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. 2020 Mar 19;11:1473. doi: 10.1038/s41467-020-15324-7

Fig. 2. Identification of N9-methyltransferase involved in converting caffeine into theacrine in tea leaves.

Fig. 2

a SDS-PAGE analysis of the recombinant CkCS, CkTbS, and CkTcS proteins. b In vitro N9-methyltransferase activity analysis of CkCS, CkTbS and CkTcS. HPLC analysis of in vitro reaction products using 1,3,7-trimethyluric acid (5) as a substrate with CkCS and denatured CkCS (dCkCS), CkTbS and denatured CkTbS (dCkTbS), CkTcS and denatured CkTcS (dCkTcS). The absorbance wavelength was set at 254 nm. c Steady state kinetic analysis of CkCS, CkTbS, and CkTcS using 1,3,7-trimethyluric acid (5) as a substrate. Initial velocities are shown as cycles and represented as mean ± SD (n = 3). The corresponding dot plots are overlaid on the figure. The blue line represents the nonlinear least-squares fit of the initial velocities versus 1,3,7-trimethyluric acid concentration to the hyperbolic Michaelis-Menten equation. Kinetic parameters were determined at a saturating concentration of 1.5 mM SAM. d In vitro N-methyltransferase activity of CkTcS and dCkTcS towards xanthosine (1), 7-methylxanthine (2), theobromine (3), caffeine (4), and 1,3,7-trimethyluric acid (5). The absorbance wavelength was set at 254 nm. In all HPLC chromatograms, a red asterisk indicates an impurity compound from the SAM reagent.