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First published online April 13, 2007; 10.1104/pp.106.094854 Plant Physiology 144:879-889 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway1,[W],[OA]European Molecular Biology Laboratory, Grenoble 38042, France (A.A.M.); and Nestlé Research and Development, Tours 37097, France (J.G.M.)
Caffeine (1,3,7-trimethylxanthine) is a secondary metabolite produced by certain plant species and an important component of coffee (Coffea arabica and Coffea canephora) and tea (Camellia sinensis). Here we describe the structures of two S-adenosyl-L-methionine-dependent N-methyltransferases that mediate caffeine biosynthesis in C. canephora ‘robusta’, xanthosine (XR) methyltransferase (XMT), and 1,7-dimethylxanthine methyltransferase (DXMT). Both were cocrystallized with the demethylated cofactor, S-adenosyl-L-cysteine, and substrate, either xanthosine or theobromine. Our structures reveal several elements that appear critical for substrate selectivity. Serine-316 in XMT appears central to the recognition of XR. Likewise, a change from glutamine-161 in XMT to histidine-160 in DXMT is likely to have catalytic consequences. A phenylalanine-266 to isoleucine-266 change in DXMT is also likely to be crucial for the discrimination between mono and dimethyl transferases in coffee. These key residues are probably functionally important and will guide future studies with implications for the biosynthesis of caffeine and its derivatives in plants.
Caffeine is a plant alkaloid known to have sensory and stimulatory effects when consumed in beverages such as coffee (Coffea arabica and Coffea canephora) and tea (Camellia sinensis). The pharmacological actions are thought to be mediated via blockade of the adenosine A1 and A2A receptors (Cauli and Morelli, 2005  ). The exact biological role of caffeine and related purine alkaloids of plants is still unclear, although there are now two main hypotheses. It has been proposed that caffeine protects young leaves and fruit from predators (chemical defense theory [Frischknecht, 1985; Hollingsworth et al., 2002]) and that the caffeine released by the seed coat prevents germination of other seeds (allelopathic or autotoxic theory [Friedman and Waller, 1983]). In agreement with these proposals, it is known that caffeine is accumulated in both the seeds and young leaves of coffee plants (Ashihara and Suzuki, 2004). Strong supporting evidence for the chemical defense theory has recently been obtained by Uefuji et al. (2005), who demonstrated that leaves of transgenic tobacco (Nicotiana tabacum) plants engineered to produce caffeine were less susceptible to insect feeding versus control leaves that did not contain caffeine.
The first N-methyltransferase from the caffeine biosynthesis pathway to be biochemically characterized was cloned from young tea leaves and designated tea caffeine synthase1 (TCS1; Kato et al., 2000
The N-methyltransferases from the caffeine biosynthetic pathway have high protein sequence homology (>80% identity), but exhibit remarkable substrate selectivity (Kato and Mizuno, 2004 ). They also belong to the wider motif B' methyltransferases specific to plants (Kato and Mizuno, 2004). This important family includes enzymes involved in the biosynthesis of small and volatile methyl esters that are proposed to act as interplant signaling molecules in plant defense (Zubieta et al., 2003). Only one member of this family, salicylic acid O-methyltransferase (SAMT), has been structurally characterized so far (Zubieta et al., 2003). In an effort to understand the subtle substrate selectivity within the family of N-methyltransferases from the caffeine biosynthetic pathway, as well as between members of the larger motif B'-containing family of methyltransferase proteins, we decided to undertake structural and biochemical characterization of XMT and DXMT from Coffea canephora ‘robusta’. Here we describe the structures of XMT and DXMT complexed with S-adenosyl-L-Cys (SAH), the demethylated product, and their respective substrates, either XR or Tb. These structures are compared to the SAMT structure complexed with salicylic acid and SAH (Zubieta et al., 2003). The structural results presented here offer several insights into the substrate specificity from this family of SAM-dependent methyltransferases.
Enzymatic Activity XMT and DXMT were expressed in Escherichia coli and highly purified for both biochemical and structural studies. Biochemical analysis of the highly purified XMT used for crystallization shows that this preparation catalyzes the addition of a methyl group to the N7 of XR and generates 7mX instead of 7mXR (Fig. 2A ). This unexpected observation is dependent on SAM because initial assays of recombinant XMT were unsuccessful due to the presence of SAH, which was added for crystallization trials. The XMT activity was restored by diafiltration into the reaction buffer containing 3 mM SAM. XMT showed no activity toward either 7mX or Tb (data not shown). DXMT is capable of converting 7mX to Tb (Fig. 2B), as well as converting Tb to caffeine (Fig. 2C), and exhibits no detectable activity toward XR (data not shown).
Structure Determination and Refinement
DXMT could be solved by molecular replacement using SAMT as a starting model. However, the subsequent refinement was problematic and the structure was eventually solved by the single anomalous diffraction method with selenomethionine (SeMet)-incorporated DXMT. The final model comprises 348 of a possible 384 residues, one SAH, two Tb molecules in different orientations, and 146 water molecules. The disordered residues comprise the N- and C-terminal residues (two and five residues, respectively) and some surface loops (residues 12–15, 82–91, 169–174, and 303–311). The DXMT dimer observed in solution (McCarthy et al., 2007
Overall Structure
The XMT and DXMT structures are nearly identical and can be superimposed with a root-mean-square derivation (rmsd) of 1.0 Å for 331 C
The biochemically characterized members of this family exist as dimers in solution (Zubieta et al., 2003 ). The dimerization interface involves residues from  4 and  3 (Fig. 3A), and leaves each of the monomer active sites independent. Dimerization buries a total of 1,080 Å2 and 1,146 Å2 of solvent-accessible surface for each monomer of XMT and DXMT, respectively. This represents 7% and 8% of the total surface area for each monomer of XMT and DXMT, respectively, which is at the low end of dimer interfaces analyzed (Jones and Thornton, 1995) and similar to that observed in SAMT (Zubieta et al., 2003). Much of the interface is hydrophobic and sequence conserved between XMT, DXMT, and SAMT, with the side chains of Phe-102, Phe-106, Phe-110, Leu-132, and Met-136 being buried between the monomers in DXMT. A number of identical hydrogen bonds are also observed, such as those between the sequence-conserved side chains Asp-105 and Asn-107 in DXMT and the two main chain hydrogen bonds made by Ala-135 in DXMT.
SAH is bound in a similar position and conformation in XMT, DXMT, and SAMT to other SAM-dependent methyltransferases (Fig. 3A). SAH/SAM binding is mediated through extensive hydrogen bonding and van der Waals interactions. Two hydrogen-bonding interactions to the adenine ring of the SAH come from the highly conserved motif C (Fig. 4). The first one is between the exocyclic amino group (N6) and the hydroxyl group of Ser-139 in DXMT and the second one occurs between N1 and the backbone amide of Phe-140 in DXMT. There is also an additional water-mediated hydrogen bond between the exocyclic amino group (N6), N7, and the carboxyl group of Leu-162 in DXMT. The adenine ring is sandwiched between the hydrophobic side chains of Leu-101 in DXMT from motif B' (Fig. 4) and Phe-140 in DXMT from motif C (Fig. 4). The adenine ring lies coplanar with the phenyl ring in a The direct hydrogen-bonding interactions between the amino tail of SAH and the backbone carboxyl residues of Gly-60 from motif A (Fig. 4) and Cys-156 in DXMT are conserved in all three structures. XMT and DXMT also make water-mediated interactions between the amino tail of SAH, the protein main chain atoms of Leu-59, Gly-60, and Cys-156, and the side chains of Asp-58 and Thr-70 in DXMT. The carboxyl tail of SAH forms a hydrogen-bonding interaction with the side chain of Tyr-18 in all three methyltransferases. An additional hydrogen bond (3.2 Å) is observed in DXMT between the carboxyl tail and Asn-66. Interestingly, this sequence-conserved Asn has moved away from the SAH carboxyl tail to a distance of approximately 4.2 Å in both XMT and SAMT. Additional water-mediated hydrogen bonds to the carboxyl tail are also observed in both XMT and DXMT.
XMT was cocrystallized in the presence of XR and it is well defined in the structure (Supplemental Fig. S1). DXMT was cocrystallized in the presence of Tb and exists in two conformations (Supplemental Fig. S1), one mimicking 7mX binding. The substrate-binding site of XMT and DXMT is located in a similar position to that found in SAMT (Fig. 5, A–D ). The substrate is properly positioned in the active site for methylation in all the methyltransferases from this family through both hydrogen bonding and van der Waals interactions. Only some of these interactions are sequence conserved and there are many differences important for both substrate recognition and catalysis.
XR makes a total of nine hydrogen bonds to the XMT protein, six with the purine ring and three with the Rib moiety (Fig. 5C). The O5' hydroxyl group from the Rib moiety of XR makes hydrogen-bonding interactions to the backbone carboxyl groups of Asn-21 and Ser-22, the hydroxyl group of Ser-316, and the amide group of Asn-25. The O5' hydroxyl group also makes an additional water-mediated hydrogen bond to the backbone carboxyl groups of Asn-21 and Ser-316. This water also makes a hydrogen bond to the O4' Rib atom in addition to a direct hydrogen bond to the Asn-25. The final XR Rib interaction is a water-mediated hydrogen bond between the O3' hydroxyl group and the carboxyl group of Tyr-297.
In SAMT, the carboxylate moiety of salicylic acid is precisely positioned via hydrogen-bonding interactions with the sequence-conserved Trp-151 (Fig. 5D), which is predicted to be important for substrate recognition in this family of methyltransferases (Zubieta et al., 2003
The largest structural difference between all the methyltransferases occurs in the loop between
In XMT, the O2 carboxylate group of XR forms a hydrogen bond with the hydroxyl groups of both Tyr-321 and Tyr-356 (Fig. 5C). Interestingly, although Tyr-333 in DXMT is structurally equivalent to Tyr-321 in XMT, this sequence-conserved Tyr is too far from either the 7mX or Tb in DXMT to form a hydrogen bond with the substrate (Fig. 5, A and B). The Tyr-356 of XMT is the sequence-conserved Tyr-368 in DXMT, which adopts a different conformation due to the movement of the
These hydrogen-bonding interactions are supplemented by hydrophobic interactions between the protein and the hydrophobic faces of the purine ring. XR is involved in hydrophobic interaction with Met-9, Tyr-18, Tyr-24, Tyr-158, Ile-227, Val-320, Tyr-321, and Tyr-356 in XMT. Val-320 caps one face of the purine ring, whereas Ile-227 and Tyr-158 abut the opposite face, with the purine ring perpendicular to the phenyl ring of Tyr-158 in a
The work presented here details the structural analysis of two very closely related enzymes involved in caffeine biosynthesis in coffee. These proteins are part of the general motif B' family of methyltransferases, which transfer the activated methyl group of SAM to different plant secondary metabolites. Biochemical studies on the purified proteins (Fig. 2) confirmed that one is an XMT and the other is a DXMT, as predicted from the analysis of their primary sequence (McCarthy et al., 2007 ). The structural studies presented here have identified a number of important loops or amino acid changes that are necessary for alterations in substrate recognition and/or catalysis.
The N-terminal part of the protein is flexible, facilitating the entry of the substrates and cofactor into the active site and the exit of the reaction products. XMT has an extra Gln residue between Asn-25 and Leu-26 (as numbered in DXMT; Fig. 4), allowing important hydrogen-bonding changes to occur. The Asn-25 of XMT makes extensive hydrogen-bonding interactions with the Rib moiety of the XR substrate (Fig. 5C). This agrees with the proposal that a shorter side chain is necessary to accommodate the larger substrates of the N-methyltransferases (Zubieta et al., 2003
Two elements important for substrate discrimination between XMT and DXMT can be identified. The first involves Ser-316 in XMT, which forms a hydrogen bond with the O5' hydroxyl group from the Rib moiety of XR (Fig. 5C). Ser-316 corresponds to a Val in DXMT and to a Cys in SAMT (Fig. 4). A Val at this position would disrupt the hydrogen bond and introduce a steric clash with the Rib moiety of XR (Fig. 3C). Ser-316 is therefore likely to be crucial for XR substrate specificity in XMT. The second occurs in the loop connecting
Another difference between the XMT and DXMT substrate-binding sites occurs at position Tyr-321 in XMT, which forms a hydrogen bond with the O2 carboxyl group of XR (Fig. 5C). In DXMT, the structurally conserved Tyr-333 is too far from any potential hydrogen-bonding partners and does not directly contribute to substrate binding. Tyr-333 instead forms a hydrogen-bonding interaction with Ser-237, orienting it for optimal hydrogen-bonding interactions with 7mX or Tb in DXMT. Ala-312 occupies this position in SAMT (Fig. 4) and is necessary to accommodate the large Trp-226, which forms part of the salicylate-binding site in SAMT (Zubieta et al., 2003
Coffee plants contain both MXMTs (or CTS1/2) and DXMTs (Kato and Mizuno, 2004
A highly purified preparation of XMT was not active on 7mX or Tb as anticipated. Surprisingly, the only clearly detectable product from the methylation of XR was 7mX (Fig. 2A). A similar activity was reported for a crude preparation of XMT from coffee (Uefuji et al., 2003
It is very clear that DXMT can methylate both 7mX and Tb (Fig. 2B). There are no residues located within the transmethylation pocket that could act as a general acid/base for the methyl transfer reaction, as observed in SAMT (Zubieta et al., 2003
The structures of XMT and DXMT presented here identify a number of key residues involved in substrate recognition and catalysis and will aid in the annotation of the many uncharacterized N-methyltransferase sequences available in GenBank. The subtle differences required for substrate recognition in the important motif B' family of methyltransferases could even be exploited to produce new compounds for the pharmaceutical industry. Our results also suggest the plausibility of engineering a single protein capable of producing caffeine from XR. Such a protein could facilitate the production of herbivore-resistant crops that are more ecologically friendly.
Purification, Crystallization, and Data Collection
Native XMT and DXMT were purified and crystallized as previously described (McCarthy et al., 2007
Six SeMet sites were located on the basis of their anomalous differences using SHELXD (Uson and Sheldrick, 1999
The crystal structure refinements of DXMT and XMT were performed using REFMAC (Murshudov et al., 1997
Reactions with purified recombinant DXMT protein were set up as follows: 30 µL of 10 mM SAM, 30 µL of 10x reaction buffer (500 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 100 mM NaCl, 1 mM DTT), and 60 µL of 5 mM substrates (XR, 7mX, and Tb) were made up to a final volume of 285 µL with water. A 100-µL aliquot was taken as a control reaction and then 15 µL of purified protein (21 µg in 1x reaction buffer) were added to the remaining reaction. Both the control and enzyme reactions were incubated at 37°C. Forty-five- or 50-µL samples were taken at various times and added to 200 µL HPLC buffer A (92% water, 8% acetonitrile, 0.1% phosphoric acid, pH 2.2) to stop the reaction. Sixty micrograms of purified XMT were first diafiltered into reaction buffer (50 mM Tris-HCl, pH 7.9, 1 mM MgCl2, 10 mM NaCl, 0.1 mM DTT, and 3 mM SAM) to give a final volume of 1 mL. Then, 240-µL aliquots of XMT in reaction buffer were added to 60 µL of 5 mM solutions of XR, 7mX, or Tb. A 50-µL control sample was immediately taken from each of the reactions and added to 200 µL HPLC buffer A. The reactions were then placed at 37°C and 50-µL samples were taken at various times and added to 200 µL HPLC buffer A to stop the reactions. The samples were then passed through a 0.2-µM filter and were analyzed by HPLC using a Waters reverse-phase C18 column (4 µM, 4.6 x 250 mm) and a gradient of 8% to 50% acetonitrile. Solvent A was 91.9% milliQ water, 8% CH3CN, and 0.1% H3PO4; solvent B was 49.9% milliQ water, 50% CH3CN, and 0.1% H3PO4. Solvents were sparged with 30% helium and the flow rate was 1 mL/min. The gradient was as follows: at 0 min, 98% A/2% B; at 5 min, 92% A/8% B; at 25 min, 50% A/50% B; at 30 min, 30% A/70% B; at 35 min, 30% A/70% B; then from 37 to 45 min, 98% A/2% B. Detection was done using a Waters photodiode array detector. The nucleotide sequences reported in this article have been deposited in the DDBJ/GenBank/EBI Data Bank with accession numbers DQ422954 (XMT) and DQ422955 (DXMT). The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rscb.org) with the accession codes 2EG5 (XMT) and 2EFJ (DXMT).
The following materials are available in the online version of this article.
We thank G. Cheminade for carrying out the HPLC analysis, the European Molecular Biology Laboratory/European Synchrotron Radiation Facility Joint Structural Biology Group for access to ID14-4, R. Ravelli for help with data collection and structural solution, and V. Petiard, R. Ravelli, and S. Cusack for their support during this work. Finally, we are grateful to C. Petosa and E. Mitchell for critically reading this manuscript. Received December 15, 2006; accepted April 5, 2007; published April 13, 2007.
1 This article is dedicated to the memory of Nicholas P. Chopey.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Andrew A. McCarthy (andrewmc{at}embl.fr).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.094854 * Corresponding author; e-mail andrewmc{at}embl.fr; fax 33–476207199.
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ABSTRACT
RESULTS
calculated for the whole dataset. Figure of merit
, where P(
was calculated as for 
, with 5% of the data omitted from the structural refinement.
-stacking interaction on one face, whereas the Leu caps the hydrophobic face on the opposite side. The Rib hydroxyls of SAH form hydrogen-bonding interactions with strictly conserved Asp-100 in DXMT from motif B' (
above the mean and within hydrogen-bonding contact to a neighboring molecule. Model building was carried out with Coot (Emsley and Cowtan, 2004
