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. 1992 Oct;100(2):998–1007. doi: 10.1104/pp.100.2.998

Molecular Analysis and Heterologous Expression of an Inducible Cytochrome P-450 Protein from Periwinkle (Catharanthus roseus L.) 1

Hans-Peter Vetter 1,2,3, Ursula Mangold 1,2,3, Gudrun Schröder 1,2,3, Franz-Josef Marner 1,2,3, Danielle Werck-Reichhart 1,2,3, Joachim Schröder 1,2,3
PMCID: PMC1075656  PMID: 16653087

Abstract

We screened cDNA libraries from periwinkle (Catharanthus roseus) cell cultures induced for indole alkaloid synthesis and selected clones for induced cytochrome P-450 (P-450) proteins by differential hybridization, size of the hybridizing mRNA, and presence of amino acid motifs conserved in many P-450 families. Four cDNAs satisfying these criteria were analyzed in detail. They were grouped in two classes (pCros1, pCros2) that represented two closely related genes of a new P-450 family designated CYP72. Antiserum against a cDNA fusion protein overexpressed in Escherichia coli recognized in C. roseus a protein band of 56 kD. Quantification of western blots showed that it represented 1.5 ± 0.5 and 6 ± 1 μg/mg of protein in the membranes from noninduced and induced cells, respectively, and analysis of the total P-450 content suggested that the cDNA-encoded protein was one of the dominant P-450 proteins. The pathway to indole alkaloids contains two known P-450 enzymes, geraniol-10-hydroxylase (GE10H) and nerol-10-hydroxylase (NE10H). The induction kinetics of the cloned P-450 protein and of GE10H activity were similar, but those of NE10H were different. Western blots with membranes from other plants suggested that P-450 CYP72 is specific for C. roseus and other plants with GE10H activity. A tentative assignment of CYP72 as GE10H is discussed. The cDNA was recloned for expression in Saccharomyces cerevisiae, and the presence of the protein was demonstrated by western blots. Assays for GE10H failed to detect enzyme activity, and the same negative result was obtained for NE10H and other P-450 enzymes that are present in C. roseus.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bairoch A. PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res. 1991 Apr 25;19 (Suppl):2241–2245. doi: 10.1093/nar/19.suppl.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bozak K. R., Yu H., Sirevåg R., Christoffersen R. E. Sequence analysis of ripening-related cytochrome P-450 cDNAs from avocado fruit. Proc Natl Acad Sci U S A. 1990 May;87(10):3904–3908. doi: 10.1073/pnas.87.10.3904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Britsch L., Grisebach H. Purification and characterization of (2S)-flavanone 3-hydroxylase from Petunia hybrida. Eur J Biochem. 1986 May 2;156(3):569–577. doi: 10.1111/j.1432-1033.1986.tb09616.x. [DOI] [PubMed] [Google Scholar]
  4. Cullin C., Pompon D. Synthesis of functional mouse cytochromes P-450 P1 and chimeric P-450 P3-1 in the yeast Saccharomyces cerevisiae. Gene. 1988 May 30;65(2):203–217. doi: 10.1016/0378-1119(88)90457-x. [DOI] [PubMed] [Google Scholar]
  5. Donaldson R. P., Luster D. G. Multiple forms of plant cytochromes p-450. Plant Physiol. 1991 Jul;96(3):669–674. doi: 10.1104/pp.96.3.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gabriac B., Werck-Reichhart D., Teutsch H., Durst F. Purification and immunocharacterization of a plant cytochrome P450: the cinnamic acid 4-hydroxylase. Arch Biochem Biophys. 1991 Jul;288(1):302–309. doi: 10.1016/0003-9861(91)90199-s. [DOI] [PubMed] [Google Scholar]
  7. Hagmann M. L., Heller W., Grisebach H. Induction and characterization of a microsomal flavonoid 3'-hydroxylase from parsley cell cultures. Eur J Biochem. 1983 Aug 15;134(3):547–554. doi: 10.1111/j.1432-1033.1983.tb07601.x. [DOI] [PubMed] [Google Scholar]
  8. Jefcoate C. R. Measurement of substrate and inhibitor binding to microsomal cytochrome P-450 by optical-difference spectroscopy. Methods Enzymol. 1978;52:258–279. doi: 10.1016/s0076-6879(78)52029-6. [DOI] [PubMed] [Google Scholar]
  9. Kalb V. F., Loper J. C., Dey C. R., Woods C. W., Sutter T. R. Isolation of a cytochrome P-450 structural gene from Saccharomyces cerevisiae. Gene. 1986;45(3):237–245. doi: 10.1016/0378-1119(86)90021-1. [DOI] [PubMed] [Google Scholar]
  10. Kalb V. F., Loper J. C. Proteins from eight eukaryotic cytochrome P-450 families share a segmented region of sequence similarity. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7221–7225. doi: 10.1073/pnas.85.19.7221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Madyastha K. M., Meehan T. D., Coscia C. J. Characterization of a cytochrome P-450 dependent monoterpene hydroxylase from the higher plant Vinca rosea. Biochemistry. 1976 Mar 9;15(5):1097–1102. doi: 10.1021/bi00650a023. [DOI] [PubMed] [Google Scholar]
  12. Madyastha K. M., Ridgway J. E., Dwyer J. G., Coscia C. J. Subcellular localization of a cytochrome P-450-dependent monogenase in vesicles of the higher plant Catharanthus roseus. J Cell Biol. 1977 Feb;72(2):302–313. doi: 10.1083/jcb.72.2.302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nebert D. W., Gonzalez F. J. P450 genes: structure, evolution, and regulation. Annu Rev Biochem. 1987;56:945–993. doi: 10.1146/annurev.bi.56.070187.004501. [DOI] [PubMed] [Google Scholar]
  14. Nebert D. W., Nelson D. R., Coon M. J., Estabrook R. W., Feyereisen R., Fujii-Kuriyama Y., Gonzalez F. J., Guengerich F. P., Gunsalus I. C., Johnson E. F. The P450 superfamily: update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Biol. 1991 Jan-Feb;10(1):1–14. doi: 10.1089/dna.1991.10.1. [DOI] [PubMed] [Google Scholar]
  15. Salaün J. P., Benveniste I., Reichhart D., Durst F. Induction and specificity of a (cytochrome P-450)-dependent laurate in-chain-hydroxylase from higher plant microsomes. Eur J Biochem. 1981 Oct;119(3):651–655. doi: 10.1111/j.1432-1033.1981.tb05657.x. [DOI] [PubMed] [Google Scholar]
  16. Schröder G., Brown J. W., Schröder J. Molecular analysis of resveratrol synthase. cDNA, genomic clones and relationship with chalcone synthase. Eur J Biochem. 1988 Feb 15;172(1):161–169. doi: 10.1111/j.1432-1033.1988.tb13868.x. [DOI] [PubMed] [Google Scholar]
  17. Strebel K., Beck E., Strohmaier K., Schaller H. Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins. J Virol. 1986 Mar;57(3):983–991. doi: 10.1128/jvi.57.3.983-991.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Urban P., Cullin C., Pompon D. Maximizing the expression of mammalian cytochrome P-450 monooxygenase activities in yeast cells. Biochimie. 1990 Jun-Jul;72(6-7):463–472. doi: 10.1016/0300-9084(90)90070-w. [DOI] [PubMed] [Google Scholar]
  19. Weber J. M., Reiser J., Käppeli O. Lanosterol 14 alpha-demethylase-encoding gene: systematic analysis of homologous overexpression in Saccharomyces cerevisiae using strong yeast promoters. Gene. 1990 Mar 15;87(2):167–175. doi: 10.1016/0378-1119(90)90298-6. [DOI] [PubMed] [Google Scholar]
  20. Werck-Reichhart D., Gabriac B., Teutsch H., Durst F. Two cytochrome P-450 isoforms catalysing O-de-ethylation of ethoxycoumarin and ethoxyresorufin in higher plants. Biochem J. 1990 Sep 15;270(3):729–735. doi: 10.1042/bj2700729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. von Lintig J., Zanker H., Schröder J. Positive regulators of opine-inducible promoters in the nopaline and octopine catabolism regions of Ti plasmids. Mol Plant Microbe Interact. 1991 Jul-Aug;4(4):370–378. doi: 10.1094/mpmi-4-370. [DOI] [PubMed] [Google Scholar]

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