Figure 1.
The four steps of the caffeine biosynthetic pathway in plants.
Step I, the conversion of xanthosine (XR) to 7-methylxanthosine (7-mXR) by xanthosine methyltransferase (CaXMT1); Step II, the hydrolysis of 7-mXR to 7-methylxanthine (7-MX) by N-methyl-nucleosidase; Step III, the methylation of 7-MX to Theboromine (Tb) by caffeine synthase (TCS1); Step IV, the methylation of Tb to caffeine (Cf) by caffeine synthase (TCS1).
Table 1.
Oligonucleoside primers for PCR.
Figure 2.
Phylogenetic tree of N-methyltransferases related to caffeine biosynthesis.
Genes, accession numbers, species nomenclature, and substrates are listed in that order as follows: MGT1, X60368, Saccharomyces cerevisiae, unknown; CaXMT1, AB048793, coffee, xanthosine; TCS1, AB031280, tea, 7-methylxanthine and theobromine; ICS1, AB056108, Camellia irrawadiensis, 7-methylxanthine; PCS1, AB207817, Camellia ptilophylla, 7-methylxanthine; BCS1, AB096699, Theobroma cacao, 7-methylxanthine. The sequences were compared and phylogenetic tree was generated by MEGA5 (Tamura, Peterson, Stecher, Nei, and Kumar 2011). The branch lengths represent numbers of substituted residues per site.
Figure 3.
SDS-PAGE analysis of recombinant proteins showing the expression of caffeine biosynthetic enzymes.
GST and the CaXMT1- and TCS1-GST fusion proteins were expressed in E. coli and prepared for gel loading. The samples were separated on a 10% (w/v) SDS-polyacrylamide gel and visualized by Coomassie Brilliant Blue staining. Arrows indicate recombinant proteins.
Figure 4.
In vitro functional analysis of CaXMT1 and TCS1 expressed in E. coli.
Extracts from E. coli expressing GST-CaXMT1 (a) and -TCS1 (b) were extracted and used for enzymatic assays. The top traces of (a) and (b) show the authentic standards run in parallel. The middle traces are negative controls. The bottom trace of (a) shows that CaXMT1 recombinant protein catalyzed the reaction from xanthosine (XR) to 7-methylxanthine (7-MX). The bottom trace of (b) presents the TCS1 catalysis of 7-MX to caffeine (Cf) via theobromine (Tb). Black arrowheads indicate reaction products.
Figure 5.
In vivo functional analysis of co-expression of CaXMT1 and TCS1 in S. cerevisiae.
The HPLC trace shows that co-expression of CaXMT1 and TCS1 can catalyze the conversion from xanthosine (XR) to caffeine (Cf). Black arrowheads indicate reaction products.
Figure 6.
Semi-quantitative RT-PCR amplification of putative nucleosidase genes.
Total RNA samples were prepared separately from different tissues of tea. Actin gene was used as the internal standard. Semi-quantitative RT-PCR analysis was performed using the gene-specific primer sets listed in Table 1.
Figure 7.
SDS-PAGE analysis of recombinant proteins.
GST and GST-ST1, -ST2 and -ST3 fusions proteins were expressed in E.coli, extracted, and resuspended in loading buffer. The samples were separated on a 10% (w/v) SDS-polyacrylamide gel and visualized by Coomassie Brilliant Blue staining. Arrows indicate recombinant proteins.
Figure 8.
The tertiary model of TCS1 with theobromine (Tb) and SAM.
(a) TCS1 model is represented as wheat cartoon; Tb and SAH (S-adenosine homocysteine) are represented as cyan and yellow sticks. (b) The close-up view of the Tb binding site of TCS1 wild type and mutants. Residues lining Tb binding sites of TCS1 wild type and mutants are colored in white and brown sticks, respectively.
Figure 9.
HPLC analysis after expression in E. coli of TCS1 or one of its putative active site mutants by using pGEX-4t-2 vector.
(a) The trace shows the authentic standards run in parallel. (b) The trace shows the original TCS1 enzymatic reaction products. (c to f) show the HPLC analysis of the reaction products from the mutants TM1 to TM4. Black arrowheads indicate reaction products.
Figure 10.
HPLC analysis after expression in E. coli of TCS1 or one of its putative active site mutants by using pMAL-c5X vector instead of pGEX-4t-2.
(a) The trace shows the authentic standards run in parallel. (b) The trace shows the original TCS1 enzymatic reaction products. (c to f) show the HPLC analysis of the reaction products from the mutants TM1 to TM4. Black arrowheads indicate reaction products.
Table 2.
Theobromine produced in enzymatic assays containing E.coli expressed TCS or TMX mutants by using pGEX-4t-2 and Rosetta (DE3).
Table 3.
Theobromine and caffeine produced in enzymatic assays containing E.coli expressed TCS or TMX mutants by using pMAL-c5X vector instead of pGEX-4t-2.
Figure 11.
HPLC analysis after co-expression in E. coli of recombinant CaXMT1 andTCS1 by using pGEX-4t-2 vector.
The top trace shows the negative control and the bottom trace shows the products of the sample enzymatic reaction. XR is substrate and SAM is added into the reaction as methyl donor.
Figure 12.
HPLC analysis after co-expression in E. coli of recombinant CaXMT1 andTCS1 by using pMAL-c5X vector instead of pGEX-4t-2.
The top trace shows the negative control and the bottom trace shows the products of the sample enzymatic reaction. XR is substrate and SAM is added into the reaction as methyl donor.