Molecular Genetics and Genomics

, Volume 275, Issue 2, pp 125–135 | Cite as

Substrate specificity of N-methyltransferase involved in purine alkaloids synthesis is dependent upon one amino acid residue of the enzyme

  • Naho Yoneyama
  • Hanayo Morimoto
  • Chuang-Xing Ye
  • Hiroshi Ashihara
  • Kouichi Mizuno
  • Misako KatoEmail author
Original Paper


Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are the major purine alkaloids in plants. To investigate the diversity of N-methyltransferases involved in purine alkaloid biosynthesis, we isolated the genes homologous for caffeine synthase from theobromine-accumulating plants. The predicted amino acid sequences of N-methyltransferases in theobromine-accumulating species in Camellia were more than 80% identical to caffeine synthase in C. sinensis. However, there was a little homology among the N-methyltransferases between Camellia and Theobroma. The recombinant enzymes derived from theobromine-accumulating plants had only 3-N-methyltransferase activity. The accumulation of purine alkaloids was, therefore, dependent on the substrate specificity of N-methyltransferase determined by one amino acid residue in the central part of the protein.


Caffeine Caffeine synthase Camellia, Theobromine Theobromine synthase Theobroma cacao 



We deeply thank Dr. Y. Takeda (National Institute of Plant Vegetable and Tea Science) for the generous supply of C. irrawadiensis. We also would like to thank Dr. A. Takeuchi (National Institute of Plant Vegetable and Tea Science) for providing cDNA library of C. sinensis and Mr. Y. Tomoda (Meiji Seika Kaisya Co Ltd.) for the supply of cacao trees. This work is supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No.14540590 to MK., No.15780076 to KM.).


  1. Ashihara H, Crozier A (1999) Biosynthesis and metabolism of caffeine and related purine alkaloids in plants. Adv Bot Res 30:117–205CrossRefGoogle Scholar
  2. Ashihara H, Gillies FM, Crozier A (1997) Metabolism of caffeine and related purine alkaloids in leaves of tea (Camellia sinensis L.). Plant Cell Physiol 38:413–419Google Scholar
  3. Ashihara H, Kato M, Chuang-xing Y (1998) Biosynthesis and metabolism of purine alkaloids in leaves of cocoa tea (Camellia ptilophylla). J Plant Res 111:599–604CrossRefGoogle Scholar
  4. Ashihara H, Kubota H (1987) Biosynthesis of purine alkaloids in Camellia plants. Plant Cell Physiol 28:535–539Google Scholar
  5. Ashihara H, Monterio AM, Gillies FM, Crozier A (1996) Biosynthesis of caffeine in leaves of coffee. Plant Physiol 111:747–753PubMedGoogle Scholar
  6. Benton WD, Davis RW (1977) Screening λgt recombinant clones by hybridization to single plaque in situ. Science 196:180–182PubMedCrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  8. Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolation of RNA from pine trees. Plant Mol Biol Rep 11:113–116Google Scholar
  9. Choi KB, Morishige T, Shitan N, Yazaki K, Sato F (2002) Molecular cloning and characterization of coclaurine N-methyltransferase from cultured cells of Coptis japonica. J Biol Chem 277:830–835CrossRefPubMedGoogle Scholar
  10. Dudareva N, Murfitt LM, Mann CJ, Gorenstein N, Kolosova N, Kish CM, Bonham C, Wood K (2000) Developmental regulation of methyl benzoate biosynthesis and emission in snapdragon flowers. Plant Cell 12:949–961CrossRefPubMedGoogle Scholar
  11. Fujimori N, Suzuki T, Ashihara H (1991) Seasonal variations in biosynthetic capacity for the synthesis of caffeine in tea leaves. Phytochemistry 30:2245–2248CrossRefGoogle Scholar
  12. Fukami H, Asakura T, Hirano H, Abe K, Shimomura K, Yamakawa T (2002) Salicylic acid carboxyl methyltransferase induced in hairy root cultures of Atropa belladonna after treatment with exogeneously added salicylic acid. Plant Cell Physiol 43:1054–1058CrossRefPubMedGoogle Scholar
  13. Gang DR, Lavid N, Zubieta C, Chen F, Beuerle T, Lewinsohn E, Noel JP, Pichersky E (2002) Characterization of phenylpropene O-methyltransferases from sweet basil: Facile change of substrate specificity and convergent evolution within plant O-methyltransferase family. Plant Cell 14:505–519CrossRefPubMedGoogle Scholar
  14. Hammerstone JF Jr, Romanczyk LJ Jr, Aitkent WM (1994) Purine alkaloid distribution within Herrania and Theobroma. Pytochemistry 35:1237–1240CrossRefGoogle Scholar
  15. Hibi N, Higashiguchi S, Hashimoto T, Yamada Y (1994) Gene expression in tobacco low-nicotine mutants. Plant Cell 6:723–735PubMedCrossRefGoogle Scholar
  16. Joshi CP, Chiang VL (1998) Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases. Plant Mol Biol 37:663–674PubMedCrossRefGoogle Scholar
  17. Kato M, Kanehara T, Shimizu H, Suzuki T, Gillies FM, Crozier A, Ashihara H (1996) Caffeine biosynthesis in young leaves of Camellia sinensis: in vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine. Physiol Plant 98:629–636CrossRefGoogle Scholar
  18. Kato M, Mizuno K (2004) Caffeine synthase and related methyltransferases in plants. Front Biosci 9:1833–1842PubMedCrossRefGoogle Scholar
  19. Kato M, Mizuno K, Fujimura T, Iwama M, Irie M, Crozier A, Ashihara H (1999) Purification and characterization of caffeine synthase from tea leaves. Plant Physiol 120:579–586CrossRefPubMedGoogle Scholar
  20. Kato M, Mizuno K, Crozier A, Fujimura T, Ashihara H (2000) Caffeine synthase gene from tea leaves. Nature 406:956–957CrossRefPubMedGoogle Scholar
  21. Koyama Y, Tomoda Y, Kato M, Ashihara H (2003) Metabolism of purine bases, nucleosides and alkaloids in theobromine-forming Theobroma cacao leaves. Plant Physiol Biochem 41:977–984CrossRefGoogle Scholar
  22. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T-4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  23. Mizuno K, Okuda A, Kato M, Yoneyama N, Tanaka H, Ashihara H, Fujimura T (2003a) Isolation of a new dual-functional caffeine synthase gene encoding an enzyme for the conversion of 7-methylxanthine to caffeine from coffee (Coffea arabica L.) FEBS Lett 534:75–81CrossRefGoogle Scholar
  24. Mizuno K, Kato M, Irino F, Yoneyama N, Fujimura T, Ashihara H (2003b) The first committed step reaction of caffeine biosynthesis: 7-methylxanthosine synthase is closely homologous to caffeine synthases in coffee (Coffea arabica L.) FEBS Lett 547:56–60CrossRefGoogle Scholar
  25. Mizuno K, Tanaka H, Kato M, Ashihara H, Fujimura T (2001) cDNA cloning of caffeine (theobromine) synthase from coffee (Coffea arabica L.,) In: International Scientific Colloquim on Coffee, ASIC, Paris, 19:815–818Google Scholar
  26. Nagata T, Sakai S (1985) Purine base pattern of Camellia irrawadiensis. Phytochemistry 24:2271–2272CrossRefGoogle Scholar
  27. Naik JP (2001) Improved high-performance liquid chromatography method to determine theobromine and caffeine in cocoa and cocoa products. J Agric Food Chem 49:3579–3583CrossRefPubMedGoogle Scholar
  28. Negishi O, Ozawa T, Imagawa H (1985) Methylation of xanthosine by tea leaf extracts and caffeine biosynthesis. Agric Biol Chem 49:887–890Google Scholar
  29. Negishi O, Ozawa T, Imagawa H (1988) N-methylnucleosidase from tea leaves. Agric Biol Chem 52:169–175Google Scholar
  30. Negre F, Kolosova N, Knoll J, Kish CM, Dudareva N (2002) Novel S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate, is not involved in floral scent production in snapdragon flowers. Arch Biochem Biophys 406:261–270CrossRefPubMedGoogle Scholar
  31. Ogawa M, Herai Y, Koizumi N, Kusano T, Sano H (2001) 7-Methylxanthine methyltransferase of coffee plants. J Biol Chem 276:8213–8218CrossRefPubMedGoogle Scholar
  32. Ross JR, Nam KH, D′Auria JC, Pichersky E (1999) S-Adenosyl-L-methionine:Salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent production and plant defense, represents a new class of plant methyltransferases. Arch Biochem Biophys 367:9–16CrossRefPubMedGoogle Scholar
  33. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedCrossRefGoogle Scholar
  34. Seo HS, Song JT, Cheong JJ, Lee YH, Iwang I, Lee JS, Choi YD (2001) Jasmonic acid carboxyl methyltransferase: a key enzyme for jasmonate-regulated plant responses. Proc Natl Acad Sci USA 98:4788–4793CrossRefPubMedGoogle Scholar
  35. Suzuki T (1972) The participation of S-adenosylmethionine in the biosynthesis of caffeine in the tea plants. FEBS Lett 24:18–20CrossRefGoogle Scholar
  36. Suzuki T, Ashihara H, Waller GR (1992) Purine and purine alkaloid metabolism in Camellia and Coffea plants. Phytochemistry 31:2575–2584CrossRefGoogle Scholar
  37. Takeuchi A, Matsumoto S, Hayatsu M (1994) Chalcone synthase from Camellia sinensis: isolation of the cDNAs and the organ-specific and sugar-responsive expression of the genes. Plant Cell Physiol 35:1011–1018PubMedGoogle Scholar
  38. Uefuji H, Ogita S, Ymaguchi Y, Koizumi N, Sano H (2003) Molecular cloning and functional characterization of three distinct N-methyltransferase involved in the caffeine biosynthetic pathway in coffee plants. Plant Physiol 132:372–380CrossRefPubMedGoogle Scholar
  39. Zubieta C, Ross JR, Koscheski P, Yang Y, Pichersky E, Noel JP (2003) Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family. Plant Cell 15:1704–1716CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Naho Yoneyama
    • 1
  • Hanayo Morimoto
    • 1
  • Chuang-Xing Ye
    • 2
  • Hiroshi Ashihara
    • 3
  • Kouichi Mizuno
    • 4
  • Misako Kato
    • 1
    Email author
  1. 1.Graduate School of Humanities and SciencesOchanomizu UniversityTokyoJapan
  2. 2.School of Life SciencesSun Yat-Sen UniversityGuangzhouChina
  3. 3.Department of Biology, Fuculty of ScienceOchanomizu UniversityTokyoJapan
  4. 4.Faculty of Bioresource of ScienceAkita Prefectural UniversityAkita CityJapan

Personalised recommendations