Structures of two natural product methyltransferases reveal the basis for substrate specificity in plant O-methyltransferases

Figure 1. Minimal phenylpropanoid biosynthetic pathway in M. sativa L (alfalfa)48. Carbon flow begins in primary metabolism with phenylalanine, which ultimately serves as the building block for a diverse class of plant secondary metabolites. The enzymes depicted are: PAL, phenylalanine-ammonia lyase; CA4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate:coenzyme A ligase; CHS, chalcone synthase; CHR, chalcone reductase; ChOMT, 4,2',4'-trihydroxychalcone 2'-hydroxyl-O-methyltransferase; CHI, chalcone isomerase; IFS, isoflavone synthase; IOMT, isoflavone-O-methyltransferase (isoflavanone-O-methyltransferase); IFOH, isoflavone 2'-hydroxylase; IFR, isoflavone reductase; and PTS, pterocarpan synthase. The reaction depicted in the solid black box occurs in vitro and likely represents a cryptic activity of IOMT, which normally would methylate an isoflavanone intermediate. The depicted dehydration step can spontaneously occur in solution over time or is catalyzed by a specific dehydratase enzyme. A-rings are derived from the head-to-tail condensation of malonyl-CoA derived acetyl groups and the B-rings are derived from the p-coumaryl moiety. Full Figure and legend (43K)

Table 1. Crystallographic data, phasing and refinement information for ChOMT Full Table

Table 2. Crystallographic data, phasing and refinement information for IOMT Full Table

Figure 2. Architecture of the ChOMT and IOMT monomers. a, C traces of the backbones of the ChOMT and IOMT monomers. Every 20th C atom is numbered and the N-termini and C-termini are labeled. The disordered loop in ChOMT between residues 160 and 173 is shown as a dashed coil. b, Stereo view of the final SIGMAA weighted 2|Fo - Fc| electron density map of the ChOMT active site encompassing the bound SAH and isoliquiritigenin (two conformations shown) molecules. Putative hydrogen bonds are shown as dashed green cylinders. Single letter amino acid code is used. The map is contoured at 1.5 . c, Stereo view of the final SIGMAA weighted 2|Fo - Fc| electron density map of the IOMT active site encompassing the bound SAH and isoformononetin molecules. The map is contoured at 1.5 . Full Figure and legend (304K)

ChOMT (Fig. 3a,b) and IOMT (Fig. 3c,d) exhibit a common tertiary structure consisting of a large C-terminal catalytic domain responsible for SAM binding and substrate methylation and a smaller N-terminal domain involved in dimerization and formation of the back wall of the substrate binding site. Due to this conservation of fold, the root mean square (r.m.s.) deviation for alignment of the catalytic domains is 1.4 Å, while both the catalytic and dimerization domains align with an r.m.s. deviation of 1.8 Å for all backbone atoms. The catalytic domain contains a core / Rossmann fold common to nucleotide binding proteins²⁸. Structural alignments with representative DNA and small molecule methyltransferases illustrate the presence of a conserved fold involved in SAM/SAH binding (Fig. 4a). Unlike most structurally characterized methyltransferases, which are monomeric, ChOMT and IOMT form homologous homodimers in their respective crystalline lattices. The monomers in both cases are related by a crystallographic two-fold axis. While ChOMT and IOMT were originally characterized as monomers, the recombinant proteins exhibit no monomer formation in solution. Dimerization appears to be critical for activity and most likely occurs in vivo as well as in vitro. The presence of a dimerization interface appears to be common to plant OMTs and intimately contributes to substrate binding.

Figure 3. Architecture of the ChOMT and IOMT dimers and active sites. a, Ribbon and molecular surface representation of the ChOMT homodimer. Monomer A is rose, monomer B is green, and the bound SAH and isoliquiritigenin molecules are shown as half-colored bonds. b, Close-up stereo view of the substrate binding site highlighting some of the hydrogen bonding and van der Waals interactions with SAH. The view is shown in the same orientation as in (a). c, Ribbon and molecular surface representation of the IOMT homodimer. Monomer A is blue, monomer B is gold and the bound SAH and isoformononetin molecules are shown as half-colored bonds. d, Close-up stereo view of the substrate binding site highlighting some of the hydrogen bonding and van der Waals interactions with SAH. The ribbon diagrams were produced with MOLSCRIPT49 and the surface diagrams with GRASP50. Both were rendered with POV-Ray51. Some side chains have been omitted for clarity. Full Figure and legend (357K)

Figure 4. Structural and sequence comparisons of representative OMTs. a, Structural comparison of IOMT, HhaI DNA C-methyltransferase (M.HhaI) and catechol O-methyltransferase ('COMT'). SAH, isoformononetin (IOMT), SAM (M.HhaI), SAM and dinitrocatechol (COMT) are shown as stick models. The conserved SAM/SAH binding domains are highlighted in gold and the nonconserved regions in blue. The reactions catalyzed by IOMT, M.HhaI and 'COMT' are illustrated with the transferred methyl group highlighted in blue. 'COMT' differs from the plant OMT called COMT, which stands for caffeic acid O-methyltransferase. b, Sequence alignment of nine representative plant O-methyltransferases. Primary (GenBank protein database) and secondary structure of IOMT from M. sativa (alfalfa; AAC49927) and ChOMT from M. sativa (alfalfa; AAB48059) and sequence alignment of caffeic acid OMT from M. sativa (alfalfa; AAB46623), scoulerine OMT from Coptis japonica (goldenthread; BAA06192), isoeugenol OMT from Clarkia breweri (fairy fans; AAC01533), hydroxymaackiain OMT from Pisum sativa (pea; AAC49856), diphenol OMT from Capsicum annum (hot pepper; AAC17455), catechol OMT from Nicotiana tabacum (tobacco; CAA52461), and flavonoid OMT from Hordeum vulgare (barley; CAA54616). -Helices of IOMT are depicted as blue cylinders and -strands as blue arrows. -Helices of ChOMT are depicted as rose cylinders and -strands as rose arrows. The numbering of each protein is in parentheses, with every 10th position dotted. Residues involved in SAM/SAH binding (pink), substrate binding (blue), substrate binding in trans from the dyad related polypeptide (green), and catalysis (yellow) are highlighted. Full Figure and legend (132K)

In ChOMT, the extensive dimerization interface buries 8,990 Ų of surface area, encompassing 30% of the available surface area of the dimer (Fig. 3a). Met 29, Thr 32 and Thr 33 insert into the catalytic domain of the neighboring molecule, thus forming the back wall of the neighboring molecule's active site. The extent of the IOMT interface is comparable, with 8,597 Ų of buried surface area at the interface, comprising 30% of the available surface area of the dimer (Fig. 3c). Tyr 25, Phe 27 and Ile 28 form the back wall of the catalytic domain of the dyad related monomer.

The structures of ChOMT and IOMT in complex with SAH clearly delineate a conserved SAH/SAM binding motif (Fig. 3b,d). The catalytic domains of ChOMT and IOMT maintain homologous / folds consisting of helices 9−13 and -strands 3−9 (Fig. 4b). In addition to conservation of the OMT tertiary structure, positional conservation of the amino acids involved in cofactor binding is evident from the crystal structures of ChOMT and IOMT as well as sequence alignments of plant OMTs (Fig. 4b). SAH binding within the active site pocket of ChOMT is mediated through a network of hydrogen bonds as well as van der Waals interactions (Fig. 3b). IOMT binds SAH through a similar set of interactions (Fig. 3d). The residues involved in hydrogen bonding and van der Waals interactions with SAM/SAH are spatially equivalent in both methyltransferases. The two structures of ChOMT and IOMT highlight the analogous orientation of the bound SAH as well as the common chemical features of the SAM/SAH binding motif.

Because of the broad structural diversity of plant phenylpropanoid compounds, most plant OMTs possess highly selective substrate and positional specificity. Efficient substrate discrimination and binding is achieved in ChOMT and IOMT through shape selectivity dictated by van der Waals interactions, including a rich set of aromatic and aliphatic side chains, and by specific hydrogen bonding patterns. In ChOMT, the isoliquiritigenin substrate adopts two conformations within the active site via a 180° rotation around the carbonyl carbon, resulting in two distinct binding modes for the B-ring of isoliquiritigenin (Fig. 3b). The position of the A-ring, which presents the 2'-hydroxyl group to SAM for methylation, is conserved in both conformers. The A-ring is bound by the thioether moieties of Met 189 and Met 329. Thr 332 and the 4'-hydroxyl moiety of the substrate are within hydrogen bonding distance, which secures the substrate within the active site and most likely ensures that the A-ring 2'-hydroxyl is firmly positioned for deprotonation followed by methylation by the putative catalytic base, His 278, and the methyl donor, SAM, respectively. The back wall of the active site consists of residues Met 29, Thr 32, and Thr 33 donated from the partner monomer (Fig. 5a).

Figure 5. ChOMT and IOMT active sites. a, ChOMT−isoliquiritigenin complex. The ribbon diagram approximates the global orientation of the ChOMT dimer used for the close-up view of the complete chalcone binding site depicted in stereo. The black box highlights the region of ChOMT shown in stereo. Bonds are color coded by atom type with isoliquiritigenin carbon atoms in charcoal and protein carbon atoms in brown. Hydrogen bonds are depicted as dashed cylinders and water molecules as green spheres. Residues labeled with (B) are contributed by the symmetric polypeptide chain. b, IOMT−isoformononetin complex (top panel) and model of a putative IOMT−(2S,3S)-2,4',7-trihydroxyisoflavanone complex (bottom panel) generated by the superposition of the B-ring of isoformononetin and the A-ring of 2,4',7-trihydroxyisoflavanone. The ribbon diagram approximates the global orientation of the IOMT dimer used for the close-up view of the isoflavone-binding site depicted in stereo. The black box highlights the region of IOMT shown in stereo. Bonds are color coded by atom type with isoflavone and isoflavanone carbon atoms in charcoal and protein carbon atoms in brown. Hydrogen bonds are depicted as dashed cylinders. Full Figure and legend (68K)

Based both upon the structures of ChOMT and IOMT and sequence alignments with the large family of plant OMTs, methylation most likely proceeds via base-assisted deprotonation of the hydroxyl group followed by a nucleophilic attack of the newly generated phenolate anion of the substrate on the reactive methyl group of SAM. In ChOMT, deprotonation of the 2'-hydroxyl group of the A-ring by His 278 sets up the subsequent attack by the hydroxyl anion on the methyl group of SAM. Because the sulfur of SAM is positively charged, the transmethylation process is easily facilitated by the deprotonation step. Glu 306 and Glu 337 bracket the catalytic His residue, and the N nitrogen of this His makes a hydrogen bonding interaction to the carboxylate group of Glu 337 (Fig. 2b). This interaction ensures the optimal orientation of the imidazole group for deprotonation of the 2'-hydroxyl of the isoliquiritigenin substrate by the N nitrogen of His 278 (Fig. 5a). Mutation of His 278 to Leu, Ala, Gln, Lys or Asn completely eliminated methyltransferase activity, further implicating His 278 as an important catalytic residue (Fig. 6).

Figure 6. Thin layer chromatography assay of ChOMT and IOMT catalytic His mutants. Lanes 1−6 of the left panel refer to wild type ChOMT, and the mutants H278L, H278A, H278Q, H278K, and H278N, respectively. 14C-methylated 4,4'-dihydroxy-2'-methoxychalcone is labeled (*). Lanes 1−6 of the right panel correspond to wild type IOMT, and the mutants H257L, H257I, H257Q, H257K, and H257D, respectively. 14C-methylated product 4'-hydroxy-7-methoxyisoflavone (isoformononetin) is labeled (**). Full Figure and legend (83K)

Catalysis in IOMT proceeds through a comparable mechanism, with His 257 serving as the base responsible for deprotonation of the 7-hydroxyl group on the A-ring of daidzein (Fig. 5b). Similarly to ChOMT, Asp 288 and Glu 318 sterically constrain His 257 and position its N nitrogen through a hydrogen bond with Glu 318. This same catalytic mechanism would be predicted for the putative physiological substrate 2,7,4'-trihydroxyisoflavanone. Mutation of His 257 to Leu, Ile, Gln or Asp eliminated the methyltransferase activity towards daidzein. Mutation of the active site His to Lys caused greatly diminished activity compared to the wild type enzyme (Fig. 6).

The alfalfa ChOMT gene (GenBank accession number L10211) and IOMT gene (GenBank accession number AF000976) were inserted into the E. coli expression vector pHIS8³⁹ (ChOMT) or pET-15b (IOMT). ChOMT and IOMT constructs were transformed into E. coli BL21(DE3). Transformed E. coli were grown at 37 °C in terrific broth (TB) containing 50 g ml^-1 kanamycin (ChOMT) or 100 g ml^-1 ampicillin (IOMT) until A_600nm = 1.0. After induction with 0.5 mM isopropyl 1-thio--galactopyranoside (IPTG), the cultures were grown for 6 h at 25 °C. Cells were pelleted, harvested, and resuspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM imidazole (pH 8.0), 20 mM -mercaptoethanol, 10% (v/v) glycerol and 1% (v/v) Tween-20). After sonication and centrifugation, the supernatant was passed over a Ni²⁺-NTA column, washed with 10 bed volumes of lysis buffer, 10 bed volumes of wash buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM imidazole (pH 8.0), 20 mM -mercaptoethanol and 10% (v/v) glycerol), then the His tagged protein was eluted with elution buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 250 mM imidazole (pH 8.0), 20 mM -mercaptoethanol, and 10% (v/v) glycerol). Incubation with thrombin during dialysis for 24 h at 4 °C against 25 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol (DTT) removed the N-terminal His tag. Dialyzed protein was reloaded onto a Ni²⁺-NTA column to remove cleaved His tag followed by thrombin depletion using a benzamidine Sepharose column. Gel filtration on a Superdex 200 column equilibrated with 25 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM DTT resulted in homogenous and active ChOMT and IOMT. Fractions containing the protein of interest were pooled and concentrated to 25 mg ml^-1 and stored at -80 °C. SeMet substituted protein was obtained from E. coli grown in minimal media with appropriate amino acids and SeMet added⁴⁰. Expression and purification steps were as above. All mutants were generated with the QuikChange (Stratagene) protocol. Automated nucleotide sequencing confirmed the fidelity of the PCR products (Salk Institute DNA sequencing facility). All mutants were expressed as described above.

Mutant enzymes were purified by Ni⁺² affinity chromatography, dialyzed against 25 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM DTT, and concentrated to 2 mg ml^-1. Qualitative activity assays were performed using 20 g of protein, 500 M substrate (2',4,4'-trihydroxychalcone for ChOMT and 4',7-dihydroxyisoflavone for IOMT), and 500 M adenosyl-l-methionine-S-(methyl-¹⁴C), in 50 l of 250 mM HEPES (pH 7.5), 100 mM NaCl. Reactions were allowed to proceed for 2 h at room temperature after which time the reaction products were extracted into ethyl acetate and applied to a Whatman LK6D silica TLC plate. Chromatograms were developed in ethyl actetate:hexane (50:50, v/v). The products were visualized by autoradiography.

Crystals of ChOMT and IOMT were grown by vapor diffusion in hanging drops containing a 1:1 mixture of protein and crystallization buffer (ChOMT, 12% (w/v) PEG 8000, 0.05 M HEPES (pH 7.5), 0.3 M ammonium acetate, 2 mM DTT at 4 °C ; IOMT, 17% (w/v) PEG 8000, 0.05 M TAPS (pH 8.25), 0.35 M lithium sulfate, 2 mM DTT, 15 °C). Crystals for both proteins grew in space group C2 with one molecule per asymmetric unit. Unit cell dimensions for ChOMT were a = 127.19 Å, b = 53.79 Å, c = 73.55 Å, = 125.55°. IOMT cell dimensions were a = 145.56 Å, b = 50.54 Å, c = 63.82 Å, = 106.69°. Diffraction data were collected from single crystals mounted in a cryoloop and flash frozen in a nitrogen stream at 105 K. All diffraction data were collected at the Stanford Synchrotron Radiation Facility, beamline 9-2 (IOMT data and ChOMT SeMet data) on a Quantum 4 CCD detector and beamline 7-1 (ChOMT−isoliquiritigenin complex) on a 30 cm MAR imaging plate. All images were indexed and scaled using DENZO⁴¹ and the reflections merged with SCALEPACK⁴¹. The ChOMT and IOMT structures were determined using multiple wavelength anomalous dispersion (MAD) phasing on the SeMet substituted protein. Initial heavy atom sites were found with SOLVE⁴². SHARP⁴³ was used to refine the initial sites and to locate additional sites. MAD phases were improved with SOLOMON⁴⁴. Subsequent complexes were solved by the difference Fourier method. All refinements were carried out using CNS⁴⁵. During refinements, structure factors obtained from intensity data were used to generate SIGMAA weighted |2F_o - F_c| and |F_o - F_c| electron density maps with phases calculated from the structure of the in-progress model. Inspection of the electron density maps and model building was performed in O⁴⁶. The quality of all models was assessed using the program PROCHECK⁴⁷. For the ChOMT−isoliquiritigenin, complex 92.6%, 6.4%, 0.7%, and 0.3% of the residues were found in the most favored, the allowed, the generously allowed, and the disallowed regions of the Ramachandran plot, respectively, with a G factor of 0.39. For the IOMT−isoformononetin complex, 91%, 8%, and 1% of the residues were found in the most favored, the allowed, and the generously allowed regions of the Ramachandran plot, respectively, with a G factor of 0.30.

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