In co¡ee and tea plants, ca¡eine is synthesized from xanthosine via a pathway that includes three methylation steps. We report the isolation of a bifunctional co¡ee ca¡eine synthase (CCS1) clone from co¡ee endosperm by reverse transcriptionpolymerase chain reaction (RT-PCR) and rapid ampli¢cation of cDNA ends (RACE) technique using previously reported sequence information for theobromine synthases (CTSs). The predicted amino acid sequences of CCS1 are more than 80% identical to CTSs and are about 40% similar to those of tea ca¡eine synthase (TCS1). Interestingly, CCS1 has dual methylation activity like tea TCS1.

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.

The ¢rst committed step reaction of ca¡eine biosynthesis:7-methylxanthosine synthase is closely homologous to ca¡eine synthases in co¡ee (Co¡ea arabica L.) Abstract In co¡ee and tea plants, ca¡eine is synthesized from xanthosine via a pathway that has three methylation steps. We identi¢ed and characterized the gene encoding the enzyme for the ¢rst methylation step of ca¡eine biosynthesis. The fulllength cDNA of co¡ee tentative ca¡eine synthase 1, CtCS1, previously isolated by the rapid ampli¢cation of cDNA ends was translated with an Escherichia coli expression system and the resultant recombinant protein was puri¢ed using Ni-NTA column. The protein renamed CmXRS1 has 7-methylxanthine synthase (xanthosine:S-adenosyl-L-methionine methyltransferase) activity. CmXRS1 was speci¢c for xanthosine and xanthosine 5P P-monophosphate (XMP) could not be used as a substrate. The K m value for xanthosine was 73.7 W WM. CmXRS1 is homologous to co¡ee genes encoding enzymes for the second and third methylation steps of ca¡eine biosynthesis.