Identification and characterization of N9-methyltransferase involved in converting caffeine into non-stimulatory theacrine in tea

Caffeine is a major component of xanthine alkaloids and commonly consumed
in many popular beverages. Due to its occasional side effects, reduction
of caffeine in a natural way is of great importance and economic
significance. Recent studies reveal that caffeine can be converted into
non-stimulatory theacrine in the rare tea plant Camellia assamica var.
kucha (Kucha), which involves oxidation at the C8 and methylation at the
N9 positions of caffeine. However, the underlying molecular mechanism
remains unclear. Here, we identify the theacrine synthase CkTcS from
Kucha, which possesses novel N9-methyltransferase activity using
1,3,7-trimethyluric acid but not caffeine as a substrate, confirming that
C8 oxidation takes place prior to N9-methylation. The crystal structure of
the CkTcS complex reveals the key residues that are required for the
N9-methylation, providing insights into how caffeine N-methyltransferases
in tea plants have evolved to catalyze regioselective N-methylation
through fine tuning of their active sites. These results may guide the
future development of decaffeinated drinks.