PMC

Tissue-specific transcript accumulation. Total RNAs were prepared from indicated tissues of coffee, and RT-PCR analysis was performed using the gene-specific primer sets. CaMTL1 was used as the internal standard ().

Caffeine biosynthetic pathway in coffee plants. Solid arrows indicate major routes, and broken arrows indicate minor or predicted routes. The first (1), third (3), and fourth (4) steps are N-methylation, and the second (2) step is Rib removal. 7 mXMP, 7-methylxanthosine 5′-monophosphate; XR, xanthosine; 7 mXR, 7-methylxanthosine; 7 mX, 7-methylxanthine; Tb, theobromine; Cf, caffeine.

In vitro reconstitution of caffeine biosynthetic pathway. A to C, Single or combined recombinant protein samples shown in Figure A 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.

Effects of substrate concentration. Recombinant proteins shown in Figure A were subjected to reaction with 1 to 500 μm methyl group acceptors and 1 to 50 μm [methyl-¹⁴C] Ado-Met (methyl group donor) for 1 h. Reaction velocity was measured with varied concentrations of the indicated substrate and a fixed concentration at maximum levels of the other substrate. When effects of Ado-Met concentration for CaXMT1, CaMXMT2, and CaDXMT1 were analyzed, the methyl group acceptors were XR, 7 mX, and Tb, respectively. The reaction products were separated by TLC and quantified with a bio-imaging analyzer system (BAS2500, Fuji Photo Film, Tokyo). The horizontal axis shows the concentration of the indicated substrate, and the vertical axis shows the velocity (v) for the indicated product (picomoles per minute per nanomole of protein). Plots are the averages of three independent measurements.

Comparison of deduced amino acid sequences of methyltransferases. A, Alignment of coffee N-methyltransferases. Genes, substrates, and accession numbers are as follows: CaXMT1, xanthosine, AB048793; CaMXMT1, 7-methylxanthine, AB048794; CaMXMT2, 7-methylxanthine, AB084126; and CaDXMT1, theobromine, AB084125. The sequences were aligned by ClustalW (). Identical residues shared by at least three proteins are shaded in black, and conserved substitutes found in at least three proteins are shaded in gray. B, Unrooted phylogenetic tree of coffee N-methyltransferases with related proteins. Genes, substrates, species nomenclatures, and accession numbers are as follows: CaMTL1, unknown, coffee, AB039725; CaMTL2, unknown, coffee, AB048792; CTS1, 7-methylxanthine, coffee, AB034700; CTS2, 7-methylxanthine, coffee, AB054841; CCS1, theobromine, coffee, AB086414; TCS1, theobromine, tea, AB031280; JMT, jasmonic acid methyltransferase, Arabidopsis, AY008434; SAMT, salicylic acid carboxyl methyltransferase, Clarkia breweri, AF133053; BAMT, benzoic acid carboxyl methyltransferase, Antirrhinum majus, AF198492; PEAMT, phosphoethanolamine N-methyltransferase, Spinacia oleracea, AF237633; CNMT, coclaurine N-methyltransferase, Coptis japonica, AB061863; and PMT1, putrescine N-methyltransferase 1, Nicotiana tabacum, BAA05867. The sequences were compared using ClustalW, and the phylogenetic tree was generated by TreeView 1.5.2 (). The branch lengths represent numbers of substituted residues per site.

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.