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.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid that is present in high concentrations in the tea plant Camellia sinensis. Caffeine synthase (CS, EC 2.1.1.160) catalyzes the S-adenosyl-L-methionine-dependent N-3- and N-1-methylation of the purine base to form caffeine, the last step in the purine alkaloid biosynthetic pathway. We studied the expression profile of the tea caffeine synthase (TCS) gene in developing leaves and flowers by means of northern blot analysis, and compared it with those of phenylalanine ammonia lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 2.3.1.74), and S-adenosyl-L-methionine synthase (SAMS, EC 2.5.1.6). The amount of TCS transcripts was highest in young leaves and declined markedly during leaf development, whereas it remained constant throughout the development of the flower. Environmental stresses other than heavy metal stress and plant hormone treatments had no effect on the expression of TCS genes, unlike the other three genes. Drought stress suppressed TCS gene expression in leaves, and the expression pattern mirrored that of the dehydrin gene. The amounts of TCS transcripts increased slightly on supply of a nitrogen source. We discuss the regulation of TCS gene expression