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Purification and Characterization of Caffeine Synthase from Tea Leaves1

Misako Kato, Kouichi Mizuno, Tatsuhito Fujimura, Masanori Iwama, Masachika Irie, Alan Crozier, and Hiroshi Ashihara*

Department of Biology, Faculty of Science, Ochanomizu University, Tokyo 112-8610, Japan (M.K., H.A.); Institute of Agricultural and Forest Engineering, University of Tsukuba, Ibaraki 305-8572, Japan (K.M., T.F.); Department of Microbiology, Hoshi College of Pharmacy, Ebara, Tokyo 142-0063, Japan (M. Iwama, M. Irie); and Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom (A.C.)

Caffeine synthase (CS), the S-adenosylmethionine-dependent N-methyltransferase involved in the last two steps of caffeine biosynthesis, was extracted from young tea (Camellia sinensis) leaves; the CS was purified 520-fold to apparent homogeneity and a final specific activity of 5.7 nkat mg-1 protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange, adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of 61 kD as estimated by gel-filtration chromatography and 41 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3- and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine, and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no 7-N-methyltransferase activity toward xanthosine and xanthosine 5'-monophosphate. The Km values of CS for paraxanthine, theobromine, 7-methylxanthine, and S-adenosylmethionine were 24, 186, 344, and 21 µM, respectively. The possible role and regulation of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence for CS showed little homology with other methyltransferases.

1   This work was supported in part by a Grant-in-Aid for Scientific Research (no. 10640627) from the Ministry of Education, Science, Sports and Culture of Japan (to H.A.) and by a grant from the Mishima Kaiun Memorial Foundation (to M.K.). A.C. received funding for travel between the United Kingdom and Japan from The Royal Society.
*   Corresponding author; e-mail ashihara@cc.ocha.ac.jp; fax 81-3-5978-5358.

Plant Physiol. (1999) 120: 579-586
Copyright Clearance Center:   0032-0889/99/120//08
© 1999 American Society of Plant Physiologists




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