Plant Physiol, May 2003, Vol. 132, pp. 372-380

Molecular Cloning and Functional Characterization of Three Distinct N-Methyltransferases Involved in the Caffeine Biosynthetic Pathway in Coffee Plants¹

Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Nara 630-0192, Japan

Caffeine is synthesized from xanthosine through N-methylation and ribose removal steps. In the present study, three types of cDNAs encoding N-methyltransferases were isolated from immature fruits of coffee (Coffea arabica) plants, 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 µM, CaMXMT2 catalyzed formation of 3,7-dimethylxanthine (theobromine) from 7-methylxanthine with a K_m of 251 µM, and CaDXMT1 catalyzed formation of 1,3,7-trimethylxanthine (caffeine) from 3,7-dimethylxanthine with a K_m of 1,222 µM. 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.