Figure 5.

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.