Abstract
Purine alkaloids producing species accumulate caffeine (1,3,7-trimethylxanthine) or theobromine (3,7-dimethylxanthine) as a major compound. Caffeine biosynthetic pathway contains three S-adenosylmethionine(SAM)-dependent methylation steps. The available data support the operation of 7-methylxanthine to caffeine via theobromine by a bifunctional caffeine synthase which catalayses the last two steps (N-1- and N-3 methylation) in caffeine-accumulated species. In this study, we focused on two N-methyltransferases. One was caffeine synthase (TCS1) from Camellia sinensis and the other was theobromine synthase (PCS1) from Camellia ptilophylla, which was theobromine-accumulated species. TCS1 was 91% identical to PCS1. We produced the recombinant enzyme introduced to the artificial modification in amino acid sequence with E. coli expression system to clarify the region related to the substrate specificity. The residues from 148 to 287 out of 369 amino acids in TCS1 determined the substrate specificity, in particular, position 221 arginine was contributed to the specificity.
