Abstract
Caffeine is synthesized from xanthine derivatives through multiple methylation. Three N-methyltransferases, CaXMT, CaMXMT and CaDXMT, were previously identified from coffee plants, and the precursor, xanthosine was successfully converted into caffeine using recombinant enzymes in vitro. To understand the catalytic mechanism of caffeine biosynthesis, we analyzed specific properties of these enzymes. Previous study reported that sequence of a salicylic acid C-methyltransferase (SbSAMT) from Clarkia breweri was similar to that of CaMXMT, and that SbSAMT formed a homo-dimer in vitro. This suggested CaMXMT to form dimer(s), and, since sequences of isolated three N-methyltransferases showed high similarity with each other, they might form hetero-dimer(s). To examine this idea, we used the bimolecular fluorescence complementation, which visualize protein-protein interaction in vivo by YFP reconstitution by two nonfluorescent fragments. Results demonstrated that CaMXMT formed a homo-dimer in cytoplasm, and hetero-dimers with respective CaXMT and CaDXMT. The effects of hetero-dimerization on catalytic activities are under investigation.
