps such as N-tert-butyl-α-phenylnitrone (PBN), 2,2-dimethyl-4-phenyl-2H-imidazole-1-oxide (DMPIO), and nitrosobenzene (NB) strongly inhibit RPE65 isomerohydrolase activity in vitro.