PMID:12746542

Citation	Uefuji, H, Ogita, S, Yamaguchi, Y, Koizumi, N and Sano, H (2003) Molecular cloning and functional characterization of three distinct N-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. Plant Physiol. 132:372-80
Abstract	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 microM, CaMXMT2 catalyzed formation of 3,7-dimethylxanthine (theobromine) from 7-methylxanthine with a K(m) of 251 microM, and CaDXMT1 catalyzed formation of 1,3,7-trimethylxanthine (caffeine) from 3,7-dimethylxanthine with a K(m) of 1,222 microM. 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.
Links	PubMed PMC166982 Online version:10.1104/pp.102.019679
Keywords	Amino Acid Sequence; Caffeine/biosynthesis; Catalytic Domain/genetics; Cloning, Molecular; Coffea/enzymology; Coffea/genetics; Culture Techniques; DNA, Complementary/chemistry; DNA, Complementary/genetics; DNA, Complementary/isolation & purification; Kinetics; Methyltransferases/genetics; Methyltransferases/metabolism; Molecular Sequence Data; Phylogeny; Plant Proteins/genetics; Plant Proteins/metabolism; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Substrate Specificity; Transcription Factors/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
COFAR:XMT1		GO:0008170: N-methyltransferase activity	ECO:0000314:		F		Based on methods: 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. Using these recombinant proteins, catalytic specificity was determined with the following substrates: XMP, xanthosine, xanthine, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, paraxanthine, theobromine, and theophylline (Fig. ​(Fig.3B–D;3B–D; Table ​TableI).I). Purified CaXMT1 solely catalyzed conversion of xanthosine to 7-methylxanthosine	complete CACAO 4251
COFAR:MXMT1		GO:0008170: N-methyltransferase activity	ECO:0000314:		F		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. Using these recombinant proteins, catalytic specificity was determined with the following substrates: XMP, xanthosine, xanthine, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, paraxanthine, theobromine, and theophylline (Fig. ​(Fig.3B–D;3B–D; Table ​TableI).I). Purified CaXMT1 solely catalyzed conversion of xanthosine to 7-methylxanthosine (Fig. ​(Fig.3B,3B, lane 2). In contrast, crude CaXMT1 catalyzed conversion of xanthosine to 7-methylxanthine, suggesting that conversion of xanthosine to 7-methylxanthosine and that of 7-methylxanthosine to 7-methylxanthine successively occurred (Fig. ​(Fig.3B,3B, lane 4). Although the former reaction was catalyzed by CaMXT1, the latter was found to be catalyzed by a nonspecific purine-nucleoside phosphorylase derived from E. coli (Fig. ​(Fig.3B,3B, lane 6).	complete CACAO 4255
COFAR:MXMT2		GO:0008170: N-methyltransferase activity	ECO:0000314:		F		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. Using these recombinant proteins, catalytic specificity was determined with the following substrates: XMP, xanthosine, xanthine, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, paraxanthine, theobromine, and theophylline (Fig. ​(Fig.3B–D;3B–D; Table ​TableI).I). CaMXMT2 catalyzed conversion of 7-methylxanthine to theobromine (Fig. ​(Fig.3C,3C, lane 2).	complete CACAO 4256

References

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