Caffeine biosynthesis in young leaves of Camellia sinensis: In vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine

The aim of this study was to investigate the S-adenosylmethionine dependent N-methyltransferase(s) (NMT) associated with the three methylation steps in the caffeine biosynthesis pathway in tea (Camellia sinensis L.). NMT activity in cell-free preparations from young leaves was purified by anion-exchange and gel-filtration column chromatography. In both systems, a single zone of NMT activity, with broad substrate specificity was detected. The N-3 position of dimethylxanthine and monomethylxanthines was methylated more readily than N-1 while comparatively little substitution occurred at the N-7 locus. When xanthosine was used as a substrate only the N-7 position was methylated These results indicate that a single NMT may participate in the conversion of xanthosine to caffeine. The apparent M(r) of the NMT, estimated by gel filtration chromatography, was 61 000. The substrate specificity of the NMT is compatible with the operation of a xanthosine --> 7-methylxanthosine --> 7-methylxanthine --> theobromine --> caffeine pathway as the main biosynthetic route to caffeine in young tea leaves. The data also indicate that the conversion of 7-methylxanthine --> paraxanthine --> caffeine may function as one of a number of minor pathways that also contribute to the production of caffeine.