Minisymposium 2: Plant Biology in the Tropics

Abs # 12003: Three N-methyltransferases that successively catalyze caffeine biosynthesis in coffee plants

Presenter:	Uefuji, Hirotaka , h-uehuzi@bs.aist-nara.ac.jp
Authors	Uefuji, Hirotaka  (A)   Ogita, Shinjiro  (A)   Ogawa, Mikihiro  (A)   Sano, Hiroshi  (A)
Affiliations:	(A): Nara Institute of Science and Technology

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid produced by a variety of plant species including coffee (Coffea arabica), tea (Camellia sinensis), maté (Ilex paraguariensis), guaraná (Paullinia cupana) and cola (Cola nitida). Accumulating in seeds, cotyledons and young leaves, its biological role appears to be in chemical defense, serving as an antiherbivory and an allelopathic compound. In human societies, it has been widely used as a stimulant and an ingredient in drugs. Caffeine is synthesized from xanthosine through N-methylation and ribose removal steps. We previously isolated a coffee N-methyltransferase cDNA, CaMXMT1 whose product catalyzes formation of theobromine (3,7-dimethylxanthine) from 7-methylxanthine. In the present study, we isolated additional three types of cDNAs encoding N-methyltransferases from immature coffee fruits and designated as CaXMT1, CaMXMT2 and CaDXMT1, respectively. The bacterially expressed encoded proteins were characterized for their catalytic properties. CaXMT1 catalyzed formation of 7-methylxanthosine from xanthosine with a K_m value of 78 mM, CaMXMT2 catalyzed formation of theobromine from 7-methylxanthine with a K_m of 251 mM, and CaDXMT1 catalyzed formation of caffeine from theobromine with a K_m of 1,222 mM. The crude extract of Escherichia coli was found to catalyze removal of the ribose moiety from 7-methylxanthosine, leading to the production of 7-methylxanthine. As a consequence, when all three recombinant proteins and E. coli extract were combined, xanthosine was successfully converted into caffeine in vitro. Transcripts for CaDXMT1 were predominantly found to accumulate in immature fruits, whereas those for CaXMT1 and CaMXMT2 were more broadly detected in sites encompassing the leaves, floral buds and immature fruits. These results suggest that the presently identified three N-methyltransferases participate in caffeine biosynthesis in coffee plants and substantiate the proposed caffeine biosynthetic pathway: xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine. Genes obtained in this study will be useful for creating not only decaffeinated coffee plants but also improved crops that produce caffeine as a pest repellent.