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Filling the Gap in Vitamin A Research

2000; Elsevier BV; Volume: 275; Issue: 16 Linguagem: Inglês

10.1074/jbc.275.16.11915

1083-351X

Johannes von Lintig, Klaus Vogt,

Retinal Development and Disorders

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. 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. 15,15′-β-carotene dioxygenase 9-cisneoxanthin-cleavage enzyme from Zea mais retinal pigment epithelium polymerase chain reaction reverse transcriptase Expressed Sequence Tag N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine high performance liquid chromatography Animals are, in general, unable to synthesize vitamin A de novo. Vitamin A (retinol) and its derivatives (retinal, retinoic acid) are C20 isoprenoids metabolically derived from the oxidative cleavage of C40 carotenoids (Fig.1). Most effects of vitamin A are exerted by retinoic acid (1.Dowling J.E. Wald G. Proc. Natl. Acad. Sci. U. S. A. 1960; 46: 587-608Crossref PubMed Google Scholar, 2.Chytil F. Livrea M.A. Vidali G. Retinoids: From Basic Science to Clinical Applications. Birkhäuser Verlag, Basel, Switzerland1994: 11-19Google Scholar). In the visual system, retinal or closely related compounds such as 3-hydroxyretinal serve as chromophores for the various visual pigments (rhodopsins) (3.Wald G. Nature. 1968; 219: 800-807Crossref PubMed Scopus (573) Google Scholar, 4.Vogt K. Z. Naturforsch. 1984; 39: 196-197Crossref Scopus (47) Google Scholar). Although the cleavage of β-carotene to form retinal in animals has been investigated in cell-free systems for about 50 years, this important enzymatic step has resisted molecular analysis (5.Moore T. Biochem. J. 1930; 24: 692-702Crossref PubMed Google Scholar, 6.Goodman D., S. Huang H.S. Science. 1965; 149: 879-880Crossref PubMed Scopus (184) Google Scholar, 7.Olson J.A. Hayaishi O. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1364-1370Crossref PubMed Scopus (258) Google Scholar, 8.Olson J.A. Am. J. Clin. Nutr. 1969; 22: 953-962Crossref PubMed Scopus (17) Google Scholar, 9.Krinsky N.I. Wang X. Tang D.G. Russel R.M. Livrea M.A. Vidali G. Retinoids: From Basic Science to Clinical Applications. Birkhäuser Verlag, Basel, Switzerland1994: 21-28Google Scholar), and the specific enzyme has yet to be purified. Despite compelling evidence in several experimental systems for a central cleavage of β-carotene yielding two molecules of retinal, there is still an ongoing debate as to the significance of eccentric cleavage of β-carotene (10.Napoli J.L. Race K.R. J. Biol. Chem. 1988; 263: 17372-17377Abstract Full Text PDF PubMed Google Scholar). Enzymatic oxidative cleavage of carotenoids is also found in bacteria and plants. In higher plants, many examples of eccentric carotenoid cleavage are found. These examples include the formation of saffron in crocus, citraurin and other apocarotenoids in citrus fruits, and most interestingly for us, the plant hormone abcisic acid, a growth regulator involved,e.g. in the autumnal fall of leaves and in seed dormancy. Abcisic acid derives from the oxidative cleavage of 9-cisepoxy carotenoids at the 11–12 carbon double bond. Recently, analysis of a maize mutant, vp14, which is defective in abcisic acid biosynthesis, has led to a better molecular understanding of this cleavage reaction. The cloning of the corresponding gene revealed that the encoded protein catalyzes this oxidative cleavage to form xanthoxin, the direct precursor of abcisic acid (11.Schwartz S.H. Tan B.C. Gage D.A. Zeevaart J.A. McCarty D.R. Science. 1997; 276: 1872-1874Crossref PubMed Scopus (703) Google Scholar). Vp14 shares weak sequence homology to several proteins of unknown function in the data bases, among which are some from animals.We wondered whether animal homologues of vp14 might also act as dioxygenases and whether oxidative cleavage of carotenoids with provitamin A activity is performed by a similar enzyme in animals. The present paper answers this question positively. We cloned and identified a β-carotene dioxygenase (β-diox)1 fromDrosophila melanogaster by expressing it into the background of an Escherichia coli strain capable of synthesizing and accumulating the direct percursor, β-carotene (provitamin A). The use of this novel test system for the first time allowed the molecular identification of a vitamin A-forming enzyme. It revealed that the protein catalyzes the centric cleavage of β-carotene, leading exclusively to the formation of C20-compounds (retinoids).RESULTSTo find homologues of vp14, the plant carotenoid-cleaving enzyme, we searched insect EST libraries and discovered a published EST fragment from D. melanogaster (GenBank™ accession numberAI063857). For cloning of the full-length cDNA and to test directly for β-carotene dioxygenase activity, an E. coli strain was constructed that was able to synthesize and accumulate β-carotene by introducing the gene set for β-carotene biosynthesis from the bacterium E. herbicola (12.Hundle B.S. Alberti M. Nievelstein V. Beyer P. Kleinig H. Armstrong G.A. Burke D.H. Hearst J.E. Mol. Gen. Genet. 1994; 245: 406-416Crossref PubMed Scopus (71) Google Scholar). This approach allowed the detection of retinoid formation by the fading of the colonies from yellow (β-carotene) to almost white (retinoids) and offered a fast and efficient test system to identify β-carotene dioxygenase activity. For this purpose, total RNA was isolated fromDrosophila heads, and cDNA was synthesized. RACE-PCR was performed with a specific oligonucleotide derived from the EST fragment and a dT17-anchor oligonucleotide. The PCR products obtained were directly cloned into the expression vector pBAD-TOPO and transformed into the described E. coli strain. After plating the bacteria on LB media containing 0.2% l-arabinose to induce the expression of the putative β-carotene dioxygenase, several almost-white colonies were found and subjected to further analysis (Fig. 2). Overnight cultures were grown under safety red light to minimize isomerization and unspecific cleavage of β-carotene by photo-oxidation. β-Carotene and retinoids were extracted and subjected to HPLC analyses. The control strain transformed with the vector alone lacked the ability to cleave β-carotene, and no traces of retinoids were detectable. However, bacteria expressing the Drosophila cDNA contained significant amounts of retinoids in addition to β-carotene (Fig.3 A). The retinoids were identified by retention time as well as co-chromatography with authentic standards and by their absorption spectra (Fig.4). The dominant retinal isomer was the all-trans form, with only ∼20% of the 13-cisisomer. Depending on the time bacteria were grown after induction, significant amounts of all-trans retinol and 13-cis retinol as well as esters of these retinol isomers could be detected. The retinoid isomers found were consistent with the isomeric composition of their β-carotene precursors, which were identified by a separate HPLC system. To confirm the formation of retinals and to improve the yield of retinoids as well as the separation of their isomers, extraction was also performed in the presence of hydroxylamine. Fig. 3 B shows that this treatment leads to the formation of the all-trans and 13-cis retinal oximes, with a corresponding blueshift of their absorption spectra. The analyses demonstrated that besides retinal, significant amounts of retinol as well as retinyl esters were formed in E. coli (Table I). Since E. coli was able to metabolize retinal to retinol, the question arose of whether retinoic acids were also formed. For the analyses of retinoic acid formation, the cells were lysed, and the extracts were analyzed on an HPLC system using an established protocol (14.Thaller C. Eichele G. Nature. 1987; 327: 625-628Crossref PubMed Scopus (745) Google Scholar). The results revealed that, under these conditions, significant amounts of retinal as well as retinol could be detected, but that no retinoic acids were formed in E. coli.Figure 2Color shift due to the cleavage of β-carotene to retinoids in E. coli. The picture shows the color shift from yellow (β-carotene) to almost white (retinoids) in β-carotene producing and accumulating E. coli caused by the expression of the β-carotene dioxygenase from D. melanogaster (E. coli (+) strain) compared with the control (E. coli (−) strain).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3HPLC analysis of carotenoids and retinoids formed in E. coli. HPLC analysis of the retinoids formed in the β-carotene-producing E. coli transformed with the plasmid (pβdiox) for the expression of the β-carotene dioxygenase cDNA from Drosophila (E. coli (+)strain) compared with the E. coli (−) strain transformed with the vector control (pBAD-TOPO). The scale bars indicate an absorbance of 0.01 at 360 nm. A, formaldehyde/chloroform extracts from E. coli (+)(upper trace) and E. coli (−) strain (lower trace). B, hydroxylamine/methanol extracts yielding the corresponding oximes (syn and anti) from the respective retinal isomers. In the upper trace, authentic standards are separated. In the middle trace, the isomeric composition of the extracts from the E. coli (+) strain, and in the lower trace, the HPLC profile of the extracts from E. coli (−)strain are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Absorbance spectra of β-carotene and retinoids. Absorbance spectra (in n-hexane) of the main substances extracted from theE. coli (+) strain compared with those of authentic standards (dotted).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IRetinoid content at E. coli stratusE. coli (−)strainE. coli (+) strainAll-transretinalND4.713-cisretinalND1.5All-trans retinolND8.013-cis retinolND2.4ND1.8ΣRetinoids18.4β-Carotene56.021.4Molar amounts (pmol/mg of dry weight) of β-carotene and retinoids in the E. coli (+) strain and in the E. coli (−) strain from bacteria cultures that have been grown for 16 h at 28 °C are shown. ND, not detectable. Open table in a new tab Taken together, these results demonstrate that the cloned cDNA encodes a β-carotene dioxygenase, and correspondingly, it was named β-diox. Since exclusively retinoids, i.e. C20compounds, were found in the E. coli test system, it must be supposed that a centric cleavage of β-carotene is catalyzed, resulting in the formation of two molecules of retinal.For further analysis of the enzymatic properties of β-diox, the cDNA was cloned in the expression vector pGEX-4T-1 and was expressed as a fusion protein. To exclude that the N-terminal fusion to the glutathione S-transferase abolishes the enzymatic activity, the construct (β-diox-gex) was transformed into the β-carotene-synthesizing E. coli strain. Using the test described above, it could be shown that retinoids were formed inE. coli test system (data not shown). After expression of β-diox-gex in E. coli, the protein was subsequently purified by affinity chromatography. The purification could be achieved without the addition of detergents, demonstrating that the fusion protein was soluble and not tightly associated to membranes. To test for enzymatic activity in vitro, one μg of the purified protein was incubated for 2 h in the presence of β-carotene in an assay containing 0.05% Triton X-100. The reaction was stopped by the addition of hydroxylamine/methanol, and the products were analyzed by HPLC after extraction (Fig. 5). For quantification of the molar amounts of the products formed, the peak integrals were normalized to defined amounts of reference substances. The analyses revealed the formation of retinal (73 pmol of retinal from 400 pmol of β-carotene) by the purified enzyme (Fig. 5 b). The addition of FeSO4/ascorbate to the assay led to an increase in the formation of the cleavage product (130 pmol of retinal from 400 pmol of β-carotene), whereas the conversion of β-carotene to retinal could be inhibited by the addition of EDTA (Fig. 5,a and c, respectively). These results indicate that the enzymatic activity of the dioxygenase depends on iron, as has been reported for several in vitro systems of animal origin (9.Krinsky N.I. Wang X. Tang D.G. Russel R.M. Livrea M.A. Vidali G. Retinoids: From Basic Science to Clinical Applications. Birkhäuser Verlag, Basel, Switzerland1994: 21-28Google Scholar). Retinal formation from β-carotene was proportional to the incubation time (up to 2 h). In the presence of FeSO4/ascorbate using varying amounts of substrate (800 pmol to 100 pmol), the apparent K m value for β-carotene was estimated to be 5 μm. Taken together, the enzymatic activity of β-diox characterized so far in the E. coli system in vivo could also be measured in vitro with the purified protein and led to the formation of the identical product.Figure 5Enzymatic activity of the β-diox-gex fusion protein under different conditions. The fusion protein β-diox-gex (approximately 1 μg) was incubated under different conditions in buffer containing 50 mm Tricine/NaOH (pH 7.6) and 100 mm NaCl. To start the reaction, 5 μl of β-carotene (80 μm) dissolved in ethanol was added. After 2 h at 30 °C, the reactions were stopped and extracted. HPLC analyses were performed, and the HPLC profiles at 360 nm are shown. The scale barindicates an absorbance of 0.005 at 360 nm. a, incubation in the presence of 5 μm FeSO4 and 10 mml-ascorbate. b, incubation without FeSO4/ascorbate. c, incubation in the presence of 10 mm EDTA. d, prior to the incubation, the fusion protein was heated for 10 min at 95 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Flies use 3-hydroxyretinals for vision. Since the visual chromophore ofDrosophila is 3-hydroxy-retinal, we tested whether β-diox was able to use zeaxhantin as a substrate to directly form hydroxylated retinoids. However, under the conditions in which we applied, the enzyme failed to catalyze this reaction. In addition, we expressed β-diox in a zeaxhantin-accumulating E. coli strain, but only the formation of nonhydroxylated retinoids could be detected. In this E. coli strain, also significant amounts of β-carotene, the direct precursor of zeaxhantin, were found that can serve as a substrate for β-diox. Although flies use 3-hydroxyretinals for vision, it has been shown that, besides 3-hydroxycarotenoids (zeaxhantin and lutein), β-carotene can serve as suitable precursor (18.Vogt K. Z. Naturforsch. 1983; 38: 329-333Crossref Scopus (66) Google Scholar, 19.Vogt K. Kirschfeld K. Naturwissenschaften. 1984; 71: 211-213Crossref Scopus (101) Google Scholar). In addition, it has been demonstrated that flies are able to hydroxylate retinal at position 3 of the β-ionone ring (20.Hardie R. Vogt K. Rudolph A. J. Insect Physiol. 1989; 35: 423-431Crossref Scopus (34) Google Scholar, 21.Seki T. Isono K. Ozaki K. Tsukahara Y. Shibata-Katsuta Y. Ito M. Irie T. Katagiri M. Eur. J. Biochem. 1998; 257: 522-527Crossref PubMed Scopus (30) Google Scholar) and to form the unusual enantiomer (3S)-3-hydroxyretinal, which is the unique chromophore of cyclorrhaph flies (22.Seki T. Isono K. Ito M. Katsuta Y. Eur. J. Biochem. 1994; 226: 691-696Crossref PubMed Scopus (19) Google Scholar, 23.Seki T. Vogt K. Comp. Biochem. Physiol. 1998; 119: 53-64Crossref Scopus (43) Google Scholar). To test whether epoxy carotenoids were cleaved, enzymatic assays were performed with 9-cis-neoxanthin (neoxanthin was a gift from P. Beyer, University of Freiburg, and was isolated from natural sources), the substrate of the plant dioxygenase vp14. However, under the conditions applied, no cleavage products of these carotenoids could be detected.The sequence analyses revealed that the cDNA encoded a protein of 620 amino acids, with a calculated molecular mass of 69.9 kDa (Fig.6). The deduced amino acid sequence shares sequence homology to the plant carotenoid dioxygenase vp14, to lignostilbene synthase from Pseudomonas paucimobilis, and to several proteins of unknown function in the Cyanobacterium Synechocystis. The highest sequence homology, however, was found to RPE65, a protein from the retinal pigment epithelium (RPE) in vertebrates, first described in bovine eyes (15.Hamel C.P. Tsilou E. Pfeffer B.A. Hooks J.J. Detrick B. Redmond T.M. J. Biol. Chem. 1993; 268: 15751-15757Abstract Full Text PDF PubMed Google Scholar, 16.Bavik C.-O. Levy F. Hellman U. Wernstedt C. Eriksson U. J. Biol. Chem. 1993; 268: 20540-20546Abstract Full Text PDF PubMed Google Scholar). RPE65 and β-diox exhibit 36.7% overall sequence identity. The alignment of the deduced amino acid sequences of β-diox, RPE65, and vp14 performed with the program Map (17.Huang X. Bioscience. 1994; 10: 227-235Google Scholar) showed a distinct pattern of conserved regions (Fig. 7). Compared with RPE65 and vp14, the insect protein possesses a long extension close to the C terminus. The N-terminal extension of the plant protein vp14 relative to its animal homologues is most probably due to a target sequence for plastid import. The sequence homologies of β-diox with bacterial and plant dioxygenases suggest that we are dealing with a new class of dioxygenases present in bacteria, plants, and animals.Figure 6cDNA sequence and deduced amino acid sequence of β-diox from D. melanogaster.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Comparison of the deduced amino acid sequences. Linear alignment of the deduced amino acid sequences of vp14 (maize), RPE65 (bovine), and β-diox (fruit fly). Identity is indicated in black, and conserved amino acids, according to the PAM250 matrix, are indicated in gray. We used visual alignment and the program Map (15.Hamel C.P. Tsilou E. Pfeffer B.A. Hooks J.J. Detrick B. Redmond T.M. J. Biol. Chem. 1993; 268: 15751-15757Abstract Full Text PDF PubMed Google Scholar). β-diox shares 36.7% overall sequence identity to RPE and 15.2% overall sequence identity to vp14.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The expression pattern of β-diox mRNA was investigated by RT-PCR. As shown in Fig. 8 the mRNA was restricted exclusively to the head, whereas in thorax and abdomen, no β-diox mRNA could be detected by this method.Figure 8mRNA-levels of β-diox in different parts of the body. The expression pattern of β-diox mRNA was investigated by RT-PCR. β-diox mRNA was only detectable in the head. The cDNAs were synthesized from total RNA preparations derived from the head, thorax, and abdomen of adult Drosophila (females and males). As a control, the mRNA levels of the ribosomal protein rp49 (FlyBase accession number FBgn0002626) was investigated in the same RNA samples using a set of intron-spanning primers. bp, base pairs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONDrosophila β-diox is the first β-carotene dioxygenase to be molecularly identified. It may provide the key to opening up a broad field for further investigation of vitamin A metabolism in animals. The β-diox cDNA encodes a protein of 620 amino acids with a calculated molecular mass of 69.9 kDa. Enzymatic activity of β-diox could be measured in β-carotene-synthesizingE. coli as well as with the purified recombinant protein.Experiments dealing with the kinetic characteristics of a purified β-carotene dioxygenase have not been published to date. The reaction velocity measured with the purified β-diox fusion protein were relatively low. However, the in vitro test system we used was quite artificial and may miss several components present in crude extracts that could contribute to higher efficiencies. In addition, it is possible that only a small fraction of the purified recombinant enzyme is enzymatically active or that the fusion part of the protein exerts a negative effect. In animals, it has been reported that β-carotene dioxygenase activity depends on iron (9.Krinsky N.I. Wang X. Tang D.G. Russel R.M. Livrea M.A. Vidali G. Retinoids: From Basic Science to Clinical Applications. Birkhäuser Verlag, Basel, Switzerland1994: 21-28Google Scholar). The addition of FeSO4/ascorbat to the assay led to an increase of the enzymatic activity, whereas the addition of EDTA decreased the formation of retinal significantly. Enzymatic activity could be measured without the addition of cofactors such as thiol reagents or electron acceptors. This indicates that β-diox depends mainly on Fe2+ and that other cofactors are not strictly required for enzymatic activity. Since β-carotene is not soluble in an aqueous environment, tests for enzymatic activity were carried out in the presence of Triton X-100. In vivo β-carotene is not freely diffusible and must be associated with lipophilic structures such as membranes or binding proteins. Therefore, the question arose whether β-diox is associated to membranes in order to interact with its lipophilic substrate. The β-diox fusion protein could be purified without the addition of detergents, and this points to its soluble state rather than to its membrane-bound topology. However, the glutathione S-transferase part of the fusion protein may contribute to its solubility, and therefore, this point needs further investigation. Taken together, we could show that β-diox catalyzes the cleavage of β-carotene, but the precise enzymatic mechanism and cofactor requirement and whether membranes or binding proteins are necessary for the delivery of the substrate have to be further investigated.The β-diox gene is located at position 87F on chromosome 3 in theDrosophila genome. Precisely in this region aDrosophila mutant, ninaB, has been mapped by cytological methods (FlyBase Map section 87). The mutant phenotype has a reduced rhodopsin content in all photoreceptor classes. However, the mutant phenotype can be rescued by the dietary supplementation of retinal but not by even high doses of β-carotene. (24.Pak W.L. Breakfield X.O. Neurogenetics: Genetic Approaches to the Nervous System. Elsevier Science Publishing Co., Inc., New York1970: 67-99Google Scholar, 25.Stephenson R.S. O'Tousa J. Scavarda N.J. Randall L.L. Pak W.L. Cosens D.J. Vince-Price D. The Biology of Photoreception. Cambridge University Press, Cambridge, UK1983: 477-501Google Scholar). Both, the availability of the visual pigment chromophores as well as the transcriptional regulation by retinoic acid of the protein moiety (opsin) of the visual pigment (26.Picking W.L. Chen D. Lee R.D. Vogt M.E. Pollizi J.L. Marietta R.G. Stark W.S. Exp. Eye Res. 1996; 63: 493-500Crossref PubMed Scopus (20) Google Scholar) depend on β-diox enzymatic activity. Thus, it seems to be likely that the ninaBphenotype is caused by a mutation in β-diox.The sequence comparison revealed that β-diox belongs to a new class of dioxygenases so far described only in bacteria and plants. The highest sequence homology of β-diox is found to RPE65, a protein first described in bovine eyes. Therefore the question arises whether RPE65 is the vertebrate equivalent to β-diox. Although the exact function of RPE65 is not yet known, a role in vitamin A metabolism has been proposed, and recently it was found that mutations in the gene are responsible for a severe form of early onset retinal dystrophy in humans (27.Gu S.M. Thompson D.A. Srikumari C.R. Lorenz B. Finckh U. Nicoletti A. Murthy K.R. Rathmann M. Kumaramanickavel G. Denton M.J. Gal A. Nat. Genet. 1997; 17: 194-197Crossref PubMed Scopus (523) Google Scholar, 28.Marlhens F. Bareil C. Griffoin J.M. Zrenner E. Amalric P. Eliaou C. Liu S.Y. Harris E. Redmond T.M. Arnaud B. Claustres M. Hamel C.P. Nat. Genet. 1997; 17: 139-141Crossref PubMed Scopus (505) Google Scholar). In the eyes of mice, where the RPE65 gene has been disrupted, all-trans vitamin A accumulates (29.Redmond T.M., Yu, S. Lee E. Bok D. Hamasaki D. Chen N. Goletz P. Ma J.X. Crouch R.K. Pfeifer K. Nat. Genet. 1998; 20: 344-351Crossref PubMed Scopus (778) Google Scholar). Therefore, it has been concluded that RPE65 takes part in the isomerization of all-trans to 11-cis vitamin A in the mammalian visual cycle. However, after removal of RPE65 from RPE membrane fractions, the isomerization of all-trans-retinol into 11-cis-retinol remained unaffected (30.Choo D.W. Cheung E. Rando R.R. FEBS Lett. 1998; 440: 195-198Crossref PubMed Scopus (18) Google Scholar). To our knowledge a β-carotene dioxygenase activity has never been reported in the RPE, nor have significant amounts of its substrate β-carotene been measured in vertebrate eyes. We cloned RPE65 by RT-PCR from the bovine RPE and expressed it in the E. coli test system. But neither the formation of retinoids nor the formation of eccentric cleavage products such as apocarotenals could be detected (data not shown). Therefore, the exact function of RPE65 remains to be further investigated, and we propose that other as yet undiscovered members of this family with different tissue specificity (small intestine, liver) are responsible for the vertebrate β-carotene dioxygenase activity. The sequence homology of β-diox with RPE65 as well as with plant and bacterial dioxygenases suggests that β-diox belongs to a new class of dioxygenases catalyzing the cleavage of a conjugated carbon double bond. The described E. coli test system by which β-diox could be molecularly identified may be a powerful tool to characterize new genes involved in retinoid formation. Furthermore, the retinoid producing E. coli strain can be used to identify further steps in vitamin A metabolism.In recent years there has been a tremendous increase in our understanding of retinoid receptors and their ligands as well as their diverse roles in development and cell differentiation. With our present findings, tissue-specific expression, the impact of the cleavage reaction on the isomeric specificity of retinoids, and the regulation of the vitamin A uptake may soon be further elucidated. Animals are, in general, unable to synthesize vitamin A de novo. Vitamin A (retinol) and its derivatives (retinal, retinoic acid) are C20 isoprenoids metabolically derived from the oxidative cleavage of C40 carotenoids (Fig.1). Most effects of vitamin A are exerted by retinoic acid (1.Dowling J.E. Wald G. Proc. Natl. Acad. Sci. U. S. A. 1960; 46: 587-608Crossref PubMed Google Scholar, 2.Chytil F. Livrea M.A. Vidali G. Retinoids: From Basic Science to Clinical Applications. Birkhäuser Verlag, Basel, Switzerland1994: 11-19Google Scholar). In the visual system, retinal or closely related compounds such as 3-hydroxyretinal serve as chromophores for the various visual pigments (rhodopsins) (3.Wald G. Nature. 1968; 219: 800-807Crossref PubMed Scopus (573) Google Scholar, 4.Vogt K. Z. Naturforsch. 1984; 39: 196-197Crossref Scopus (47) Google Scholar). Although the cleavage of β-carotene to form retinal in animals has been investigated in cell-free systems for about 50 years, this important enzymatic step has resisted molecular analysis (5.Moore T. Biochem. J. 1930; 24: 692-702Crossref PubMed Google Scholar, 6.Goodman D., S. Huang H.S. Science. 1965; 149: 879-880Crossref PubMed Scopus (184) Google Scholar, 7.Olson J.A. Hayaishi O. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1364-1370Crossref PubMed Scopus (258) Google Scholar, 8.Olson J.A. Am. J. Clin. Nutr. 1969; 22: 953-962Crossref PubMed Scopus (17) Google Scholar, 9.Krinsky N.I. Wang X. Tang D.G. Russel R.M. Livrea M.A. Vidali G. Retinoids: From Basic Science to Clinical Applications. Birkhäuser Verlag,