Identification and characterization of N9-methyltransferase involved in converting caffeine into non-stimulatory theacrine in tea

Abstract

Caffeine is a major component of xanthine alkaloids and commonly consumed in many popular beverages. Due to its occasional side effects, reduction of caffeine in a natural way is of great importance and economic significance. Recent studies reveal that caffeine can be converted into non-stimulatory theacrine in the rare tea plant Camellia assamica var. kucha (Kucha), which involves oxidation at the C8 and methylation at the N9 positions of caffeine. However, the underlying molecular mechanism remains unclear. Here, we identify the theacrine synthase CkTcS from Kucha, which possesses novel N9-methyltransferase activity using 1,3,7-trimethyluric acid but not caffeine as a substrate, confirming that C8 oxidation takes place prior to N9-methylation. The crystal structure of the CkTcS complex reveals the key residues that are required for the N9-methylation, providing insights into how caffeine N-methyltransferases in tea plants have evolved to catalyze regioselective N-methylation through fine tuning of their active sites. These results may guide the future development of decaffeinated drinks.

Fig. 1. Qualitative and quantitative analysis of major xanthine alkaloids in Puer and Kucha.

a The main biosynthetic pathway of caffeine (4) from xanthosine (1). b Conversion of caffeine to 1,3,7-trimethyluric acid does not progress to theacrine in Puer leaves. c The content of caffeine (4), 1,3,7-trimethyluric acid (5) and theacrine (6) in Puer and Kucha leaves. The left panel shows the HPLC analysis of 4 and 6 at absorbance wavelength of 254 nm. The right three panels show the quantification of 4, 5, and 6 by HPLC-MS. Data represents mean ± SD (n = 4). The corresponding dot plots are overlaid on the figure. d Conversion of caffeine to 1,3,7-trimethyluric acid then to theacrine in Kucha leaves.

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

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.

Fig. 3. Transcriptional level analysis of CkTcS gene in Kucha and Puer.

a Qualitative analysis of CkTcS and GAPDH expression in Puer and Kucha. PL1, PL2, PL3, and KL1, KL2, KL3 represent three different leaves from Pure tea plant and Kucha tea plant, respectively. b Quantitative analysis of CkTcS and GAPDH expression in Puer and Kucha by quantitative real-time PCR. The gene expression in each leaf was analyzed at three replicates. The PCR was run for 40 cycles. Expression of CkTcS relative to GAPDH in three Pure leaves versus two Kucha leaves is shown.

Fig. 4. Crystal structure of CkTcS.

a Crystal structure of CkTcS dimer. SAH and 1,3,7-trimethyluric acid (5) are shown in ball-and-stick representation. b Structural overlap of CkTcS (pink) with DXMT (blue) and XMT (grey), with the major structural difference around the substrate binding pocket between CkTcS, DXMT, and XMT highlighted. c A close-up view of the CkTcS-1,3,7-trimethyluric acid interactions. The hydrogen bonds are shown in red dashed lines. d A close-up view of the π–π-stacking interactions. e In vitro methylation assay of wild-type (WT) or mutants of CkTbS using 1,3,7-trimethyluric acid as substrate, with CkTcS as positive control. Data represent mean ± SD (n = 3). The corresponding dot plots are overlaid on the figure. The experiment was repeated twice. Differences were assessed statistically by two-tailed Student’s t-test, ^****P < 0.0001, ^*P < 0.05.