THE STRUCTURE OF TWO N -METHYLTRANSFERASES FROM THE CAFFEINE BIOSYNTHETIC PATHWAY

XR, xanthosine; Cf, caffeine; Caffeine (1,3,7-trimethylxanthine) is a secondary metabolite produced by certain plant species and an important component of coffee and tea. Here we describe the structures of two S-adenosyl- L -methione (SAM) dependant N -methyltransferases that mediate caffeine biosynthesis in Coffea canephora (robusta), xanthosine methyltransferase (XMT) and 1,7 dimethylxanthine methyltransferase (DXMT). Both were co-crystallized with the demethylated cofactor, S-adenosyl- L -cysteine (SAH), and substrate, either xanthosine (XMT) or theobromine (DXMT). Our structures reveal several elements that appear critical for substrate selectivity. S316 in XMT appears central to the recognition of xanthosine. Likewise, a change from Q161 in XMT to H160 in DXMT is likely to have catalytic consequences. A F266 to I266 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. Finally, we propose an enzymatic mechanism whereby XMT could potentially generate 7-methylxanthine from xanthosine.

6 step ( Fig. 1), the ribose removal step being carried out by a currently unidentified 7methylxanthosine nucleosidase.
The N-methyltransferases from the caffeine biosynthetic pathway have a 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, salicyclic acid O-methyltransferase (SAMT), has been structurally characterised 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 a structural and biochemical characterisation of XMT and DXMT from C. canephora (robusta). Here we describe the structures of XMT and DXMT complexed with SAH, the demethylated product, and their respective substrates, either xanthosine (XR) or theobromine (Tb). These structures are compared to the SAMT structure complexed with salicyclic acid and SAH (Zubieta et al., 2003). The structural results presented here offer several insights into the substrate specificity from this family of SAM dependant 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 catalyses the addition of a methyl group to the N7 of xanthosine (XR) and generates 7-methylxanthine (7mX) instead of 7-methylxanthosine (7mXR) ( Fig. 2A). This unexpected observation is dependant 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 3mM SAM. XMT showed no activity towards either 7mX or Tb, data not shown. DXMT is capable of converting 7mX to Tb (Fig. 2B), as well as converting Tb to Cf (Fig. 2C), and exhibits no detectable activity towards XR (data not shown). 7

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 (SAD) method with selenomethine-incorporated (SeMet) 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 (2 and 5 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) is preserved in the crystal lattice, where each monomer is related by a crystallographic two fold axis along B. XMT was solved by molecular replacement using a model derived from the DXMT structure with flexible loops or insertions deleted. The final model comprises of 4 XMT molecules in the asymmetric unit and 499 water molecules. Each of the XMT molecules contained 344 of a possible 372 residues, one SAH, and one XR. The 13 N-terminal residues have very weak electron density but were excluded, and there was no discernable density for two surface loops (residues 85-89 and 303-306) or the 5 C-terminal residues of each molecule. The four molecules in the asymmetric unit form two dimer pairs. All the crystallographic information is summarised in Table 1. The R and R free values for both DXMT and XMT are larger than those for comparable structures found in the PDB and this is mainly due to the generous cutoff criteria used for the determination of the highresolution limit.

Overall Structure
The XMT and DXMT structures are nearly identical and can be superimposed with an r.m.s.d. of 1.0Å for 331 CA atoms from a possible 356. XMT and DXMT consist of two domains, the core SAM-dependent methyltransferase domain (residues 23-162, 190-227, 266-295 and 367-379 for DXMT) and an α-helical cap domain (residues 1-22, 163-189, 228-265 and 296-366 for DXMT) (Fig 3A and Fig. 4) 8 structural differences between all three of these structures occur in loop regions at or proximal to the active site ( Fig. 3B and C). The first involves the deletion of one amino acid between residues S22-L28 in DXMT (S22-I29 in XMT), resulting in a structural difference for this region between DXMT, XMT and SAMT. The second occurs in the loop region between β5 and α6, where there is a structural difference between all three methyltransferases, (XMT, DXMT and SAMT), and includes an extra 4 residues in SAMT (Fig. 4). The third difference occurs in the loop region between β6 and α9, which corresponds to a large insertion in XMT and DXMT (Fig.   4), of which D303-A311 and I303-D305 are disordered in DXMT and XMT respectively. The final structural difference occurs in the loop region between α10 and β7.
The biochemically characterized members of this family exist as dimers in solution (Zubieta et al., 2003). The dimerization interface involves residues from α4 and β3, (Fig. 3A), and leaves each of the monomer active sites independent.
Dimerization buries a total of 1080Å 2 and 1146Å 2 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 F102, F106, F110, L132, and M136 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 D105 and N107 in DXMT, and the two main chain hydrogen bonds made by A135 in DXMT.

SAH/SAM binding site
SAH is bound in a similar position and conformation in XMT, DXMT and SAMT to other SAM-dependant 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 S139 in DXMT and the second one occurs between N1 and the backbone amide of F140 in DXMT. There is also an additional water mediated hydrogen bond between the exocyclic amino group (N6), N7 and the carboxyl group of L162 in DXMT. The adenine ring is sandwiched between the hydrophobic side chains of a leucine (L101 in DXMT) from motif B' (Fig. 4) and a phenylalanine (F140 in DXMT) from motif C (Fig. 4). The adenine ring lies coplanar with the phenyl ring in a π-stacking interaction on one face, while the leucine caps the hydrophobic face on the opposite side. The ribose hydroxyls of SAH form hydrogenbonding interactions with a strictly conserved aspartate (D100 in DXMT) from motif B' (Fig. 4). There is also an additional sulfhydryl hydrogen bond between the cysteine C158 in DXMT (C159 in XMT) and the ribose O4' of SAH in XMT and DXMT.
The direct hydrogen-bonding interactions between the amino tail of SAH and the backbone carboxyl residues of G60 from motif A (Fig. 4) and C156 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 L59, G60 and C156, and the side chains of D58 and T70 in DXMT. The carboxyl tail of SAH forms a hydrogen bonding interaction with the side chain of Y18 in all three methyltransferases. An additional hydrogen hydrogen bond (3.2Å) is observed in DXMT between the carboxyl tail and N66. Interestingly, this sequence conserved asparagine has moved away from the SAH carboxyl tail to a distance of ~4.2Å in both XMT and SAMT. Additional water-mediated hydrogen bonds to the carboxyl tail are also observed in both XMT and DXMT.

Substrate binding site
XMT was co-crystallized in the presence of XR and it is well defined in the structure (Supplemental Fig. S1). DXMT was co-crystallized in the presence of Tb and exists in two conformations (Fig. S1), one mimicking 7-methylxanthine (7mX) binding. The substrate-binding site of XMT and DXMT is located in a similar position to that found in SAMT ( Fig. 5A-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 9 hydrogen bonds to the XMT protein, six with the purine ring and three with the ribose moiety (Fig. 5C) ribose moiety of XR makes hydrogen-bonding interactions to the backbone carboxyl groups of N21 and S22, the hydroxyl group of S316 and the amide group of N25. The O5' hydroxyl group also makes an additional water mediated hydrogen bond to the backbone carboxyl groups of N21 and S316. This water also makes a hydrogen bond to the O4' ribose atom, in addition to a direct hydrogen bond to the N25. The final XR ribose interaction is a water mediated hydrogen bond between the O3' hydroxyl group and the carboxyl group of Y297.
In SAMT the carboxylate moiety of salicylic acid is precisely positioned via hydrogen bonding interactions with the sequence conserved W151 (Fig. 5D), which is predicted to be important for substrate recognition in this family of methyltransferases  5B). S237 is mutated to an alanine in XMT resulting in a loss of hydrogen bonding interaction and a loop movement away from the binding site of ~1.6Å relative to DXMT (Fig. 3B). In SAMT this loop moves by ~3.6Å relative to DXMT and forms part of the salicylic hydrophobic binding pocket.
In XMT the O2 carboxylate group of XR forms a hydrogen bond with the hydroxyl groups of both Y321 and Y356 (Fig. 5C). Interestingly, although Y333 in DXMT is structurally equivalent to Y321 in XMT, this sequence conserved tyrosine is too far from either the 7mX or Tb in DXMT to form a hydrogen bond with the substrate (Fig. 5A and B). The Y356 of XMT is the sequence conserved Y368 in DXMT, which adopts a different conformation due to the movement of the β5-α6 loop ( Fig. 3B and C).
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 M9, Y18, Y24, Y158, I227, V320, Y321 and Y356 in XMT. V320 caps one face of the purine ring, while I227 and Y158 abut the opposite face, with the purine ring perpendicular to the phenyl ring of Y158 in a π-stacking interaction (Fig. 5C). Tb forms hydrophobic interactions with M9, L26, F27, Y157, I226, M238, I266, V328, I332, Y333 and Y368 in DXMT. In particular, I332 forms a hydrophobic interaction with one face, while F27 and Y157 abut the opposite face, with the purine ring again perpendicular to the sequence conserved Y157 in a π-stacking orientation ( Fig. 5A and B).

DISCUSSION
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.

N-terminus
The N-terminal part of the protein is flexible, facilitating the entry of the substrates and co-factor into the active site, and the exit of the reaction products.
XMT has an extra glutamine residue between N25 and L26 (as numbered in DXMT) ( Fig. 4) 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). The carboxylate group of N25 in XMT is only 2.9Å away from C8, indicating that it may also form an aromatic hydrogen bond to XR (Fig. 5C). In contrast to XMT, N25 of DXMT is not involved in hydrogen bonding interactions with either 7mX or Tb and actually points away from the active site (Fig. 3B). The space vacated by the N25 movement is occupied by four well ordered water molecules, which form an extensive hydrogen bonding network between DXMT and 7mX or Tb. The additional space also allows for the closer approach of Y18 to the substrate in DXMT, enabling it to hydrogen bond with the O6 carboxyl group in Tb (Fig. 5B). Y18 is strictly conserved in the Motif B' methyltransferases and its hydroxyl group is in close proximity to the positively charged S-atom of SAM. A similarly positioned tyrosine (Y21) in an unrelated N-methyltransferase, glycine N-methyltransferase, has been implicated in its methyltransfer mechanism (Takata et al., 2003) and hence a catalytic role for this residue in motif B' methyltransferases appears likely.

Substrate discrimination between XMT and DXMT
Two elements important for substrate discrimination between XMT and DXMT can be identified. The first involves S316 in XMT, which forms a hydrogen bond with the O5' hydroxyl group from the ribose moiety of XR (Fig. 5C). S316 corresponds to a valine in DXMT and to a cysteine in SAMT (Fig. 4). A valine at this position would disrupt the hydrogen bond and introduce a steric clash with the ribose moiety of XR (Fig. 3C). S316 is therefore likely to be crucial for the XR substrate specificity in XMT. The second occurs in the loop connecting α10 to β7 (Fig 3B), where large conformational differences are observed between the three methyltransferases in this family. The Y356 of XMT adopts a different conformation from the corresponding sequence conserved Y368 of DXMT, (Fig. 3C). These differences result in the Y356 of XMT hydrogen bonding with the O2 carboxyl group of XR (Fig. 5C), while the Y368 in DXMT adopts a conformation close to the ribose site and probably distorts the site enough to eliminate its potential to accept a ribose (Fig. 3C). The alternate conformations of these tyrosine side-chains are mainly due to a movement of the β5-α6 loop away from the substrate binding site in XMT. This is probably due to the sequence divergence observed between XMT and DXMT (Fig. 4) www 13 and specifically where a change from a proline (P235 in DXMT) to an arginine (R236 in XMT) allows the β5-α6 loop in each structure to adopt a different conformation.
Another difference between the XMT and DXMT substrate binding sites occurs at position Y321 in XMT, which forms a hydrogen bond with the O2 carboxyl group of XR (Fig. 5C). In DXMT, the structurally conserved tyrosine (Y333) is too far from any potential hydrogen bonding partners and does not directly contribute to substrate binding. Y333 instead forms a hydrogen bonding interaction with S237, orienting it for optimal hydrogen bonding interactions with 7mX or Tb in DXMT. An alanine (A312) occupies this position in SAMT (Fig.4), and is necessary to accommodate the large W226, which forms part of the salicylate binding site in SAMT (Zubieta et al., 2003).

Substrate specificity between MXMTs and DXMTs
Coffee plants contain both MXMTs (or coffee theobromine synthases (CTS1/2)) and DXMTs (Kato and Mizuno, 2004). It is still unclear why both enzymes are required, as the DXMTs can readily catalyse both methyl transfers (Fig. 2) (Uefuji et al., 2003). However, while multiple isoforms of the MXMTs are expressed in various tissue types in coffee plants, the DXMTs are predominantly expressed in immature fruit (Uefuji et al., 2003). This observation may have important consequences for caffeine production in the immature fruit. There are fifteen residues within 5Å of Tb in DXMT. Only three of these are of significance when comparing the sequences of MXMT with DXMT, as eight are identical and four involve very conservative changes. The first involves the substitution of F27 in DXMT by an alanine in MXMT (Fig. 4). There is no obvious reason from our DXMT structure why this substitution would favour the 7mX substrate, unless there is a conformational change. The next involves a substitution of S237 in DXMT for a proline in MXMT (Fig. 4). This would disrupt the hydrogen bond to either the O6 carboxyl group of 7mX or the N9 of Tb (Fig. 5A and B), so it is unclear how this change would discriminate between 7mX and Tb. The last involves the substitution of I266 in DXMT by a phenylalanine in the MXMTs (Fig. 4). A phenylalanine at this position in the MXMTs would result in a steric clash with a methyl group on the N3 position, precluding the binding of Tb and its subsequent methylation (Fig. 5B) MXMTs. However, it should be noted that the sequences of a MXMT from Camellia plants (PCS1) (Yoneyama et al., 2006) and a DXMT from tea (TCS1) (Kato et al., 2000) vary quite significantly in these regions (Fig. 4). So while this isoleucine to phenylalanine substitution is likely to be important for substrate discrimination in coffee it is unlikely to account for the substrate selectivity observed between all the MXMT and DXMT proteins.

Dual activity for XMT
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 C. arabica (Uefuji et al., 2003) and the 7mX nucleosidase activity was reportedly due to a nonspecific E. coli purine-nucleoside phosphorylase (PNP) (Uefuji et al., 2003). However, it is highly unlikely that PNP would uniquely act on 7mXR.
The structure and mechanism of PNP is well established (Koellner et al., 2002), (Bennett et al., 2003) and XR, with its free N7 atom, would be a far better substrate.
The reaction is dependant on SAM, as no product peaks were observed in XMT assays when high concentrations of SAH were still present and we believe that the methyl transfer and nucleoside cleavage may indeed be coupled (Fig. 1). This also explains why transgenic tobacco plants expressing XMT, MXMT and DXMT are able to produce caffeine Ogita et al., 2005). Similar reactions and mechanisms involving the formation of an oxocarbenium intermediate are observed in the N-ribohydrolases (Degano et al., 1998;Lee et al., 2005), and phosphorylases (Bennett et al., 2003). A plausible mechanism for XMT ribose cleavage would commence with the methylation of N7, resulting in a partial positive charge on the purine ring moiety. This positive charge would induce the flow of electrons from the ribose moiety, resulting in the carbenium intermediate. A nucleophilic attack by water on the oxocarbenium intermediate could then occur. However, nucleophilic activation may not be required as the ribose moiety is partially solvent exposed and oxocarbenium ions are an extremely reactive species in aqueous solution (Amyes and Jencks, 1989). It is also possible that the Y18 maybe capable of activating a water via a charge-dipole interaction with the SAM carboxylate. From the XMT structure there are two other residues, Y24 and N25 that may also be important for nucleosidase activity. The carboxyl group of the N25 side-chain in XMT is within 2.9Å of the C8 atom in XR and maybe involved in stabilizing a positive charge on the purine ring.
Any lengthening of the C1'-N9 bond and subsequent movement of the ribose ring would allow the aromatic ring of Y24 to form a stabilizing π-cation interaction with the ribose carbenium ion.

Catalytic residues involved in N-methyltransferase activity
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 substrates for the O-methyltransferases are expected to be fully or predominantly deprotonated at cellular pH values and should only require their correct positioning for methylation to occur (Zubieta et al., 2003). However, the conserved tyrosine (Y18) noted earlier, could form a charge-dipole interaction with the positively charged S-atom of SAM, facilitating the methyl transfer reaction in this family of methyltransferases, as observed in GNMT (Takata et al., 2003). The Nmethyltransferases may also require some additional help for methylation, as nitrogen is not as electronegative as oxygen. The H160 side chain in DXMT forms a hydrogen bond with O2 in both 7mX and Tb substrate binding ( Fig. 5A and B). Both 7mX and Tb can exist as enolate ions (Fig. 1) and it is quite likely that the H160 in DXMT could stabile the enolate ion, making either N1 or N3 more electronegative for subsequent methylation. H160 is conserved in all the MXMTs and DXMTs, but not in the XMTs or SAMT (Fig4), where its position would not aid in catalysis. The importance of this amino acid sequence in the substrate specificity of Nmethyltransferases was previously postulated (Ogawa et al., 2001;Mizuno et al., 2003), and here we also predict its potential catalytic importance.

CONCLUSION
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 the protein database. 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 xanthosine. 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). SeMet DXMT protein was produced via inhibition of the methionine metabolism pathway (Doublie, 1997) and prepared using the same expression and purification protocols as the native DXMT (McCarthy et al., 2007).

The SeMet DXMT crystallized in similar conditions, 23-28% PEG 3350, 200mM
Li 2 SO 4 , 100mM Tris-HCl, pH=8.5-8.7 containing 2mM DTT, 2mM SAH and 2mM Tb, to the native DXMT with a plate like morphology in 1-3 days at 20 o C. The crystals were then flash frozen at 100K after transferring them to identical crystallisation conditions containing 38% PEG 3350. These crystals were orthorhombic, space group C222 1 , with one molecule in the asymmetric unit and a 2.0 Å data set was collected. A highly redundant data set to 2.66 Å was collected from a SeMet DXMT crystal at the peak of the Se-Met signal, as measured by X-ray fluorescence. XMT in complex with SAH and XR crystallised in space group P2 1 with 4 molecules per asymmetric unit and a 2.2 Å data set was collected. All X-ray data were collected on beamline ID14-4 at the European Synchrotron Radiation Facility (ESRF). All X-ray data were integrated and scaled using the XDS suite (Kabsch, 1993) and a summary of the data statistics is given in Table 1.

Structure Determination and refinement
Six Se-Met sites were located on the basis of their anomalous differences using SHELXD (Uson and Sheldrick, 1999). The sites were refined and experimental phases to 2.66Å were calculated using the single anomalous dispersion (SAD) procedure in SHARP and further improved with the density modification package SOLOMON in SHARP (de La Fortelle and Bricogne, 1997). These phases were input into ARP/wARP (Perrakis et al., 1999) and resulted in a fragmented polyalanine model of 281 residues. Subsequent rounds of model building followed by phased refinement using the SHARP phases in REFMAC (Murshudov et al., 1997)  the majority of the model to be built. XMT was solved by molecular replacement using PHASER (McCoy et al., 2005) and the DXMT structure as a search model.
The crystal structure refinements of DXMT and XMT were performed using REFMAC (Murshudov et al., 1997), with a randomly chosen subset of 5% of reflections for the calculation of the free R-factor. All the crystallographic information is summarized in Table 1. A well-ordered SAH was easily modelled into both structures in the early stages of refinement. A Tb molecule in two orientations was modelled into DXMT (Fig S1A and B) and a well ordered XR molecule was easily modelled into XMT ( fig S1C). Ordered water molecules were added at locations were there was |F O |-|F C | density greater than 3σ above the mean and within hydrogen bonding contact to a neighbouring molecule. Model building was carried out with Coot (Emsley and Cowtan, 2004) and the stereochemical quality of the protein molecules were validated with PROCHECK (Laskowski et al., 1993). Restraints for XR were generated by using the Dundee PRODRG server (Schuttelkopf and van Aalten, 2004). Sequence alignments were done with ClustalW (Thompson et al., 1994) and ESPript (Gouet et al., 1999), and the figures were prepared with PYMOL (DeLano, 2002).

Determination of enzymatic activity
The reactions with purified recombinant DXMT protein were set up as follows; 30 µl of 10 mM SAM, 30 µl of 10 x reaction buffer (500 mM Tris-HCl pH 7.9, 10 mM MgCl 2 , 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 1 x reaction buffer) was added to the remaining reaction. Both the control and enzyme reactions were incubated at 37°C. 45 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. 60 µg of purified XMT was first diafiltered into reaction buffer (50 mM Tris-HCl pH 7.9, 1 mM MgCl 2 , 10 mM NaCl, 0.1 mM DTT and 3 mM SAM) to give a final volume of 1ml. Then, 240 µl aliquots of XMT in reaction buffer were added to 60 µl of 5 mM solutions of either XR, 7mX or Tb. A 50 µl control sample was immediately taken from each of the reactions and added to 200 µl HPLC Solvents were sparged with 30% helium and the flow rate was 1 ml/minute. The gradient was as follows; at 0 min, 98% A -2% B; at 5 min, 92% A -8% B; at 25min, 50% A -50% B; at 30min, 30% A -70% B; at 35min, 30% A -70% B; then from 37-45 minutes, 98% A -2% B. Detection was done using a Waters photodiode array detector.

Accession numbers
The nucleotide sequences reported in this paper have been deposited in the DDBJ/Genbank/EBI Data Bank with accession numbers DQ422954 (XMT) and  XMT reaction activity with XR as substrate, with a sample taken at T=1min (top) and T=180mins (bottom). B: DXMT reaction with 7mX as substrate, with a sample taken at T=0min before recombinant enzyme was added (top) and T=30min (bottom). C: DXMT reaction with Tb as substrate, with a sample taken at T=0min before recombinant enzyme was added (top) and T=30min (bottom). (C) Superposition of the XMT (blue) and DXMT (gold) active sites with important substrate interacting residues highlighted, Tb is in grey and XR is in green.