Figure 3.
N-methyltransferase activity. A, SDS-PAGE analysis of recombinant proteins. GST and CaXMT1, CaMXMT2, and CaDXMT1 fusion proteins with GST (XMT1, MXMT2, and DXMT1) were expressed in Escherichia coli and prepared as crude and purified samples. The samples were separated on a 9% (w/v) SDS-polyacrylamide gel and visualized by Coomassie Brilliant Blue staining. Asterisks indicate recombinant proteins. Lane numbers are given under the panel. B to D, TLC analysis of reaction products. Each recombinant protein was subjected to reaction with 500 μm methyl group acceptor and 16 μm [methyl-14C] Ado-Met for 16 h. Reaction mixtures were subjected to TLC analysis, and methylated products were visualized by autoradiography. Proteins and methyl-acceptors are indicated above the panel. Identity of methylated products and Ado-Met are indicated on the left of the panel. Nonindicated spots are impurities. In B, [methyl-14C] 7-methylxanthosine (7 mXR) produced by purified CaXMT1 was further applied to the Rib removal reaction by non-transformed E. coli crude extract (E. coli) and the extraction buffer (None) for 16 h. In D, concentrated samples are shown in the lanes marked with asterisks. XR, Xanthosine; 7 mXR, 7-methylxanthosine; 7 mX, 7-methylxanthine; Px, paraxanthine; Tb, theobromine; Cf, caffeine. Lane numbers referred to in the text are given under each panel.