In vertebrates, symmetric versus asymmetric cleavage of ␤-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 ␤,␤-carotene-15,15-dioxygenase from the fruit fly Drosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving ␤-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 ␤-carotene at the 9,10 double bond of ␤-carotene and resulting in the formation of ␤-apo-10-carotenal and ␤-ionone, a substance known as a floral scent from roses, for example. Besides ␤-carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the ␤,␤-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.

Most important, certain carotenoids are the precursors (provitamins) for the formation of vitamin A in animals. This vitamin is needed for vision in the entire animal kingdom. The visual pigments (rhodopsins) of animals are composed of a retinoid chromophore (vitamin A derivative) bound to a protein moiety (opsin) embedded in the photoreceptor membranes (3, 4). Light activation of the visual pigments triggers a G protein-coupled receptor cascade leading to changes in the permeability of the photoreceptor cell membranes. Besides being crucial for vision, in vertebrates vitamin A is also important in development and cellular differentiation processes. Here, the vitamin A derivative retinoic acid, together with its nuclear receptors, is involved in the regulation of diverse target genes; consequently, complete vitamin A deficiency leads to early embryonic death (5).To become biologically active, dietary carotenoids must first be absorbed, then delivered to the site of action in the body and, in the case of provitamin A function, metabolically converted. Despite the general importance of carotenoids in animals, their metabolism is still poorly understood (6). Invertebrates like Drosophila represent excellent models for the genetic dissection of the pathway leading from dietary carotenoids to vitamin A. Here, this vitamin is only needed for vision; therefore, its deficiency has no fatal consequences. Among the various Drosophila mutants affected in their visual performance (4), the two mutants ninaB and ninaD lack the visual chromophore of the fly, 3-hydroxyretinal, when raised on standard media with carotenoids as the sole source for vitamin A formation (7). By analyzing the molecular basis of the blindness of ninaB mutants, we already showed that the phenotype is caused by mutations in a gene coding a carotene-15,15Ј-oxygenase and molecularly identified the key enzyme for carotenoid conversion to vitamin A in animals (8, 9). By sequence identity, orthologs to this insect gene were cloned from several vertebrate species including man, showing that the enzymes catalyzing vitamin A formation are evolutionarily well conserved (10-13). In Drosophila, mRNA expression of ninaB was exclusively found in the head, in agreement with retinoids being restricted in their distribution to the eyes (8,14). In vertebrates (with vitamin A needed also for cellular differentiation processes), the vitamin A-forming enzyme is expressed in a variety of different tissues including reproductive tissues and the eyes (10, 12, 13). After dietary absorption, carotenoids must be distributed to these tissues to be converted to vitamin A.In the second chromophore-less Drosophila mutant, ninaD, the carotenoid content was shown to be specifically and significantly altered compared with wild-type (wt) flies and was ineffective at mediating visual pigment synthesis (14). This phenotype is presumably caused by a defect in the body distribution of dietary carotenoids and makes the ninaD gene an interesting candidate for a molecular player necessary for ...

The blind Drosophila mutant ninaD lacks the visual chromophore. Genetic evidence that the molecular basis is a defect in carotenoid uptake which causes vitamin A deficiency exists. The ninaD gene encodes a scavenger receptor that is significantly homologous in sequence with the mammalian scavenger receptors SR-BI (scavenger receptor class B type I) and CD36 (cluster determinant 36), yet NinaD has not been characterized in functional detail. Therefore, we established a Drosophila S2 cell culture system for biochemically characterizing the ninaD gene products. We show that the two splice variant isoforms encoded by ninaD exhibit different subcellular localizations. NinaD-I, the long protein variant, is localized at the plasma membrane, whereas the short variant, NinaD-II, is localized at intracellular membranes. Only NinaD-I could mediate the cellular uptake of carotenoids from micelles in this cell culture system. Carotenoid uptake was concentration-dependent and saturable. By in vivo analyses of different mutant and transgenic fly strains, we provide evidence of an essential role of NinaD-I in the absorption of dietary carotenoids to support visual chromophore synthesis. Moreover, our analyses suggest a role of NinaD-I in tocopherol metabolism. Even though Drosophila is a sterol auxotroph, we found no evidence of a contribution of NinaD-I to the uptake of these compounds. Together, our study establishes an evolutionarily conserved connection between class B scavenger receptors and the numerous functions of fat soluble vitamins in animal physiology.

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 pro-vitamin 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.

Visual pigments (rhodopsins) are composed of a chromophore (vitamin A derivative) bound to a protein moiety embedded in the retinal membranes. Animals cannot synthesize the visual chromophore de novo but rely on the uptake of carotenoids, from which vitamin A is formed enzymatically by oxidative cleavage. Despite its importance, the enzyme catalyzing the key step in vitamin A formation resisted molecular analyses until recently, when the successful cloning of a cDNA encoding an enzyme with ␤,␤-carotene-15,15-dioxygenase activity from Drosophila was reported. To prove its identity with the key enzyme for vitamin A formation in vivo, we analyzed the blind Drosophila mutant ninaB. In two independent ninaB alleles, we found mutations in the gene encoding the ␤,␤-carotene-15,15-dioxygenase. These mutations lead to a defect in vitamin A formation and are responsible for blindness of these flies.

Visual pigments (rhodopsins) are composed of a chromophore (vitamin A derivative) bound to a protein moiety embedded in the retinal membranes. Animals cannot synthesize the visual chromophore de novo but rely on the uptake of carotenoids, from which vitamin A is formed enzymatically by oxidative cleavage. Despite its importance, the enzyme catalyzing the key step in vitamin A formation resisted molecular analyses until recently, when the successful cloning of a cDNA encoding an enzyme with ␤,␤-carotene-15,15-dioxygenase activity from Drosophila was reported. To prove its identity with the key enzyme for vitamin A formation in vivo, we analyzed the blind Drosophila mutant ninaB. In two independent ninaB alleles, we found mutations in the gene encoding the ␤,␤-carotene-15,15-dioxygenase. These mutations lead to a defect in vitamin A formation and are responsible for blindness of these flies.