RPE65 is essential for isomerization of vitamin A to the visual chromophore. Mutations in RPE65 cause early-onset blindness, and Rpe65-deficient mice lack 11-cis-retinal but overaccumulate alltrans-retinyl esters in the retinal pigment epithelium (RPE). RPE65 is proposed to be a substrate chaperone but may have an enzymatic role because it is closely related to carotenoid oxygenases. We hypothesize that, by analogy with other carotenoid oxygenases, the predicted iron-coordinating residues of RPE65 are essential for retinoid isomerization. To clarify RPE65's role in isomerization, we reconstituted a robust minimal visual cycle in 293-F cells. Only cells transfected with RPE65 constructs produced 11-cisretinoids, but coexpression with lecithin:retinol acyltransferase was needed for high-level production. Accumulation was significant, amounting to >2 nmol of 11-cis-retinol per culture. Transfection with constructs harboring mutations in residues of RPE65 homologous to those required for interlinked enzymatic activity and iron coordination in related enzymes abolish this isomerization. Iron chelation also abolished isomerization activity. Mutating cysteines implicated in palmitoylation of RPE65 had generally little effect on isomerization activity. Mutations associated with Leber congenital amaurosis͞early-onset blindness cause partial to total loss of isomerization activity in direct relation to their clinical effects. These findings establish a catalytic role, in conjunction with lecithin:retinol acyltransferase, for RPE65 in synthesis of 11-cisretinol, and its identity as the isomerohydrolase.11-cis-retinoids ͉ Leber congenital amaurosis ͉ retinal pigment epithelium R egeneration of 11-cis-retinal, the chromophore of all visual pigments (opsins), occurs by a process in the retinal pigment epithelium (RPE) termed the visual cycle that involves isomerization of all-trans-retinyl esters to 11-cis-retinol. In outline, lecithin:retinol acyltransferase (LRAT) (1, 2) esterifies incoming all-trans-retinol to all-trans-retinyl esters, the substrate for the putative isomerohydrolase (IMH) (3, 4). IMH is postulated to perform a concerted hydrolysis and isomerization yielding 11-cis-retinol, which is trapped by cellular retinaldehyde-binding protein (CRALBP) and then oxidized to 11-cis-retinal by 11-cis-retinol dehydrogenase͞RDH5 (11cisRDH). An alternate mechanism proposes generation of 11-cis-retinol via a retinyl carbocation intermediate (5). Mutations of RDH5, RPE65, LRAT, and CRALBP in the human (6 -12) and cognate disruptions in the mouse (13-16) result in mild to severe blindness due to derangement of RPE retinoid metabolism. CRALBP and RDH5 disruptions do not block the visual cycle but reduce its efficiency (13, 16). Although mutation or loss of LRAT and RPE65 each block the visual cycle, they do so differently. With LRAT loss, no vitamin A accumulates in the RPE, obviating retinoid metabolism (15). With RPE65 loss, all-trans-retinyl ester accumulates to very high levels, but 11-cis-retinoids are not formed (14).Howev...
We have identified from mouse the first mammalian -carotene 15,15-dioxygenase (-CD), a crucial enzyme in development and metabolism that governs the de novo entry of vitamin A from plant-derived precursors. -CD is related to the retinal pigment epithelium-expressed protein RPE65 and belongs to a diverse family that includes the plant 9-cis-epoxycarotenoid dioxygenase and bacterial lignostilbene dioxygenases. -CD expression in Escherichia coli cells engineered to produce -carotene led to the accumulation of all-trans-retinal at the expense of -carotene, confirming that -CD catalyzed the central cleavage of this vitamin A precursor. Purified recombinant -CD protein cleaves -carotene in vitro with a V max of 36 pmol of retinal/mg of enzyme/ min and a K m of 6 M. Non-provitamin A carotenoids were also cleaved, although with much lower activity. By Northern analysis, a 2.4-kilobase (kb) message was observed in liver, kidney, small intestine, and testis, tissues important in retinoid/carotenoid metabolism. This message encoded a 63-kDa cytosolic protein expressed in these tissues. A shorter transcript of 1.8 kb was found in testis and skin. Developmentally, the 2.4-kb mRNA was abundant at embryonic day 7, with lower expression at embryonic days 11, 13, and 15, suggesting a critical role for this enzyme in gastrulation. Identification of -CD in an accessible model organism will create new opportunities to study vitamin A metabolism.In vertebrates, vitamin A in its various oxidative and isomeric forms is essential for embryonic development (1), pattern formation (2, 3), and vision (4). Retinoic acid, through its interaction with the nuclear retinoic acid receptor and retinoid X receptor, profoundly affects cell differentiation and development. Because animals are unable to synthesize vitamin A de novo from endogenous isoprenoid precursors, they must instead derive it from cleavage of -carotene and certain other carotenoids with an unsubstituted -ring (e.g. ␥-and ␣-carotenes, -zeacarotene, and -cryptoxanthin). It is generally accepted that central cleavage of -carotene by a putative dioxygenase gives rise to two molecules of all-trans-retinal, whereas eccentric cleavage with subsequent processing leading to a single molecule of retinoic acid from an apocarotenal is quantitatively far less important (5). -Carotene cleavage activity is reported highest in the intestinal mucosa, but is found at high activity levels in liver, kidney, lung, and fat tissues, among other sites. However, an inability to purify the protein catalyzing this reaction has hindered thorough investigation of this crucial first step in vitamin A metabolism.Because of a loose similarity between the mammalian protein RPE65 and the neoxanthin cleavage enzymes of plants, our laboratories have considered the hypothesis that the putative -CD 1 would belong to an emerging family of carotenoidcleaving dioxygenases known mainly from examples in plants (6), but with members also in bacteria and Metazoa. The first described representative was a ba...
C57BL/6J-c(2J) (c2J) albino mice showed much less damage to their photoreceptors after exposure to prolonged light than BALB/c mice and seven other albino strains tested. There were no gender differences, and preliminary studies suggested that the c2J relative protective effect was a complex trait. A genome-wide scan using dinucleotide repeat markers was carried out for the analysis of 194 progeny of the backcross (c2J x BALB/c)F(1) x c2J and the thickness of the outer nuclear layer (ONL) of the retina was the quantitative trait reflecting retinal damage. Our results revealed a strong and highly significant quantitative trait locus (QTL) on mouse Chromosome (Chr) 3 that contributes almost 50% of the c2J protective effect, and three other very weak but significant QTLs on Chrs 9, 12, and 14. Interestingly, the Chrs 9 and 12 QTLs corresponded to relative susceptibility alleles in c2J (or relative protection alleles in BALB/c), the opposite of the relative protective effect of the QTLs on Chrs 3 and 14. We mapped the Rpe65 gene to the apex of the Chr 3 QTL (LOD score = 19.3). Northern analysis showed no difference in retinal expression of Rpe65 message between c2J and BALB/c mice. However, sequencing of the Rpe65 message revealed a single base change in codon 450, predicting a methionine in c2J and a leucine in BALB/c. When the retinas of aging BALB/c and c2J mice reared in normal cyclic light were compared, the BALB/c retinas showed a small but significant loss of photoreceptor cells, while the c2J retinas did not. Finding light damage-modifying genes in the mouse may open avenues of study for understanding age-related macular degeneration and other retinal degenerations, since light exposures may contribute to the course of these diseases.
Alignment of sequences of vertebrate 405 , and Glu 457 in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are essential for catalytic activity. To test this hypothesis, we produced mutant forms of mouse BCMO1 by replacing the conserved histidines and acidic residues as well as four histidines and one glutamate nonconserved in the overall family with alanines by sitedirected mutagenesis. Our in vitro and in vivo data showed that mutation of any of the four conserved histidines and Glu 405 caused total loss of activity. However, mutations of non-conserved histidines or any of the other conserved acidic residues produced impaired although enzymatically active proteins, with a decrease in activity mostly due to changes in V max . The iron bound to protein was determined by inductively coupled plasma atomic emission spectrometry. Bound iron was much lower in preparations of inactive mutants than in the wild-type protein. Therefore, the conserved histidines and Glu 405 are absolutely required for the catalytic mechanism of BCMO1. Because the mutant proteins are impaired in iron binding, these residues are concluded to coordinate iron required for catalytic activity. These data are discussed in the context of the predicted structure for the related eubacterial apocarotenal oxygenase. -Carotene 15,15Ј-monooxygenase-1 (BCMO1)1 is the initial enzyme step in the biosynthesis of vitamin A in animals, symmetrically cleaving -carotene to produce two molecules of alltrans-retinal (1). In the last several years, a number of BCMO1 proteins have been cloned and biochemically characterized (2-7). BCMO1 belongs to a family of oxygenases of diverse activities, including lignostilbene dioxygenase in bacteria (8 -10); an epoxycarotenoid-cleaving enzyme required for abscisic acid biosynthesis in plants (11); and mammalian RPE65, a retinal pigment epithelial protein required for the production of the visual chromophore 11-cis-retinal in the visual cycle (12). The most recent addition to the characterized carotenoid oxygenases is cyanobacterial apocarotenoid 15,15Ј-oxygenase PCC 6803 (ACO) (13). Despite their unique functionalities and the obvious importance of these proteins, structural studies on this family have been hindered by technical difficulties, and only recently has the crystal structure of eubacterial ACO been published (14).Originally, Drosophila BCMO1 was identified by homology to a previously characterized plant 9-cis-epoxycarotenoid dioxygenase (15) and by its ability to cleave -carotene (4). Subsequently, mouse BCMO1 was identified by our laboratory based on its homology to mammalian RPE65 (3). Because the biochemical function and mechanism of BCMO1 have been studied in detail (3, 5, 7), it is functionally the best characterized mammalian member of the family and could serve as a model for studying putative vertebrate oxygenases of more elusive function such as RPE65 and BCMO2 (16).Although there is a weak overall identity (Ͻ10%) am...
The isomerization of all-trans-retinyl ester to 11-cis-retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle and is essential for normal vision. Recently, we have established that protein RPE65 is the isomerohydrolase catalyzing this reaction. The present study investigated if metal ions are required for the isomerohydrolase activity of RPE65. The conversion of alltrans-[3 H]retinol to 11-cis-[ 3 H]retinol was used as the measure for isomerohydrolase activity. Metal chelators 2,2-bipyridine and 1,10-phenanthroline both showed dose-dependent inhibitions of the isomerohydrolase activity in bovine RPE microsomes, with IC 50 values of 0.5 and 0.2 mM, respectively. In the same reaction systems, however, lecithin-retinol acyltransferase (LRAT) activity was not affected by these metal chelators. The isomerohydrolase activity inhibited by the metal chelators was restored by FeSO 4 but not by CuSO 4 , ZnCl 2 , or MgCl 2 . Moreover, addition of Fe(III) citrate or FeCl 3 did not restore the activity, indicating that Fe 2؉ is the metal ion essential for the isomerohydrolase activity. To confirm this result in recombinant RPE65, we expressed RPE65 in a 293A cell line stably expressing LRAT. In vitro activity assay showed that both metal chelators inhibited isomerohydrolase activity of recombinant RPE65. The addition of FeSO 4 restored the enzymatic activity of the recombinant RPE65. Further, two specific iron-staining methods showed that purified RPE65 contains endogenous iron. Inductively coupled plasma mass spectrometry measurements showed that bovine RPE65 binds iron ion with a stoichiometry of 0.8 ؎ 0.1. These results indicate that RPE65 is an iron-dependent isomerohydrolase in the visual cyclePhoton absorption by the visual pigments results in isomerization of 11-cis-retinal, the chromophore, to all-trans-retinal and, subsequently, triggers a phototransduction cascade (1). Obviously, an efficient regeneration of 11-cis-retinal is necessary for the continuous phototransduction process and normal vision. 11-cis-Retinal is regenerated through a series of sequential reactions termed the visual cycle (2). The most critical step of the visual cycle is the isomerization of all-trans-retinol to 11-cis-retinol in the retinal pigment epithelium (RPE).3 It has been shown that all-trans-retinol must be first acylated by lecithin-retinol acyltransferase (LRAT), and the resultant retinyl ester serves as a substrate for the enzyme termed isomerohydrolase (3, 4). The isomerohydrolase is postulated to concomitantly hydrolyze and isomerize alltrans-retinyl ester to generate 11-cis-retinol (2). The isomerohydrolase activity was known for almost 20 years, but the enzyme has only been identified recently (5, 6).RPE65 is a microsomal protein predominantly expressed in the RPE (7,8). Mutations in RPE65 are associated with some forms of retinal dystrophies such as retinitis pigmentosa and Leber's congenital amaurosis (9 -12). Evidence from the RPE65 gene knock-out mouse has shown that RPE65 is essential for the...
Beta-carotene 15,15'-monooxygenase (BCM) catalyzes the first step of vitamin A biosynthesis from provitamin A carotenoids. We wished to determine the factors underlying the transcriptional regulation of this gene. After cloning of the 40 kilobase pair (kbp) mouse Bcm gene and determination of its genomic organization, analysis of the 2 kb 5'-flanking region showed several putative transcription factor binding sites including TATA box, a peroxisome proliferator response element (PPRE), AP2, and bHLH. The 2 kb fragment drove specific luciferase gene expression in vitro only in cell lines that express BCM (TC7, PF11, and monkey retinal pigment epithelium). Nucleotides -41 to +163, and -60 to +163 drove basal and specific Bcm transcriptional activity, respectively. Site-directed mutagenesis and gel shift experiments demonstrate that PPRE was essential for Bcm promoter specificity and that the peroxisome proliferator activated receptor (PPAR) gamma (PPARgamma) specifically binds to this element. Furthermore, cotransfection experiments and pharmacological treatments in vitro, using the specific PPARgamma agonists LY17883 and ciglitazone, demonstrate that the PPRE element confers peroxisome proliferator responsiveness via the PPARgamma and retinoid X receptor-alpha heterodimer. Treatment of mice with the PPARalpha/gamma agonist WY14643 increases BCM protein expression in liver. Thus PPAR is a key transcription factor for the transcriptional regulation of the Bcm gene, suggesting a broader function for PPARs in the regulation of carotenoid metabolism metabolism that is consistent with their established role in neutral lipid metabolism and transport.
The mechanism of retinol isomerization in the vertebrate retina visual cycle remains controversial. Does the isomerase enzyme RPE65 operate via nucleophilic addition at C 11 of the all-trans substrate, or via a carbocation mechanism? To determine this, we modeled the RPE65 substrate cleft to identify residues interacting with substrate and/or intermediate. We find that wild-type RPE65 in vitro produces 13-cis and 11-cis isomers equally robustly. All Tyr-239 mutations abolish activity. Trp-331 mutations reduce activity (W331Y to ϳ75% of wild type, W331F to ϳ50%, and W331L and W331Q to 0%) establishing a requirement for aromaticity, consistent with cation-carbocation stabilization. Two cleft residues modulate isomerization specificity: Thr-147 is important, because replacement by Ser increases 11-cis relative to 13-cis by 40% compared with wild type. Phe-103 mutations are opposite in action: F103L and F103I dramatically reduce 11-cis synthesis relative to 13-cis synthesis compared with wild type. Thr-147 and Phe-103 thus may be pivotal in controlling RPE65 specificity. Also, mutations affecting RPE65 activity coordinately depress 11-cis and 13-cis isomer production but diverge as 11-cis decreases to zero, whereas 13-cis reaches a plateau consistent with thermal isomerization. Lastly, experiments using labeled retinol showed exchange at 13-cis-retinol C 15 oxygen, thus confirming enzymatic isomerization for both isomers. Thus, RPE65 is not inherently 11-cis-specific and can produce both 11-and 13-cis isomers, supporting a carbocation (or radical cation) mechanism for isomerization. Specific visual cycle selectivity for 11-cis isomers instead resides downstream, attributable to mass action by CRALBP, retinol dehydrogenase 5, and high affinity of opsin apoproteins for 11-cis-retinal.A sequence of metabolic events, termed the visual cycle (1, 2), keeps retinal visual pigments, such as rhodopsin, in a state capable of responding to light. In brief, 11-cis-retinal bound to rhodopsin is photo-isomerized to all-trans-retinal, activating rhodopsin. To regenerate rhodopsin, all-trans-retinal is released, reduced to all-trans-retinol that is transported to the retinal pigment epithelium (RPE), 3 and esterified to all-trans-retinyl esters, the substrate for the retinol isomerase (3). All-transretinyl esters are enzymatically isomerized to yield 11-cis-retinol that is oxidized to 11-cis-retinal and returned to the photoreceptors (3, 4). Recently, the RPE protein RPE65 (5) has been identified as the isomerase central to this cycle (6 -8). The importance of RPE65 in chromophore regeneration had been well established by Rpe65 knock-out mice, which display extreme chromophore starvation (no rhodopsin) in the photoreceptors concurrent with overaccumulation of the all-transretinyl ester substrate of RPE65 in the RPE (9). Consequently, Rpe65 Ϫ/Ϫ mice are extremely insensitive to light. Mutations in the human RPE65 gene cause Leber congenital amaurosis 2, a condition of severe early onset blindness (10 -13), which has been the subject...
In order to maintain visual sensitivity at all light levels, the vertebrate eye possesses a mechanism to regenerate the visual pigment chromophore 11-cis retinal in the dark enzymatically, unlike in all other taxa, which rely on photoisomerization. This mechanism is termed the visual cycle and is localized to the retinal pigment epithelium (RPE), a support layer of the neural retina. Speculation has long revolved around whether more primitive chordates, such as tunicates and cephalochordates, anticipated this feature. The two key enzymes of the visual cycle are RPE65, the visual cycle all-trans retinyl ester isomerohydrolase, and lecithin:retinol acyltransferase (LRAT), which generates RPE65’s substrate. We hypothesized that the origin of the vertebrate visual cycle is directly connected to an ancestral carotenoid oxygenase acquiring a new retinyl ester isomerohydrolase function. Our phylogenetic analyses of the RPE65/BCMO and N1pC/P60 (LRAT) superfamilies show that neither RPE65 nor LRAT orthologs occur in tunicates (Ciona) or cephalochordates (Branchiostoma), but occur in Petromyzon marinus (Sea Lamprey), a jawless vertebrate. The closest homologs to RPE65 in Ciona and Branchiostoma lacked predicted functionally diverged residues found in all authentic RPE65s, but lamprey RPE65 contained all of them. We cloned RPE65 and LRATb cDNAs from lamprey RPE and demonstrated appropriate enzymatic activities. We show that Ciona ß-carotene monooxygenase a (BCMOa) (previously annotated as an RPE65) has carotenoid oxygenase cleavage activity but not RPE65 activity. We verified the presence of RPE65 in lamprey RPE by immunofluorescence microscopy, immunoblot and mass spectrometry. On the basis of these data we conclude that the crucial transition from the typical carotenoid double bond cleavage functionality (BCMO) to the isomerohydrolase functionality (RPE65), coupled with the origin of LRAT, occurred subsequent to divergence of the more primitive chordates (tunicates, etc.) in the last common ancestor of the jawless and jawed vertebrates.
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