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The enzyme β-carotene oxygenase 2 (BCO2) converts carotenoids into more polar metabolites. Studies in mammals, fish, and birds revealed that BCO2 controls carotenoid homeostasis and is involved in the pathway for vitamin A production. However, it is controversial whether BCO2 function is conserved in humans, because of a 4-amino acid long insertion caused by a splice acceptor site polymorphism. We here show that human BCO2 splice variants, BCO2a and BCO2b, are expressed as pre-proteins with mitochondrial targeting sequence (MTS). The MTS of BCO2a directed a green fluorescent reporter protein to the mitochondria when expressed in ARPE-19 cells. Removal of the MTS increased solubility of BCO2a when expressed in Escherichia coli and rendered the recombinant protein enzymatically active. The expression of the enzymatically active recombinant human BCO2a was further improved by codon optimization and its fusion with maltose-binding protein. Introduction of the 4-amino acid insertion into mouse Bco2 did not impede the chimeric enzyme's catalytic proficiency. We further showed that the chimeric BCO2 displayed broad substrate specificity and converted carotenoids into two ionones and a central C14-apocarotendial by oxidative cleavage reactions at C9,C10 and C9',C10'. Thus, our study demonstrates that human BCO2 is a catalytically competent enzyme. Consequently, information on BCO2 becomes broadly applicable in human biology with important implications for the physiology of the eyes and other tissues.

Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates. The iron centers of CCDs are typically five-coordinate in their resting states, with solvent occupying an exchangeable site. The involvement of this iron-bound solvent in CCD catalysis has not been experimentally addressed, but computational studies suggest two possible roles. 1) Solvent dissociation provides a coordination site for O(2), or 2) solvent remains bound to iron but changes its equilibrium position to allow O(2) binding and potentially acts as a proton source. To test these predictions, we investigated isotope effects (H(2)O versus D(2)O) on two stilbenoid-cleaving CCDs, Novosphingobium aromaticivorans oxygenase 2 (NOV2) and Neurospora crassa carotenoid oxygenase 1 (CAO1), using piceatannol as a substrate. NOV2 exhibited an inverse isotope effect (k(H)/k(D) ∼ 0.6) in an air-saturated buffer, suggesting that solvent dissociates from iron during the catalytic cycle. By contrast, CAO1 displayed a normal isotope effect (k(H)/k(D) ∼ 1.7), suggesting proton transfer in the rate-limiting step. X-ray absorption spectroscopy on NOV2 and CAO1 indicated that the protonation states of the iron ligands are unchanged within pH 6.5–8.5 and that the Fe(II)–aquo bond is minimally altered by substrate binding. We pinpointed the origin of the differential kinetic behaviors of NOV2 and CAO1 to a single amino acid difference near the solvent-binding site of iron, and X-ray crystallography revealed that the substitution alters binding of diffusible ligands to the iron center. We conclude that solvent-iron dissociation and proton transfer are both associated with the CCD catalytic mechanism.

Carotenoids can be enzymatically converted to apocarotenoids by carotenoid cleavage dioxygenases. Insect genomes encode only one member of this ancestral enzyme family. We cloned and characterized the ninaB genes from the silk worm (Bombyx mori) and the flour beetle (Tribolium castaneum). We expressed BmNinaB and TcNinaB in E. coli and analyzed their biochemical properties. Both enzymes catalyzed a conversion of carotenoids into cis-retinoids. The enzymes catalyzed a combined trans to cis isomerization at the C11, C12 double bond and oxidative cleavage reaction at the C15, C15' bond of the carotenoid carbon backbone. Analyses of the spatial and temporal expression patterns revealed that ninaB genes were differentially expressed during the beetle and moth life cycles with high expression in reproductive organs. In Bombyx mori, ninaB was almost exclusively expressed in female reproductive organs of the pupa and adult. In Tribolium castaneum, low expression was found in reproductive organs of females but high expressions in male reproductive organs of the pupa and imagoes. We performed RNAi experiments to characterize the role of NinaB in insect reproduction. We observed that RNAi treatment significantly decreased the expression levels of BmninaB and TcninaB and reduced the egg laying capacity of both insects. Together, our study revealed that NinaB's unique enzymatic properties are well conserved among insects and implicate NinaB function in insect reproduction.

The photochemistry of vision employs opsins and geometric isomerization of their covalently bound retinylidine chromophores. In different animal classes, these light receptors associate with distinct G proteins that either hyperpolarize or depolarize photoreceptor membranes. Vertebrates also use the acidic form of chromophore, retinoic acid, as the ligand of nuclear hormone receptors that orchestrate eye development. To establish and sustain these processes, animals must acquire carotenoids from the diet, transport them, and metabolize them to chromophore and retinoic acid. The understanding of carotenoid metabolism, however, lagged behind our knowledge about the biology of their receptor molecules. In the past decades, much progress has been made in identifying the genes encoding proteins that mediate the transport and enzymatic transformations of carotenoids and their retinoid metabolites. Comparative analysis in different animal classes revealed how evolutionary tinkering with a limited number of genes evolved different biochemical strategies to supply photoreceptors with chromophore. Mutations in these genes impair carotenoid metabolism and induce various ocular pathologies. This review summarizes this advancement and introduces the involved proteins, including the homeostatic regulation of their activities.

Carotenoids exert a rich variety of physiological functions in mammals and are beneficial for human health. These lipids are acquired from the diet and metabolized to apocarotenoids, including retinoids (vitamin A and its metabolites). The small intestine is a major site for their absorption and bioconversion. From here, carotenoids and their metabolites are distributed within the body in triacylglycerol-rich lipoproteins to support retinoid signaling in peripheral tissues and photoreceptor function in the eyes. In recent years, much progress has been made in identifying carotenoid metabolizing enzymes, transporters, and binding proteins. A diet-responsive regulatory network controls the activity of these components and adapts carotenoid absorption and bioconversion to the bodily requirements of these lipids. Genetic variability in the genes encoding these components alters carotenoid homeostasis and is associated with pathologies. We here summarize the advanced state of knowledge about intestinal carotenoid metabolism and its impact on carotenoid and retinoid homeostasis of other organ systems, including the eyes, liver, and immune system. The implication of the findings for science-based intake recommendations for these essential dietary lipids is discussed. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.

A family of enzymes collectively referred to as carotenoid cleavage oxygenases is responsible for oxidative conversion of carotenoids into apocarotenoids, including retinoids (vitamin A and its derivatives). A member of this family, the β-carotene 9,10-dioxygenase (BCO2), converts xanthophylls to rosafluene and ionones. Animals deficient in BCO2 highlight the critical role of the enzyme in carotenoid clearance as accumulation of these compounds occur in tissues. Inactivation of the enzyme by a four-amino acid-long insertion has recently been proposed to underlie xanthophyll concentration in the macula of the primate retina. Here, we focused on comparing the properties of primate and murine BCO2s. We demonstrate that the enzymes display a conserved structural fold and subcellular localization. Low temperature expression and detergent choice significantly affected binding and turnover rates of the recombinant enzymes with various xanthophyll substrates, including the unique macula pigment meso-zeaxanthin. Mice with genetically disrupted carotenoid cleavage oxygenases displayed adipose tissue rather than eye-specific accumulation of supplemented carotenoids. Studies in a human hepatic cell line revealed that BCO2 is expressed as an oxidative stress-induced gene. Our studies provide evidence that the enzymatic function of BCO2 is conserved in primates and link regulation of BCO2 gene expression with oxidative stress that can be caused by excessive carotenoid supplementation.

Recent evidence suggests an important role for outer retinal cells in the pathogenesis of diabetic retinopathy (DR). Here we investigated the effect of the visual cycle inhibitor retinylamine (Ret-NH2) on the development of early DR lesions. Wild-type (WT) C57BL/6J mice (male, 2 months old when diabetes was induced) were made diabetic with streptozotocin, and some were given Ret-NH2 once per week. Lecithin-retinol acyltransferase (LRAT)-deficient mice and P23H mutant mice were similarly studied. Mice were euthanized after 2 (WT and Lrat(-/-)) and 8 months (WT) of study to assess vascular histopathology, accumulation of albumin, visual function, and biochemical and physiological abnormalities in the retina. Non-retinal effects of Ret-NH2 were examined in leukocytes treated in vivo. Superoxide generation and expression of inflammatory proteins were significantly increased in retinas of mice diabetic for 2 or 8 months, and the number of degenerate retinal capillaries and accumulation of albumin in neural retina were significantly increased in mice diabetic for 8 months compared with nondiabetic controls. Administration of Ret-NH2 once per week inhibited capillary degeneration and accumulation of albumin in the neural retina, significantly reducing diabetes-induced retinal superoxide and expression of inflammatory proteins. Superoxide generation also was suppressed in Lrat(-/-) diabetic mice. Leukocytes isolated from diabetic mice treated with Ret-NH2 caused significantly less cytotoxicity to retinal endothelial cells ex vivo than did leukocytes from control diabetics. Administration of Ret-NH2 once per week significantly inhibited the pathogenesis of lesions characteristic of early DR in diabetic mice. The visual cycle constitutes a novel target for inhibition of DR.

Background: Nonalcoholic fatty liver disease is positively associated with obesity and cardiovascular disease risk. Apo-10′-lycopenoic acid (APO10LA), a potential oxidation product of apo-10′-lycopenal that is generated endogenously by β-carotene-9′,10′-oxygenase (BCO2) cleavage of lycopene, inhibited hepatic steatosis in BCO2-expressing mice. Objective: The present study evaluated lycopene and APO10LA effects on hepatic steatosis in mice without BCO2 expression. Methods: Male and female BCO2-knockout (BCO2-KO) mice were fed a high saturated fat diet (HSFD) with or without APO10LA (10 mg/kg diet) or lycopene (100 mg/kg diet) for 12 wk. Results: Lycopene or APO10LA supplementation reduced hepatic steatosis incidence (78% and 72%, respectively) and severity in BCO2-KO male mice. Female mice did not develop steatosis, had greater hepatic total cholesterol (3.06 vs. 2.31 mg/g tissue) and cholesteryl ester (1.58 vs. 0.86 mg/g tissue), but had lower plasma triglyceride (TG) (229 vs. 282 mg/dL) and cholesterol (97.1 vs. 119 mg/dL) than male mice. APO10LA-mitigated steatosis in males was associated with reduced hepatic total cholesterol (18%) and activated sirtuin 1 signaling, which resulted in reduced fatty acids (FAs) and TG synthesis markers [stearoyl-coenzyme A (CoA) desaturase protein, 71%; acetyl-CoA carboxylase phosphorylation, 79%; AMP-activated protein kinase phosphorylation, 67%], and elevated cholesterol efflux genes (cytochrome P450 family 7A1, 65%; ATP-binding cassette transporter G5/8, 11%). These APO10LA-mediated effects were not mimicked by lycopene supplementation. Intriguingly, steatosis inhibition by lycopene induced peroxisome proliferator–activated receptor (PPAR)α- and PPARγ-related genes in mesenteric adipose tissue (MAT) that increases mitochondrial uncoupling [cell death–inducing DNA fragmentation factor, α subunit-like effector a, 55%; PR domain-containing 16, 47%; uncoupling protein 3 (Ucp3), 55%], FA β-oxidation (PPARα, 53%; very long chain acyl-CoA dehydrogenase, 38%), and uptake (FA transport protein 4, 29%; lipoprotein lipase 43%). Expressions of 10 MAT PPAR-related genes were inversely correlated with steatosis score, suggesting that lycopene reduced steatosis by increasing MAT FA utilization. Conclusions: Our data suggest that lycopene and APO10LA inhibit HSFD-induced steatosis in BCO2-KO male mice through differential mechanisms. Sex disparity of BCO2-KO mice was observed in the outcomes of HSFD-induced liver steatosis and plasma lipids.

Carotenoids and their metabolic derivatives serve critical functions in both prokaryotic and eukaryotic cells, including pigmentation, photoprotection and photosynthesis as well as cell signaling. These organic compounds are also important for visual function in vertebrate and non-vertebrate organisms. Enzymatic transformations of carotenoids to various apocarotenoid products are catalyzed by a family of evolutionarily conserved, non-heme iron-containing enzymes named carotenoid cleavage oxygenases (CCOs). Studies have revealed that CCOs are critically involved in carotenoid homeostasis and essential for the health of organisms including humans. These enzymes typically display a high degree of regio- and stereo-selectivity, acting on specific positions of the polyene backbone located in their substrates. By oxidatively cleaving and/or isomerizing specific double bonds, CCOs generate a variety of apocarotenoid isomer products. Recent structural studies have helped illuminate the mechanisms by which CCOs mobilize their lipophilic substrates from biological membranes to perform their characteristic double bond cleavage and/or isomerization reactions. In this review, we aim to integrate structural and biochemical information about CCOs to provide insights into their catalytic mechanisms.

Mammalian genomes encode two provitamin A-converting enzymes as follows: the β-carotene-15,15'-oxygenase (BCO1) and the β-carotene-9',10'-oxygenase (BCO2). Symmetric cleavage by BCO1 yields retinoids (β-15'-apocarotenoids, C20), whereas eccentric cleavage by BCO2 produces long-chain (C20) apocarotenoids. Here, we used genetic and biochemical approaches to clarify the contribution of these enzymes to provitamin A metabolism. We subjected wild type, Bco1(-/-), Bco2(-/-), and Bco1(-/-)Bco2(-/-) double knock-out mice to a controlled diet providing β-carotene as the sole source for apocarotenoid production. This study revealed that BCO1 is critical for retinoid homeostasis. Genetic disruption of BCO1 resulted in β-carotene accumulation and vitamin A deficiency accompanied by a BCO2-dependent production of minor amounts of β-apo-10'-carotenol (APO10ol). We found that APO10ol can be esterified and transported by the same proteins as vitamin A but with a lower affinity and slower reaction kinetics. In wild type mice, APO10ol was converted to retinoids by BCO1. We also show that a stepwise cleavage by BCO2 and BCO1 with APO10ol as an intermediate could provide a mechanism to tailor asymmetric carotenoids such as β-cryptoxanthin for vitamin A production. In conclusion, our study provides evidence that mammals employ both carotenoid oxygenases to synthesize retinoids from provitamin A carotenoids.

Low dietary intake of β-carotene is associated with chronic disease and vitamin A deficiency. β-Carotene is converted to vitamin A in the intestine by the enzyme β-carotene-15,15'-monoxygenase (BCMO1) to support vision, reproduction, immune function, and cell differentiation. Considerable variability for this key step in vitamin A metabolism, as reported in the human population, could be related to genetics and individual vitamin A status, but it is unclear how these factors influence β-carotene metabolism and vitamin A homeostasis. Here we show that the intestine-specific transcription factor ISX binds to the Bcmo1 promoter. Moreover, upon induction by the β-carotene derivative retinoic acid, this ISX binding decreased expression of a luciferase reporter gene in human colonic CaCo-2 cells indicating that ISX acts as a transcriptional repressor of BCMO1 expression. Mice deficient for this transcription factor displayed increased intestinal BCMO1 expression and produced significantly higher amounts of vitamin A from supplemental β-carotene. The ISX binding site in the human BCMO1 promoter contains a common single nucleotide polymorphism that is associated with decreased conversion rates and increased fasting blood levels of β-carotene. Thus, our study establishes ISX as a critical regulator of vitamin A production and provides a mechanistic explanation for how both genetics and diet can affect this process.

Vitamin A deficiency is a major public health problem in developing countries. Some studies also implicate a suboptimal vitamin A intake in certain parts of the population of the industrialized world. Provitamin A carotenoids such as β-carotene are the major source for retinoids (vitamin A and its derivatives) in the human diet. However, it is still controversial how much β-carotene intake is required and safe. An important contributor to this uncertainty is the lack of knowledge about the biochemical and molecular basis of β-carotene metabolism. Recently, key players of provitamin A metabolism have been molecularly identified and biochemically characterized. Studies in knockout mouse models showed that intestinal β-carotene absorption and conversion to retinoids is under negative feedback regulation that adapts this process to the actual requirement of vitamin A of the body. These studies also showed that in peripheral tissues a conversion of β-carotene occurs and affects retinoid-dependent physiologic processes. Moreover, these analyses provided a possible explanation for the adverse health effects of carotenoids by showing that a pathologic accumulation of these compounds can induce oxidative stress in mitochondria and cell signaling pathways related to disease. Genetic polymorphisms in identified genes exist in humans and also alter carotenoid homeostasis. Here, the advanced knowledge of β-carotene metabolism is reviewed, which provides a molecular framework for understanding the role of this important micronutrient in health and disease.

Vitamin A (all-trans-retinol) must be adequately distributed within the mammalian body to produce visual chromophore in the eyes and all-trans-retinoic acid in other tissues. Vitamin A is transported in the blood bound to retinol-binding protein (holo-RBP), and its target cells express an RBP receptor encoded by the Stra6 (stimulated by retinoic acid 6) gene. Here we show in mice that cellular uptake of vitamin A from holo-RBP depends on functional coupling of STRA6 with intracellular lecithin:retinol acyltransferase (LRAT). Thus, vitamin A uptake from recombinant holo-RBP exhibited by wild type mice was impaired in Lrat−/− mice. We further provide evidence that vitamin A uptake is regulated by all-trans-retinoic acid in non-ocular tissues of mice. When in excess, vitamin A was rapidly taken up and converted to its inert ester form in peripheral tissues, such as lung, whereas in vitamin A deficiency, ocular retinoid uptake was favored. Finally, we show that the drug fenretinide, used clinically to presumably lower blood RBP levels and thus decrease circulating retinol, targets the functional coupling of STRA6 and LRAT to increase cellular vitamin A uptake in peripheral tissues. These studies provide mechanistic insights into how vitamin A is distributed to peripheral tissues in a regulated manner and identify LRAT as a critical component of this process. Background: Cellular uptake of retinol bound to its serum binding protein depends on a cell surface receptor. Results: Functional coupling of this receptor with lecithin:retinol acyltransferase is required for the regulated cellular uptake of retinol. Conclusion: The lecithin: retinol acyltransferase is critical for retinol uptake and homeostasis. Significance: Blood retinol homeostasis is associated with blinding retinopathies and diabetes.

Humans depend on a dietary intake of lipids to maintain optimal health. Among various classes of dietary lipids, the physiological importance of carotenoids is still controversially discussed. On one hand, it is well established that carotenoids, such as β,β-carotene, are a major source for vitamin A that plays critical roles for vision and many aspects of cell physiology. On the other hand, large clinical trials have failed to show clear health benefits of carotenoids supplementation and even suggest adverse health effects in individuals at risk of disease. In recent years, key molecular players for carotenoid metabolism have been identified, including an evolutionarily well conserved family of carotenoid-oxygenases. Studies in knockout mouse models for these enzymes revealed that carotenoid metabolism is a highly regulated process and that this regulation already takes place at the level of intestinal absorption. These studies also provided evidence that β,β-carotene conversion can influence retinoid-dependent processes in the mouse embryo and in adult tissues. Moreover, these analyses provide an explanation for adverse health effects of carotenoids by showing that a pathological accumulation of these compounds can induce oxidative stress in mitochondria and cell signaling pathways related to disease. Advancing knowledge about carotenoid metabolism will contribute to a better understanding of the biochemical and physiological roles of these important micronutrients in health and disease. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.

Two enzymes have been identified for the oxidative metabolism of carotenoids in mammals. Carotene-15,15'-monooxygenase (CMO-I) primarily centrally cleaves β,β-carotene to form vitamin A. We hypothesize that carotene-9',10'-monooxygenase (CMO-II) plays a key role in metabolism of acyclic nonprovitamin A carotenoids such as lycopene. We investigated carotenoid bioaccumulation in young adult, male, wild-type (WT) mice or mice lacking CMO-II (CMO-II KO). Mice were fed an AIN-93G diet or identical diets supplemented with 10% tomato powder, 130 mg lycopene/kg diet (10% lycopene beadlets), or placebo beadlets for 4 or 30 d. Lycopene preferentially accumulated in CMO-II KO mouse tissues and serum compared with WT mouse tissues. β-Carotene preferentially accumulated in some CMO-II KO mouse tissues compared with WT mouse tissues. Relative tissue mRNA expression of CMO-I and CMO-II was differentially expressed in mouse tissues, and CMO-II, but not CMO-I, was expressed in mouse prostate. In conclusion, the loss of CMO-II expression leads to increased serum and tissue concentrations of lycopene in tomato-fed mice.

Hepatic stellate cells (HSCs) are responsible for storing 90–95% of the retinoid present in the liver. These cells have been reported in the literature also to accumulate dietary β-carotene, but the ability of HSCs to metabolize β-carotene in situ has not been explored. To gain understanding of this, we investigated whether β-carotene-15,15′-monooxygensase (Bcmo1) and β-carotene-9′,10′-monooxygenase (Bcmo2) are expressed in HSCs. Using primary HSCs and hepatocytes purified from wild type and Bcmo1-deficient mice, we establish that Bcmo1 is highly expressed in HSCs; whereas Bcmo2 is expressed primarily in hepatocytes. We also confirmed that HSCs are an important cellular site within the liver for accumulation of dietary β-carotene. Bcmo2 expression was found to be significantly elevated for livers and hepatocytes isolated from Bcmo1-deficient compared to wild type mice. This elevation in Bcmo2 expression was accompanied by a statistically significant increase in hepatic apo-12′-carotenal levels of Bcmo1-deficient mice. Although apo-10′-carotenal, like apo-12′-carotenal, was readily detectable in livers and serum from both wild type and Bcmo1-deficient mice, we were unable to detect either apo-8′- or apo-14′-carotenals in livers or serum from the two strains. We further observed that hepatic triglyceride levels were significantly elevated in livers of Bcmo1-deficient mice fed a β-carotene-containing diet compared to mice receiving no β-carotene. Collectively, our data establish that HSCs are an important cellular site for β-carotene accumulation and metabolism within the liver.

Increasing evidence has been provided for a connection between retinoid metabolism and the activity of peroxisome proliferator receptors (Ppars) in the control of body fat reserves. Two different precursors for retinoids exist in the diet as preformed vitamin A (all-trans-retinol) and provitamin A (beta,beta-carotene). For retinoid production, beta,beta-carotene is converted to retinaldehyde by beta,beta-carotene monooxygenase 1 (Bcmo1). Previous analysis showed that Bcmo1 knock-out mice develop dyslipidemia and are more susceptible to diet-induced obesity. However, the role of Bcmo1 for adipocyte retinoid metabolism has yet not been well defined. Here, we showed that Bcmo1 mRNA and protein expression are induced during adipogenesis in NIH 3T3-L1 cells. In mature adipocytes, beta,beta-carotene but not all-trans-retinol was metabolized to retinoic acid (RA). RA decreased the expression of Ppar gamma and CCAAT/enhancer-binding protein alpha, key lipogenic transcription factors, and reduced the lipid content of mature adipocytes. This process was inhibited by the retinoic acid receptor antagonist LE450, showing that it involves canonical retinoid signaling. Accordingly, gavage of beta,beta-carotene but not all-trans-retinol induced retinoid signaling and decreased Ppar gamma expression in white adipose tissue of vitamin A-deficient mice. Our study identifies beta,beta-carotene as a critical physiological precursor for RA production in adipocytes and implicates provitamin A as a dietary regulator of body fat reserves.

Recently, much progress has been made in elucidating the chemistry and metabolism of retinoids and carotenoids, as well as the structures of processing proteins related to vision. Carotenoids and their retinoid metabolites are isoprenoids, so only a limited number of chemical transformations are possible, and just a few of these occur naturally. Although there is an intriguing evolutionary conservation of the key components involved in the production and recycling of chromophores, these genes have also adapted to the specific requirements of insect and vertebrate vision. These 'ancestral footprints' in animal genomes bear witness to the common origin of the chemistry of vision, and will further stimulate research across evolutionary boundaries.

In animals, visual pigments are essential for photoreceptor function and survival. These G-protein-coupled receptors consist of a protein moiety (opsin) and a covalently bound 11-cis-retinylidene chromophore. The chromophore is derived from dietary carotenoids by oxidative cleavage and trans-to-cis isomerization of double bonds. In vertebrates, the necessary chemical transformations are catalyzed by two distinct but structurally related enzymes, the carotenoid oxygenase beta-carotenoid-15,15'-monooxygenase and the retinoid isomerase RPE65 (retinal pigment epithelium protein of 65 kDa). Recently, we provided biochemical evidence that these reactions in insects are catalyzed by a single enzyme family member named NinaB. Here we show that in the fly pathway, carotenoids are mandatory precursors of the chromophore. After chromophore formation, the retinoid-binding protein Pinta acts downstream of NinaB and is required to supply photoreceptors with chromophore. Like ninaE encoding the opsin, ninaB expression is eye-dependent and is activated as a downstream target of the eyeless/pax6 and sine oculis master control genes for eye development. The requirement for coordinated synthesis of chromophore and opsin is evidenced by analysis of ninaE mutants. Retinal degeneration in opsin-deficient photoreceptors is caused by the chromophore and can be prevented by restricting its supply as seen in an opsin and chromophore-deficient double mutant. Thus, our study identifies NinaB as a key component for visual pigment production and provides evidence that chromophore in opsin-deficient photoreceptors can elicit retinal degeneration.

For vertebrate development, vitamin A (all-trans retinol) is required in quantitative different amounts and spatiotemporal distribution for the production of retinoic acid, a nuclear hormone receptor ligand, and 11-cis retinal, the chromophore of visual pigments. We show here for zebrafish that embryonic retinoid homeostasis essentially depends on the activity of a leci-thin:retinol acyltransferase (Lratb). During embryogenesis, lratb is expressed in mostly non-overlapping domains opposite to retinal dehydrogenase 2 (raldh2), the key enzyme for retinoic acid synthesis. Blocking retinyl ester formation by a targeted knock down of Lratb results in significantly increased retinoic acid levels, which lead to severe embryonic patterning defects. Thus, we provide evidence that a balanced competition between Lratb and Raldh2 for yolk vitamin A defines embryonic compartments either for retinyl ester or retinoic acid synthesis. This homeostatic mechanism dynamically adjusts embryonic retinoic acid levels for gene regulation, concomitantly sequestering excess yolk vitamin A in the form of retinyl esters for the establishment of larval vision later during development.

Rod and cone visual pigments use 11-cis-retinal, a vitamin A derivative, as their chromophore. Light isomerizes 11-cis- into all-trans-retinal, triggering a conformational transition of the opsin molecule that initiates phototransduction. After bleaching all-trans-retinal leaves the opsin, and light sensitivity must be restored by regeneration of 11-cis-retinal. Under bright light conditions the retinal G protein-coupled receptor (RGR) was reported to support this regeneration by acting as a photoisomerase in a proposed photic visual cycle. We analyzed the contribution of RGR to rhodopsin regeneration under different light regimes and show that regeneration, during light exposure and in darkness, is slowed about 3-fold in Rgr(-/-) mice. These findings are not in line with the proposed function of RGR as a photoisomerase. Instead, RGR, independent of light, accelerates the conversion of retinyl esters to 11-cis-retinal by positively modulating isomerohydrolase activity, a key step in the "classical" visual cycle. Furthermore, we find that light accelerates rhodopsin regeneration, independent of RGR.

Vitamin A derivatives (retinoids) are essential components in vision; they contribute to pattern formation during development and exert multiple effects on cell differentiation with important clinical implications. All naturally occurring vitamin A derives by enzymatic oxidative cleavage from carotenoids with provitamin A activity. To become biologically active, these plant-derived compounds must first be absorbed, then delivered to the site of action in the body, and metabolically converted to the real vitamin. Recently, molecular players of this pathway were identified by the analysis of blind Drosophila mutants. Similar genome sequences were found in vertebrates. Subsequently, these homologous genes were cloned and their gene products were functionally characterized. This review will summarize the advanced state of knowledge about the vitamin A biosynthetic pathway and will discuss biochemical, physiological, developmental and medical aspects of carotenoids and their numerous derivatives.

Vitamin A and its derivatives (retinoids) are essential components in vision; they contribute to pattern formation during development and exert multiple effects on cell differentiation. It has been known for 70 y that the key step in vitamin A biosynthesis is the oxidative cleavage of a carotenoid with provitamin A activity. While a detailed biochemical characterization of the respective enzymes could be achieved in cell-free homogenates, their molecular nature has remained elusive for a long time. Recent research led to the identification of genes encoding two different types of carotene oxygenases from animal species. The molecular cloning of these different types of animal carotene oxygenases establishes the existence of a family of carotenoid metabolizing enzymes in animals heretofore described in plants. With these tools in hands, old questions in vitamin A research can be definitively addressed on the molecular levels contributing to a mechanistic understanding of the regulation of vitamin A homeostasis or tissue specificity of vitamin A formation, with impact on animal physiology and human health.

Carotenoid cleavage products (apocarotenoids) are widespread in living organisms and exert key biological functions. In animals, retinoids function as vitamins, visual pigments and signalling molecules. In plants, apocarotenoids play roles as hormones, pigments, flavours, aromas and defence compounds. The first step in their biosynthesis is the oxidative cleavage of a carotenoid catalysed by a non-heme iron oxygenase. A novel family of enzymes, which can cleave different carotenoids at different positions, has been characterized.

In vertebrates, symmetric versus asymmetric cleavage of beta-carotene in the biosynthesis of vitamin A and its derivatives has been controversially discussed. Recently we have been able to identify a cDNA encoding a metazoan beta,beta-carotene-15,15'-dioxygenase from the fruit fly Drosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving beta-carotene to give two molecules of retinal. Mutations in the corresponding gene are known to lead to a blind, vitamin A-deficient phenotype. Orthologs of this enzyme have very recently been found also in vertebrates and molecularly characterized. Here we report the identification of a cDNA from mouse encoding a second type of carotene dioxygenase catalyzing exclusively the asymmetric oxidative cleavage of beta-carotene at the 9',10' double bond of beta-carotene and resulting in the formation of beta-apo-10'-carotenal and beta-ionone, a substance known as a floral scent from roses, for example. Besides beta-carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the beta,beta-carotene-15,15'-dioxygenases, and the two enzyme types have several conserved motifs. To establish its occurrence in different vertebrates, we then attempted and succeeded in cloning cDNAs encoding this new type of carotene dioxygenase from human and zebrafish as well. As regards their possible role, the apocarotenals formed by this enzyme may be the precursors for the biosynthesis of retinoic acid or exert unknown physiological effects. Thus, in contrast to Drosophila, in vertebrates both symmetric and asymmetric cleavage pathways exist for carotenes, revealing a greater complexity of carotene metabolism.

Beta-carotene 15,15'-dioxygenase cleaves beta-carotene into two molecules of retinal and is the key enzyme in the metabolism of carotene to vitamin A. Although the enzyme has been known for more than 40 years, all attempts to purify the protein to homogeneity or to clone its gene have failed until recently, when the successful cloning and sequencing of cDNAs encoding enzymes with beta-carotene 15,15'-dioxygenase activity from Drosophila (J. von Lintig and K. Vogt, 2000, J. Biol. Chem. 275, 11915-11920) and chicken (A. Wyss et al., 2000, Biochem. Biophys. Res. Commun. 271, 334-336) were reported. Very soon it became clear, that we have cloned two members of a new family of carotenoid cleaving enzymes. Overall homologies are very high, certain amino acid stretches almost identical. Thus, beta-carotene 15,15'-dioxygenase can be considered as evolutionarily well conserved. These findings open up wide perspectives for further analysis of this important biosynthetic pathway, concerning basic and medical research as well as biotechnological aspects related to vitamin A supply, which are discussed here.

Vitamin A and its derivatives (retinoids) are essential components in vision; they contribute to pattern formation during development and exert multiple effects on cell differentiation with important clinical implications. It has been known for 50 years that the key step in the formation of vitamin A is the oxidative cleavage of β-carotene; however, this enzymatic step has resisted molecular analysis. A novel approach enabled us to clone and identify a β-carotene dioxygenase from Drosophila melanogaster,expressing it into the background of a β-carotene (provitamin A)-synthesizing and -accumulating Escherichia coli strain. The carotene-cleaving enzyme, identified here for the first time on the molecular level, is the basis of the numerous branches of vitamin A action and links plant and animal carotene metabolism.