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Phytochemistry Reviews

, Volume 5, Issue 2–3, pp 433–444 | Cite as

Cytochrome P450 oxygenases of Taxol biosynthesis

  • Rüdiger Kaspera
  • Rodney CroteauEmail author
Original Paper

Abstract

Cytochrome P450 monooxygenases play a prominent role in the biosynthesis of the diterpenoid anticancer drug Taxol, as they appear to constitute about half of the 19 enzymatic steps of the pathway in yew (Taxus) species. A combination of classical biochemical and molecular methods, including cell-free enzyme studies and differential-display of mRNA-reverse transcription polymerase chain reaction (RT-PCR) combined with a homology-based searching and random sequencing of a cDNA library from induced T. cuspidata cells, led to the discovery of six novel cytochrome P450 taxoid (taxane diterpenoid) hydroxylases. These genes show unusually high sequence similarity with each other (>70%) but low similarity (<30%) to, and significant evolutionary distance from, other plant P450s. Despite their high similarity, functional analysis of these hydroxylases demonstrated distinctive substrate specificities responsible for an early bifurcation in the biosynthetic pathway after the initial hydroxylation of the taxane core at C5, leading into a biosynthetic network of competing, but interconnected, branches. The use of surrogate substrates, in cases where the predicted taxoid precursors were not available, led to the discovery of two core oxygenases, the 2α- and the 7β-hydroxylase. This general approach could accelerate the functional analysis of candidate cDNAs from the extant family of P450 genes to identify the remaining oxygenation steps of this complex pathway.

Keywords

Taxoid hydroxylases Taxane diterpenoid Taxadiene Taxusin Taxus 

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Notes

Acknowledgement

We thank Christopher J.D. Mau and Raymond E.B. Ketchum for critical reading of the manuscript, and Robert M. Long for helpful discussions. The work from the authors’ laboratory described in this paper was supported by National Institutes of Health Grant CA-55254 and by Project 0967 from The Agricultural Research Center, Washington State University.

References

  1. Abe I, Prestwich GD (1999) Squalene epoxidase and oxidosqualene:lanosterol cyclase-key enzymes in cholesterol biosynthesis. In: Cane DE (ed) Comprehensive natural product chemistry. Elsevier, Amsterdam, NL, pp 267–298Google Scholar
  2. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  3. Baloglu E, Kingston DGI (1999) The taxane diterpenoids. J Nat Prod 62:1448–1472PubMedCrossRefGoogle Scholar
  4. Brown DT (2003) Preclinical and clinical studies of the taxanes. In: Itokawa H, Lee KH (eds) Taxus – The Genus Taxus. Taylor & Francis, London. UK, pp 387–435Google Scholar
  5. Chapple C (1998) Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases. Ann Rev Plant Physiol Plant Mol Biol 49:311–343CrossRefGoogle Scholar
  6. Chau M, Croteau R (2004) Molecular cloning and characterization of a cytochrome P450 taxoid 2α-hydroxylase involved in Taxol biosynthesis. Arch Biochem Biophys 427:48–57PubMedCrossRefGoogle Scholar
  7. Chau M, Jennewein S, Walker K, Croteau R (2004) Taxol biosynthesis: molecular cloning and characterization of a cytochrome P450 taxoid 7β-hydroxylase. Chem Biol 11:663–672PubMedGoogle Scholar
  8. Chen Y, Zhu W, Wu Y, Hu Q (1999) Effects of fungus elicitors on taxol production in suspension cells of Taxus yunnanensis. Shengwu Gongcheng Xuebao 15:522–524Google Scholar
  9. Croom EM Jr (1995) Taxus for taxol and taxoids. In: Suffness M (ed), Taxol – science and applications. CRC Press, Boca Raton, USA, pp 37–70Google Scholar
  10. Croteau R, Ketchum REB, Long RM, Kaspera R, Wildung MR (2006) Taxol biosynthesis and molecular genetics. Phytochem. Rev. in pressGoogle Scholar
  11. Daniewski WM, Gumulka M, Anczemski M, Masnyk M, Bioszyk E, Gupta KK (1998) Why the yew tree (Taxus baccata) is not attacked by insects. Phytochemistry 49:1279–1282CrossRefGoogle Scholar
  12. De Jong JM, Liu Y, Bollon AP, Long RM, Jennewein S, Williams D, Croteau R (2006) Genetic engineering of Taxol biosynthetic genes in Saccharomyces cerevisiae. Biotechnol Bioeng 93:212–224CrossRefGoogle Scholar
  13. Elmer WH, Mattine MJI, MacEachern GI (1994) Sensitivity of plant pathogenic fungi to taxane extracts from ornamental yews. Phytopathology 84:1179–1185Google Scholar
  14. Feldmann KA, Choe S, Kim H, Park J-H (2002) Functional genomics of cytochromes P450 in plants. Rec Adv Phytochem 36:125–143Google Scholar
  15. Floss HG, Mocek U (1995) Biosynthesis of taxol. In: Suffness M (ed) Taxol – science and applications. CRC Press, Boca Raton, USA, pp 191–208Google Scholar
  16. Funk C, Croteau R (1994) Diterpenoid resin acid biosynthesis in conifers – characterization of two cytochrome P450-dependent monooxygenases and an aldehyde dehydrogenase involved in abietic acid biosynthesis. Arch Biochem Biophys 308:258–266PubMedCrossRefGoogle Scholar
  17. Gibson DM, Ketchum REB, Hirasuna TJ, Shuler ML (1995) Potential of plant cell cultures for taxane production. In: Suffness M (ed) Taxol – Science and Applications. CRC Press, Boca Raton, USA, pp 71–95Google Scholar
  18. Gotoh O (1992) Substrate recognition sites in cytochrome P450 Family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. J Biol Chem 267:83–90PubMedGoogle Scholar
  19. Hefner J, Rubenstein SM, Ketchum REB, Gibson DM, Williams RM, Croteau R (1996) Cytochrome P450-catalyzed hydroxylation of taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5α-ol: the first oxygenation step in taxol biosynthesis. Chem Biol 3:479–489PubMedCrossRefGoogle Scholar
  20. Helliwell CA, Poole A, Peacock J, Dennis ES (1999) Arabidopsis ent-kaurene oxidase catalyzes three steps of gibberellin biosynthesis. Plant Physiol 119:507–510PubMedCrossRefGoogle Scholar
  21. Hezari M, Lewis NG, Croteau R (1995) Purification and characterization of taxa-4(5),11(12)-diene synthase from Pacific yew (Taxus brevifolia) that catalyzes the first commited step of Taxol biosynthesis. Arch Biochem Biophys 322:437–444PubMedCrossRefGoogle Scholar
  22. Itokawa H (2003) Taxoids occuring in the genus Taxus. In: Itokawa H, Lee KH (eds) Taxus – The Genus Taxus. Taylor & Francis, London, UK, pp 35–78Google Scholar
  23. Jennewein S, Long R, Williams RM, Croteau R (2004a) Cytochrome P450 taxadien 5α-hydroxylase, a mechanistically unusual monooxygenase catalyzing the first oxygenation step of Taxol biosynthesis. Chem Biol 11:379–387CrossRefGoogle Scholar
  24. Jennewein S, Park H, DeJong JM, Long RM, Bollon AP, Croteau RB (2005) Coexpression in yeast of Taxus cytochrome P450 reductase with cytochrome P450 oxygenases involved in Taxol biosynthesis. Biotechnol Bioeng 89:588–598PubMedCrossRefGoogle Scholar
  25. Jennewein S, Rithner CD, Williams RM, Croteau R (2001) Taxol biosynthesis: taxane 13α-hydroxylase is a cytochrome P450-dependent monooxygenase. Proc Natl Acad Sci USA 98:13595–13600PubMedCrossRefGoogle Scholar
  26. Jennewein S, Rither CD, Williams RM, Croteau R (2003) Taxoid metabolism: taxoid 14β-hydroxylase is a cytochrome P450-dependent monooxygenase. Arch Biochem Biophys 413:262–270PubMedCrossRefGoogle Scholar
  27. Jennewein S, Wildung MR, Chau M, Walker K, Croteau R (2004b) Random sequencing of an induced Taxus cell cDNA library for identification of clones involved in Taxol biosynthesis. Proc Natl Acad Sci USA 101:9149–9154CrossRefGoogle Scholar
  28. Kagawa N, Waterman MR (1995) Regulation of steroidogenic and related P450s. In: Ortiz de Montellano PR (ed) Cytochrome P450: structure, mechanism, and biochemistry. Plenum Press, New York, USA, pp 419–442Google Scholar
  29. Kahn RA, Durst F (2000) Function and evolution of plant cytochrome P450. Rec Adv Phytochem 34:151–189CrossRefGoogle Scholar
  30. Ketchum REB, Croteau R (2006) The Taxus metabolome and the elucidation of the Taxol® biosynthetic pathway in cell suspension cultures. In: Saito K, Dixon R, Willmetzer L (eds) Plant metabolomics (biotechnology in agriculture and forestry). Vol 57. Springer, Heidelberg, D, pp 291–309Google Scholar
  31. Ketchum REB, Rithner CD, Qiu D, Kim YS, Williams RM, Croteau RB (2003) Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus × media cell cultures. Phytochemistry 62:901–909PubMedCrossRefGoogle Scholar
  32. Ketchum REB, Tandon M, Gibson DM, Begley T, Shuler ML (1999) Isolation of labeled 9-dihydrobaccatin III and related taxoids from cell cultures of Taxus canadensis elicited with methyl jasmonate. J Nat Prod 62:1395–1398PubMedCrossRefGoogle Scholar
  33. Kikuchi Y, Yatagai M (2003) The commercial cultivation of Taxus species and production of taxoids. In: Itokawa H, Lee KH (eds) Taxus – The genus Taxus. Taylor & Francis, London, UK, pp 151–178Google Scholar
  34. Kim G-T, Tsukaya H (2002) Regulation of the biosynthesis of plant hormones by cytochrome P450s. J Plant Res 115:169–177PubMedCrossRefGoogle Scholar
  35. Kingston DGI (1995) Recent advances in the chemistry and structure-activity relationships of paclitaxel. Am Chem Soc Symp Ser 583:203–216Google Scholar
  36. Koepp AE, Hezari M, Zajicek J, Vogel BS, Lafever RE, Lewis NG, Croteau R (1995) Cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene is the committed step of Taxol biosynthesis in Pacific yew. J␣Biol Chem 270:8686–8690PubMedCrossRefGoogle Scholar
  37. Kutchan TM, Bock A, Dittrich H (1994) Heterologous expression of the plant proteins strictosidine synthase and berberine bridge enzyme in insect cell culture. Phytochemistry 35:353–360PubMedCrossRefGoogle Scholar
  38. Long RM, Croteau R (2005) Preliminary assessment of the C13-side chain 2′-hydroxylase involved in Taxol biosynthesis. Biochem Biophys Res Comm 338:410–417PubMedCrossRefGoogle Scholar
  39. Lovy Wheeler A, Long RM, Ketchum REB, Rither CD, Williams RM, Croteau R (2001) Taxol biosynthesis: differential transformations of taxadien-5α-ol and its acetate ester by cytochrome P450 hydroxylases from Taxus suspension cultures. Arch Biochem Biophys 390:265–278CrossRefGoogle Scholar
  40. McCoy M (2004) Lining up to make a cancer drug. Chem Eng News 82:12–14Google Scholar
  41. Nelson DR (1999) Cytochrome P450 and the individuality of species. Arch Biochem Biophys 369:1–10PubMedCrossRefGoogle Scholar
  42. Odgen L (1998) Taxus (Yews) - A highly toxic plant. Vet Hum Toxicol 30:563–564Google Scholar
  43. Ohta D, Mizutani M (2004) Redundancy or flexibility: molecular diversity of the electron transfer components for P450 monooxygenases in higher plants. Front Biosci 9:1587–1597PubMedGoogle Scholar
  44. Pichersky E, Gang DR (2000) Genetics and biochemistry of secondary metabolites in plants: an evolutionary perspective. Trends Plant Sci 5:439–445PubMedCrossRefGoogle Scholar
  45. Pompon D, Louerat B, Bronine A, Urban P (1996) Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol 272:51–64PubMedGoogle Scholar
  46. Poulos TL, Johnson EF (2005) Structures of cytochrome P450 enzymes. In: Ortiz de Montellano PR (ed) Cytochrome P450: structure, mechanism, and biochemistry. Kluwer, New York, USA, pp 87–114Google Scholar
  47. Ro DK, Arimura GL, Lau SYW, Piers E, Bohlmann J (2005) Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP720B1) is a multifunctional, multisubstrate cytochrome P450 monooxygenase. Proc Natl Acad Sci USA 102:8060–8065PubMedCrossRefGoogle Scholar
  48. Schoendorf A, Rither CD, Williams RM, Croteau RB (2001) Molecular cloning of a cytochrome P450 taxane 10β-hydroxylase cDNA from Taxus and functional expression in yeast. Proc Natl Acad Sci USA 98:1501–1506PubMedCrossRefGoogle Scholar
  49. Schuler MA (1996) Plant cytochrome P450 monooxygenases. Crit Rev Plant Sci 15:235–284Google Scholar
  50. Schuler MA, Werck-Reichhart D (2003) Functional genomics of P450s. Ann Rev Plant Biol 54:629–667CrossRefGoogle Scholar
  51. Stierle A, Strobel G, Stierle D, Grothaus P, Bignami G (1995) The search for a Taxol-producing microorganism among the endophytic fungi of the Pacific yew, Taxus brevifolia. J Nat Prod 58:1315–1324PubMedCrossRefGoogle Scholar
  52. Suffness M, Wall ME (1995) Discovery and development of taxol. In: Suffness M (ed) Taxol – science and␣applications. CRC Press, Boca Raton, USA, pp 3–25Google Scholar
  53. Takeya K (2003) Plant tissue culture of taxoids. In: Itokawa H, Lee KH (eds) Taxus – The genus Taxus. Taylor & Francis, London, UK, pp 134–150Google Scholar
  54. Tu J, Zhu P, Cheng KD, Meng C (2004) Cloning and sequencing of hydroxylase genes involved in taxol biosynthesis. Z Naturforsch 59c:561–564Google Scholar
  55. von Wachenfeldt CA, Johnson EF (1995) Structures of eucaryotic cytochrome P450 enzymes. In: Ortiz de Montellano PR (ed) Cytochrome P450: structure, metabolism, and biochemistry. Plenum Press, New York USA, pp 183–223Google Scholar
  56. Wade RC, Winn PJ, Schlichting I, Sudarko (2004) A survey of active site access channels in cytochrome P450. J Inorgan Biochem 98: 1175–1182CrossRefGoogle Scholar
  57. Walker K, Ketchum REB, Hezari M, Gatfield D, Goleniowski M, Barthol A, Croteau R (1999) Partial purification and characterization of acetyl coenzyme A: taxa-4(20),11(12)-dien-5α-ol O-acetyl transferase that catalyzes the first acylation step of Taxol biosynthesis. Arch Biochem Biophys 364: 273–279PubMedCrossRefGoogle Scholar
  58. Walker K, Fujisaki S, Long R, Croteau R (2002) Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis. Proc Nat Acad Sci USA 99:12715–12720PubMedCrossRefGoogle Scholar
  59. Walker KD, Klettke K, Akiyama T, Croteau R (2004) Cloning, heterologous expression, and characterization of a phenylalanine aminomutase involved in taxol biosynthesis. J Biol Chem 279:53947–53954PubMedCrossRefGoogle Scholar
  60. Wall ME (1998) Camptothecin and taxol: discovery to clinic. Med Res Rev 18:299–314PubMedCrossRefGoogle Scholar
  61. Wang X, Itokawa H, Lee K-H (2003) Structure-activity relationships of taxoids. In: Itokawa H, Lee KH (eds) Taxus – The Genus Taxus. Taylor & Francis, London, UK, pp 298–386Google Scholar
  62. Wüst M, Little DB, Schalk M, Croteau R (2001) Hydroxylation of limonene enantiomers and analogs by recombinant (-)-limonene 3- and 6-hydroxylases from mint (Mentha) species: evidence for catalysis within sterically constrained active sites. Arch Biochem Biophys 387:125–136PubMedCrossRefGoogle Scholar
  63. Yuan Y-J, Li C, Hu Z-D, Wu J-C (2002) A double oxidative burst for taxol production in suspension cultures of Taxus chinensis var. mairei induced by oligosaccharide from Fusarium oxysporum. Enz Microbial Technol 30:774–778CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  1. 1.Institute of Biological ChemistryWashington State UniversityPullmanUSA

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