Plant Molecular Biology

, Volume 34, Issue 6, pp 935–948

Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don

  • Felipe Vazquez-Flota
  • Emidio De Carolis
  • Anne-Marie Alarco
  • Vincenzo De Luca
Article

Abstract

A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the 4-hydroxylation of desacetoxyvindoline was purified to homogeneity. Three oligopeptides isolated from a tryptic digest of the purified protein were microsequenced and one oligopeptide showed significant homology to hyoscyamine 6β-hydroxylase from Hyoscyamus niger. A 36-mer degenerate oligonucleotide based on this peptide sequence was used to screen a Catharanthus roseus cDNA library and three clones, cD4H-1 to -3, were isolated. Although none of the three clones were full-length, the open reading frame on each clone encoded a putative protein containing the sequence of all three peptides. Primer extension analysis suggested that cD4H-3, the longest cDNA clone, was missing 156 bp at the 5′ end of the clone and sequencing of the genomic clone, gD4H-8, confirmed these results. Southern blot analysis suggested that d4h is present as a single-copy gene in C. roseus which is a diploid plant, and the significant differences in the sequence of the 3′-UTR between cD4H-1 and -3 suggest that they represent dimorphic alleles of the same hydroxylase. The identity of the clone was further confirmed when extracts of transformed Escherichia coli expressed D4H enzyme activity. The D4H clone encoded a putative protein of 401 amino acids with a calculated molecular mass of 45.5 kDa and the amino acid sequence showed a high degree of similarity with those of a growing family of 2-oxoglutarate-dependent dioxygenases of plant and fungal origin. The similarity was not restricted to the dioxygenase protein sequences but was also extended to the gene structure and organization since the 205 and 1720 bp introns of d4h were inserted around the same highly conserved amino acid consensus sequences as those for e8 protein, hyoscyamine-6β-hydroxylase and ethylene-forming enzyme. These results provide further support that a common ancestral gene is responsible for the appearance of this family of dioxygenases.

Hydroxylase assays and RNA blot hybridization studies showed that enzyme activity followed closely the levels of d4h transcripts, occurring predominantly in young leaves and in much lower levels in stems and fruits. In contrast, etiolated seedlings which contained considerable levels of d4h transcripts had almost undetectable hydroxylase activity, whereas exposure of seedlings to light resulted in a rapid increase of enzyme activity without a significant further increase in d4h transcripts over those detected in dark-grown seedlings. These results suggest that the activating effect of light may occur at a point downstream of transcription which remains to be elucidated.

Catharanthus roseus dioxygenases indole alkaloids molecular regulation secondary metabolism 

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References

  1. 1.
    Aebersol RH, Leavitt S, Saavedra RA, Hood LE, Kent SBH: Internal amino acid sequence analysis of protein separated by one-or two-dimensional electrophoresis after in situprotease digestion on nitrocellulose. Proc Natl Acad Sci USA 84: 6970- 69-74 (1987).PubMedGoogle Scholar
  2. 2.
    Aerts R, Alarco AM, De Luca V: Auxins induce tryptophan decarboxylase activity in radicles of Catharanthusseedlings. Plant Physiol 100: 1014-1019 (1992).Google Scholar
  3. 3.
    Aerts R, De Luca V: Phytochrome is involved in the light-regulation of vindoline biosynthesis in Catharanthus. Plant Physiol 100: 1029-1032 (1992).Google Scholar
  4. 4.
    Aerts R, Gisi D, De Carolis E, De Luca V, Baumann TW: Methyl jasmonate vapor increases the developmentally controlled synthesis of alkaloids in Catharanthusand Cinchonaseedlings. Plant J 5: 635-643 (1994).Google Scholar
  5. 5.
    Alvarez-Fernandez J, De Luca V: Ubiquitin-mediated degradation of tryptophan decarboxylase from Catharanthus roseus. Phytochemistry 36: 1123-1128 (1994).CrossRefGoogle Scholar
  6. 6.
    Alvarez-Fernandez J, Owens TG, Kurz WGW, De Luca V: Immunological detection and quantitation of tryptophan decarboxylase in developing Catharanthus roseusseedlings. Plant Physiol 91: 79-84 (1989).Google Scholar
  7. 7.
    Balsevich J, De Luca V, Kurz WGW: Altered alkaloid pattern in dark grown seedlings of Catharanthus roseus. The isolation and characterization of 4-desacetoxyvindoline: a novel indole alkaloid an proposed precursor of vindoline. Heterocycles 24: 2415-2421 (1986).Google Scholar
  8. 8.
    Bristsch L, Dedio J, Saedler H, Forkmann G: Molecular characterization of flavanone 3β-hydroxylases. Consensus sequence, comparison with related enzyme and the role of conserved histidine residues. Eur J Biochem 217: 745-754 (1993).PubMedGoogle Scholar
  9. 9.
    Chen VJ, Frolik CA, Orville AM, Harpel MR, Lipscornb JD, Surerus KK, Münck E: Spectroscopic studies of isopenicillin N synthase.Amononuclear nonheme Fe2+ oxidase withmetal coordination sites for small molecules and substrate. J Biol Chem 264: 21677-21681 (1989).PubMedGoogle Scholar
  10. 10.
    Constabel F, Gaudet LaPraire P, Kurz WGW, Kutney JP: Alkaloid production in Catharanthus roseuscell cultures XII. Biosynthetic capacity of callus from original explants and regenerated shoots. Plant Cell Rep 1: 139-142 (1982).CrossRefGoogle Scholar
  11. 11.
    Corpet F: Multiple sequence alignment with hierarchical clustering. Nucl Acid Res 16: 10881-10890 (1988).Google Scholar
  12. 12.
    Davis KM: A MaluscDNA with homology to Anthirrhinum candidaand Zea A2genes. Plant Physiol 103: 1015 (1993).CrossRefPubMedGoogle Scholar
  13. 13.
    De Carolis E: Enzymology of vindoline biosynthesis. Purification, characterization and molecular cloning of a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus. Ph. D. thesis, Universit é de Montréal, Montréal, Canada (1994).Google Scholar
  14. 14.
    De Carolis E, Chan F, Balsevich J, De Luca V: Isolation and characterization of a 2-oxoglutarate dependent dioxygenase involved in the second-to-last step in vindoline biosynthesis. Plant Physiol 94: 1323-1329 (1990).Google Scholar
  15. 15.
    De Carolis E, De Luca V: Purification, characterization, and kinetic analysis of a 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis from Catharanthus roseus. J Biol Chem 268: 5504-5511 (1993).PubMedGoogle Scholar
  16. 16.
    De Carolis E, Luca V: 2-oxoglutarate-dependent dioxygenase and related enzymes: Biochemical characterization. Phytochemistry 36: 1093-1107 (1994).CrossRefPubMedGoogle Scholar
  17. 17.
    Deikman J, Fischer RL: Interaction of a DNA binding factor with the 5′-flanking region of an ethylene-responsive fruit ripening gene from tomato. EMBO J 7: 3315-3320 (1988).PubMedGoogle Scholar
  18. 18.
    De Luca V, Alvarez-Fernández JA, Campbell D, Kurz WGW: Developmental regulation of enzymes of indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol 86: 447-450 (1988).Google Scholar
  19. 19.
    De Luca V, Balsevich J, Kurz WGW: Acetyl coenzyme A: deacetoxyvindoline O-acetyltransferase, a novel enzyme from Catharanthus. J Plant Physiol 121: 417-428 (1985).Google Scholar
  20. 20.
    De Luca V, Balsevich J, Tyler RT, Eilert U, Panchuk BD, Kurz WGW: Biosynthesis of indole alkaloids: Developmental regulation of the biosynthetic pathway from taberonine to vindoline in Catharanthus roseus. J Plant Physiol 125: 147-156 (1986).Google Scholar
  21. 21.
    De Luca V, Balsevich J, Tyler RT, Kurz WGW: Characterization of a novel N-methyltransferase (NMT) from Catharanthus roseus. Plant Cell Rep 6: 458-461 (1987).Google Scholar
  22. 22.
    De Luca V, Cutler AJ: Subcellular localization of enzymes involved in indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol 85: 1099-1102 (1987).Google Scholar
  23. 23.
    De Luca V, Marineau C, and Brisson N: Molecular cloning and analysis of cDNA encoding a plant tryptophan decarboxylase: Comparison with animal dopa decarboxylases. Proc Natl Acad Sci USA 88: 9969-9973 (1989).Google Scholar
  24. 24.
    Dethier M, De Luca V: Partial purification of an Nmethyltransferase involved in vindoline biosynthesis in Catharanthus roseus. Phytochemistry 32: 673-678 (1993).CrossRefGoogle Scholar
  25. 25.
    Eilert U, Constabel F, Kurz WGW: Elicitor stimulation of monoterpene indole alkaloid formation in suspension cultures of Catharanthus roseus. J Plant Physiol 126: 11-22 (1986).Google Scholar
  26. 26.
    Feinberg AP, Vogelstein B: Atechnique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137: 266-267 (1984).PubMedGoogle Scholar
  27. 27.
    French DL, Laskov R, Scharff MD: The role of somatic hypermutation in the generation of antibody diversity. Science 244: 152-1157 (1989).Google Scholar
  28. 28.
    Gamborg OL, Miller RA, Ojima K: Nutrient requirements of suspension cultures of soybean root cell. Exp Cell Res 50: 151-158 (1968).PubMedGoogle Scholar
  29. 29.
    Goddijn O, Lohman FP, de Kam RJ, Schilperoort RA, Hoge JHC: Nucleotide sequence of the tryptophan decarboxylase gene of Catharanthus roseusand expression of a tdc-gusAgene fusion in Nicotiana tabacum. Mol Gen Genet 242: 217-225 (1994).PubMedGoogle Scholar
  30. 30.
    Gubler U, Hoffman BJ: A simple and very efficient method for generating cDNA libraries. Gene 25: 263-269 (1983).CrossRefPubMedGoogle Scholar
  31. 31.
    Han S, Eltis LD, Timmis KN, Muchmore SW, Bolin JT: Crystal structure of the biphenyl-cleaving extradiol dioxygenase from a PCB-degrading pseudomonad. Science 270: 976-980 (1995).PubMedGoogle Scholar
  32. 32.
    Holdsworth MJ, Bird CR, Ray J, Schuch W, Grierson D: Structure and expression of an ethylene-related Mrna from tomato. Nucl Acid Res 15: 731-739 (1987).Google Scholar
  33. 33.
    Holdsworth MJ, Schuch W, Grieson D: Nucleotide sequence of an ethylene-related gene from tomato. Nucl Acid Res 15: 10600 (1987).Google Scholar
  34. 34.
    Holton TA, Brugleira F, Tanaka Y: Cloning and expression of flavonol synthase from Petunia hybrida. Plant J 4: 1003-1010 (1993).CrossRefPubMedGoogle Scholar
  35. 35.
    Jia S, Van Dusen WJ, Diehl RE, Dixon RA, Elliston KO, Stern AM, Friedman PA: cDNA cloning and expression of bovine aspartyl (asparinyl)β-hydroxylase. J Biol Chem 267: 14322- 14327 (1992).PubMedGoogle Scholar
  36. 36.
    Johnson IS, Wright HF, Svoboda GH, Vlantis J: Antitumor principles derived from Vinca roseaLinn I. Vincaleukoblastine and leurosine. Cancer Res 20: 1016-1022 (1960).PubMedGoogle Scholar
  37. 37.
    Jones JDG, Dunsmuir P, Bedbrook J: High level expression of introduce chimeric gene. EMBO J 4: 2411-2418 (1985).Google Scholar
  38. 38.
    Joshi CP: An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucl Acid Res 15: 6643-6653 (1987).Google Scholar
  39. 39.
    Joshi CP: Putative polyadenilation signal in nuclear genes of higher plants: a compilation and analysis. Nucl Acid Res 15: 9627-9640 (1987).Google Scholar
  40. 40.
    Kanegae T, Kajiya H, Amano Y, Hashimoto T, Yamada Y: Species-dependent expression of the hyoscyamine 6β-hydroxylase gene in the pericycle. Plant Physiol 105: 483-490 (1994).CrossRefPubMedGoogle Scholar
  41. 41.
    Kurz WGW, Chatson KB, Constabel F, Kutney JP, Choi LSL, Kolodziejczyk P, Sleigh SK, Stuart KL, Worth BR: Alkaloid production in Catharanthus roseuscell cultures: Initial studies on cell lines and their alkaloid content. Phytochemistry 19: 2583-2587 (1980).CrossRefGoogle Scholar
  42. 42.
    Kutchan TM, Hampp N, Lottspeich F, Beyreuther K, Zenk MH: The cDNAclone for strictosidine synthase from Rauvolfia serpentina. DNA sequence determination and expression in Escherichia coli. FEBS Lett 237: 40-44 (1988).CrossRefPubMedGoogle Scholar
  43. 43.
    Kutney JB, Choi LSL, Kolodziejczyk P, Sleigh SK, Worth BR, Kurz WGW, Chatson KB, Constabel F: Alkaloid production in Catharanthus roseuscell cultures: Isolation and characterization of alkloids from one cell line. Phytochemistry 19: 2589-2595 (1980).CrossRefGoogle Scholar
  44. 44.
    Lipman DJ, Pearson WR: Rapid and sensitive protein similarity searches. Science 227: 1435-1441 (1985).PubMedGoogle Scholar
  45. 45.
    Madyastha KM, Meehan TD, Coscia CJ: Characterization of a cytochrome P-450 dependent monoterpene hydroxylase from the higher plant Vinca rosea. Biochemistry 15: 1097-102 (1976).PubMedGoogle Scholar
  46. 46.
    Matsuda J, Okabe S, Hashimoto T, Yamada Y: Molecular cloning of hyoscyamine 6β-hydroxylase, a 2-oxoglutaratedependent dioxygenase from cultured roots of Hyoscyamus niger. J Biol Chem 226: 9460-9464 (1991).Google Scholar
  47. 47.
    McKnight TD, Bergey DR, Burnett RJ, Nessler CL: Expression of an enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants. Planta 185: 148-152 (1991).CrossRefGoogle Scholar
  48. 48.
    McKnight TD, Roessner CA, Devagupta R, Scott AI, Nessler CL: Nucleotide sequence of a cDNA enconding the vacuolar protein strictosidine synthase from Catharanthus roseus. Nucl Acids Res 18: 4939 (1990).PubMedGoogle Scholar
  49. 49.
    Ming L-J, Que L Jr, Kriauciunas A, Frolick CA, Chen VJ: NMR studies of the active site of isopenicillin-N-synthase, a non-heme iron (II) enzyme. Biochemistry 30: 11653-11659 (1991).PubMedGoogle Scholar
  50. 50.
    Moreno PRH, van der Heijden R, Verpoorte R: Cell and tissue culture of Catharanthus roseus: a literature survey II. Updating from 1988-1993. Plant Cell Tissue Organ Cult 42: 1-25 (1995).Google Scholar
  51. 51.
    Murray M, Thompson WF: Rapid isolation of high molecular weight plant DNA. Nucl Acids Res 8: 4321-4325 (1980).PubMedGoogle Scholar
  52. 52.
    Nakai C, Uyeyama H, Kagayama H, Nakasawa T, Inouye S, Kishi F, Kakazawa A, Nuzaki M: Cloning, DNA sequencing and aminoacid sequencing of catechol 1,2-dioxygenases (pyrocatechase) from Pseudomonas putidamt-2 and Pseudomonas arvillaC-1. Arch Biochem Biophys 321: 353-362 (1995).CrossRefPubMedGoogle Scholar
  53. 53.
    Pasquali G, Goddjin OJM, de Waal A, Verpoorte R, Schilperoot RA, Hoge JHC, Memelink J: Coordinate regulation of two indole alkaloid biosynthetic genes from Catharanthus roseusby auxin and elicitors. Plant Mol Biol 18: 1121-1131 (1992).PubMedGoogle Scholar
  54. 54.
    Phillips A, Ward DA, Ukness S, Appleford NEJ, Lange T, Huttly AK, Gaskin P, Graebe JE, Hedden P: Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis. Plant Physiol 108: 1049-1057 (1995).CrossRefPubMedGoogle Scholar
  55. 55.
    Power R, Kurz WGW, De Luca V: Purification and characterization of acetylcoenzyme A: deacetylvindoline 4-O-acetyltransferase from Catharanthus roseus. Arch Biochem Biophys 279: 370-376 (1990).PubMedGoogle Scholar
  56. 56.
    Prescott AG, John P: Dioxygenases: molecular structure and role in plant metabolism. Annu Rev Plant Physiol Plant Mol Biol 47: 245-271 (1996).CrossRefPubMedGoogle Scholar
  57. 57.
    Ramon D, Carramolino L, Patiño C, Sánchez F, Peñalva MA: Cloning and characterization of the isopenicillin N synthase gene mediating for formation of the β-lactam ring in Aspergillus nidulans. Gene 57: 171-181 (1987).CrossRefPubMedGoogle Scholar
  58. 58.
    Roach PL, Clifton IJ, Fülöp V, Harlos K, Barton GJ, Hajdu J, Andersson I, Schofield CJ, Baldwin JE: Crystal structure of isopenicillin N synthase is the first from a newstructural family of enzymes. Nature 375: 700-704 (1994).CrossRefGoogle Scholar
  59. 59.
    Roewer IA, Clouthier N, Nessler C, De Luca V: Transient induction of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) genes in cell suspension cultures of Catharanthus roseus. Plant Cell Rep 11: 86-89 (1992).CrossRefGoogle Scholar
  60. 60.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).Google Scholar
  61. 61.
    Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 83: 8073-8076 (1977).Google Scholar
  62. 62.
    Stearn WT: A synopsis of the genus Catharanthus(Apocynaceae). In: Taylor WI, Farnsworth NR (eds) The CatharanthusAlkaloids: Botany, Chemistry, Pharmacology and Clinical Uses, pp. 9-44. Marcel Dekker, New York (1975).Google Scholar
  63. 63.
    St-Pierre B, De Luca V: A cytochrome P-450 monooxygenase catalyzes the first step in the conversion of tabersonine to vindoline in Catharanthus roseus. Plant Physiol 109: 131-139 (1995).PubMedGoogle Scholar
  64. 64.
    Svoboda GH, Blake DA: The phytochemistry and pharmacology of Catharanthus roseus. In: Taylor WI, Farnsworth NR (eds) The CatharanthusAlkaloids: Botany, Chemistry, Pharmacology and Clinical Uses, pp. 45-83. Marcel Dekker, New York (1975).Google Scholar
  65. 65.
    Tan DSH, Sim T-S: Functional analysis of conserved histidine residues in Cephalosporium acremoniumisopenicillin N synthase by site-directed mutagenesis. J Biol Chem 271: 889-894 (1996).PubMedGoogle Scholar
  66. 66.
    Van der Heijden R, Verpoorte R, ten Hoopen HJG: Cell and tissue cultures of Catharanthus roseus(L.) G. Don: a literature survey. Plant Cell Tissue Organ Cult 18: 231-280 (1989).Google Scholar
  67. 67.
    Westerkemper P, Wieczorek U, Gueritte F, Langlois N, Langlois Y, Potier P, Zenk MH: Radioimmunoassay for the determination of the indole alkaloid vindoline in Catharanthus. Planta Med 39: 24-37 (1980).Google Scholar

Copyright information

© Kluwer Academic Publishers 1997

Authors and Affiliations

  • Felipe Vazquez-Flota
    • 1
  • Emidio De Carolis
    • 1
  • Anne-Marie Alarco
    • 1
  • Vincenzo De Luca
    • 1
  1. 1.Institut de Recherche en Biologie Végétale, Département de Sciences BiologiquesUniversité de MontréalMontréalCanada
  2. 2.Pharmaceutical DivisionPfizer Canada Inc.KirklandCanada
  3. 3.Institut de Recherches Cliniques de MontréalMontréalCanada

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