In vitro reconstitution of caffeine biosynthetic
pathway. A to C, Single or combined recombinant protein samples shown
in Figure 3A were subjected to reaction with 500 μm
xanthosine as the sole methyl group acceptor (starting material) and
1.5 mm Ado-Met for 16 h, and the reaction products
were identified by HPLC analysis. The recombinant protein samples were
crude CaXMT1 alone (A), crude CaXMT1 and purified CaMXMT2 (B), and
crude CaXMT1, purified CaMXMT2, and purified CaDXMT1 (C). Authentic
standards were also run in parallel (D). Black arrowheads indicate
reaction products and standards, and white arrowheads indicate
impurities.