Abstract
A DNA fragment which has been isolated previously from an Anabaena DNA expression library was subcloned. The corresponding protein was overexpressed in Escherichia coli. The recombinant enzyme was fully active in converting ζ-carotene into lycopene in vitro with neurosporene as an intermediate. A smaller fragment which still contained the active enzyme was sequenced. An open reading frame of 1497 bp was found coding for a protein consisting of 499 amino acids with the calculated molecular weight of 56 740. In a computer search of nucleotide sequences contained in the EMBL nucleotide sequence library, all the best-fitting comparisons were carotenoid desaturases. The highest similarity was found with the crtI phytoene desaturase genes of bacteria and the al-1 gene from Neurospora crassa. A much lower similarity was found with the pds genes coding for phytoene desaturase from cyanobacteria and higher plants. It is shown in protein similarity plots that the amino acid similarity of ζ-carotene desaturase to the latter is mainly limited to the N terminus of the polypeptides. In contrast, the protein similarity plots and a comparison of a conserved region clearly demonstrate that there is a strong relationship between ζ-carotene desaturase and the phytoene desaturases from various bacteria and fungi. Therefore we propose that the ζ-carotene desaturase gene is homologous to the crt I phytoene desaturase genes of bacteria and fungi.
Similar content being viewed by others
References
Armstrong GA, Leach F, Alberti M, Hearst JE: Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus. Mol Gen Genet 216: 254–268 (1989).
Armstrong GA, Alberti M, Hearst JE: Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotolsynthetic and photosynthetic prokaryotes. Proc Natl Acad Sci USA 87: 9975–9979 (1990).
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidmann JG, Smith JA, Struhl K: Current Protocols in Molecular Biology, vol. 1, John Wiley, New York (1987).
Bartley GE, Schmidhauser TJ, Yanofsky C, Scolnik PA: Carotenoid desaturases from Rhodobacter capsulatus and Neurospora crassa are structurally and functionally conserved and contain domains homologous to flavoprotein disulfide oxidoreductases. J Biol Chem 265: 16020–16024 (1990).
Bartley GE, Viitanen PV, Pecker I, Chamovitz D, Hirschberg J, Scolnik PA: Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci USA 88: 6532–6536 (1991).
Britton G: Carotenoid biosynthesis: an overview. In: Krinsky NI, Mathews-Roth MM, Taylor RF (eds) Carotenoids: Chemistry and Biology, pp. 167–194. Plenum Press, New York (1990).
Chamovitz D, Pecker I, Hirschberg J: The molecular basis of resistance to the herbicide norflurazon. Plant Mol Biol 16: 967–974 (1991).
Foote C: Photosensitized oxidation and singlett oxygen. In: Pryor WA (ed) Free Radicals in Biology, vol. 2, pp. 85–112. Academic Press, New York (1976).
Fraser PD, Misawa N, Linden H, Yamano S, Kobayashi K, Sandmann G. Expression in E. coli, purification and reactivation of the recombinant Erwinia uredovora phytoene desaturase. J Biol Chem 267: 19891–19895 (1992).
Fraser PD, Linden H, Sandmann G: Purification and reactivation of recombinant Synechococcus phytoene desaturase from an overexpressing strain of Escherichia coli. Biochem J 291: 687–692 (1993).
Goodwin TW: The Biochemistry of the Carotenoids, vol. 1, Chapman and Hall, London (1980).
Hanukoglu I, Gutfinger T: cDNA sequence of adrenodoxin reductase: identification of NADP-binding sites in oxidoreductases. Eur J Biochem 180: 479–484 (1989).
Henikoff S: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351–359 (1984).
Hugueney P, Roemer S, Kuntz M, Camara B: Characterisation and molecular cloning of a flavoprotein catalyzing the synthesis of phytoene and ζ-carotene in Capsicum chromoplasts. Eur J Biochem 209: 399–407 (1992).
Krinsky NI: Non-photosynthetic functions of carotenoids. Phil Trans R Soc Lond B 284: 581–590 (1978).
Laemmli UK: Cleavage of structural proteins during the assembley of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Linden H, Misawa N, Chamovitz D, Pecker I, Hirschberg J, Sandmann G: Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated carotenes. Z Naturforsch 46c: 1045–1051 (1991).
Linden A, Vioque A, Sandmann G: Isolation of a carotenoid biosynthesis gene coding for ζ-carotene desaturase from Anabaena PCC 7120 by heterologous complementation. FEMS Microbiol Lett 106: 99–104 (1993).
Martínez-Férez IM, Vioque A: Nucleotide sequence of the phytoene desaturase gene from Synechocystis sp. PCC 6803 and characterization of a new mutation which confers resistance to the herbicide norflurazon. Plant Mol Biol 18: 981–983 (1992).
McClure WR: Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem 54: 171–204 (1985).
Misawa N, Nakagawa M, Kobayashi K, Yamano S, Izawa Y, Nakamura K, Harashima K: Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J Bact 172: 6704–6712 (1990).
Pecker I, Chamovitz D, Linden H, Sandmann G, Hirschberg J: A single polypeptide catalyzing the conversion of phytoene to ζ-carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962–4966 (1992).
Platt T: Transcription termination and the regulation of gene expression. Annu Rev Biochem 55: 339–372 (1986).
Rogers SO, Bendich AJ: Extraction of DNA of plant tissues. In: Gelvin SB, Schilperoort RA (eds) Plant Molecular Biology Manual, pp. A6/1–10, Kluwer Academic Publishers, Dordrecht (1988).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Sandmann G, Kowalczyk S: In vitro carotenogenesis and characterization of the phytoene desaturase reaction in Anacystis. Biochem Biophys Res Commun 163: 916–921 (1989).
Sandmann G: Biosynthesis of cyclic carotenoids: biochemistry and molecular genetics of the reaction sequence. Physiol Plant 83: 186–193 (1991).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Schmidhauser TJ, Lauter FR, Russo VEA, Yanofsky C: Cloning, sequence, and photoregulation of al-1, a carotenoid biosynthetic gene of Neurospora crassa. Mol Cell Biol 10: 5064–5070 (1970).
Schneider GJ, Lang JD, Haselkorn R: Promotor recognition by the RNA polymerase from vegetative cell of the cyanobacterium Anabaena 7120. Gene 105: 51–60 (1991).
Scrutton NS, Berry A, Perham RN: Redesign of the co-enzyme specifity of a dehydrogenase by protein engineering. Nature 343: 38–43 (1990).
Shine J, Dalgarno L: The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementary to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71: 1342–1346 (1974).
Siefermann-Harms D: The light-harvesting and protective functions of carotenoids in photosynthetic membranes. Physiol Plant 69: 561–568 (1987).
Vieira J, Messing J: Production of a single-stranded plasmid DNA. Meth Enzymol 153: 3–11 (1987).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Linden, H., Misawa, N., Saito, T. et al. A novel carotenoid biosynthesis gene coding for ζ-carotene desaturase: functional expression, sequence and phylogenetic origin. Plant Mol Biol 24, 369–379 (1994). https://doi.org/10.1007/BF00020174
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00020174