Structure-function relationships of wheat flavone O-methyltransferase: Homology modeling and site-directed mutagenesis
- Jian-Min Zhou†1,
- Eunjung Lee†2,
- Francesca Kanapathy-Sinnaiaha1,
- Younghee Park2,
- Jack A Kornblatt3,
- Yoongho Lim2 and
- Ragai K Ibrahim1Email author
© Zhou et al; licensee BioMed Central Ltd. 2010
Received: 1 April 2010
Accepted: 29 July 2010
Published: 29 July 2010
Wheat (Triticum aestivum L.) O-methyltransferase (TaOMT2) catalyzes the sequential methylation of the flavone, tricetin, to its 3'-methyl- (selgin), 3',5'-dimethyl- (tricin) and 3',4',5'-trimethyl ether derivatives. Tricin, a potential multifunctional nutraceutical, is the major enzyme reaction product. These successive methylations raised the question as to whether they take place in one, or different active sites. We constructed a 3-D model of this protein using the crystal structure of the highly homologous Medicago sativa caffeic acid/5-hydroxyferulic acid O-methyltransferase (MsCOMT) as a template with the aim of proposing a mechanism for multiple methyl transfer reactions in wheat.
This model revealed unique structural features of TaOMT2 which permit the stepwise methylation of tricetin. Substrate binding is mediated by an extensive network of H-bonds and van der Waals interactions. Mutational analysis of structurally guided active site residues identified those involved in binding and catalysis. The partly buried tricetin active site, as well as proximity and orientation effects ensured sequential methylation of the substrate within the same pocket. Stepwise methylation of tricetin involves deprotonation of its hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of SAM-methyl groups. We also demonstrate that Val309, which is conserved in a number of graminaceous flavone OMTs, defines the preference of TaOMT2 for tricetin as the substrate.
We propose a mechanism for the sequential methylation of tricetin, and discuss the potential application of TaOMT2 to increase the production of tricin as a nutraceutical. The single amino acid residue in TaOMT2, Val309, determines its preference for tricetin as the substrate, and may define the evolutionary differences between the two closely related proteins, COMT and flavone OMT.
The structural diversity of flavonoid compounds in plants is the result of a number of enzyme-catalyzed substitution reactions . Of these, enzymatic O-methylation is mediated by a family of substrate-specific, position oriented O-methyltransferases (OMTs; EC 2.1.1-). Substrate methylation confers significant changes to the physiochemical properties of methyl acceptor molecules by altering their solubility, reactivity and interaction with cellular targets. Several plant OMTs have been characterized both at the biochemical and molecular levels , and most of the enzymes involved in flavonoid biosynthesis, including flavonoid OMTs, were recently studied at the structural level .
TaOMT2 and its analogues constitute a distinct flavone OMT gene family that has recently been characterized in a number of cereal species, including rice , barley and maize , as well as a few other graminaceous species (Additional file 1). In fact, alignment of their amino acid sequences shows that the putative residues involved in substrate binding and catalysis are strictly conserved . Several members of this OMT family have previously been mis-annotated as caffeic acid/5-hydroxyferulic acid 3/5-OMTs (COMTs), possibly because of their high amino acid sequence similarity/identity to flavonoid OMTs and the structural analogy between their phenylpropanoid moiety and the flavonoid B-ring with its 3-C side chain (Fig. 1). In fact, TaOMT2 accepts 5-hydroxyferulic acid (5HFA) as an alternate substrate with ~78% relative activity, but with > 2-fold lower affinity and a 4-fold lower turnover than its preferred substrate, tricetin .
There are > 100 functionally diverse MTs that have been structurally characterized [3, 14], including several plant OMTs [15–17] that belong to Class I structures. Regardless of the level of overall sequence identity, these enzymes share a common conserved S-adenosyl-L-methionine (SAM) binding domain with a core α/ß Rossman fold  and a unique α-helical cap that forms the top of the active site cavity. Except for the mammalian catechol OMT  and caffeoyl CoA OMT  whose reaction mechanisms invoke a divalent cation-dependent process, that of other OMTs is thought to proceed via direct transfer of the SAM-methyl group to the substrate with inversion of symmetry in a SN2-like mechanism  and the removal of a proton before, during or after methyl transfer . The fact that TaOMT2 catalyzes the methylation of three different substrates: tricetin, selgin and tricin (Fig. 1) raised the question as to whether this enzyme protein possesses one substrate binding pocket for the three substrates, or three different sites.
To circumvent the difficulties we encountered in obtaining high quality crystals, we resorted to homology modeling of TaOMT2 using Medicago sativa MsCOMT  as a template; the two proteins share 63% sequence identity. In addition, the fact that the reaction product of COMT, 5HFA, is structurally similar to ring B and its 3-C side chain of selgin, the first methylated intermediate of TaOMT2 (Fig. 1), suggests a close evolutionary relationship between these two enzyme proteins.
The aim of this article was to study the architecture of the active site of TaOMT2 in relation to the sequential methylation of tricetin, and propose a mechanism whereby the single protein can catalyze three successive methylations. Furthermore, the proposed structural model allowed us to investigate the role of Val309 in defining the substrate preference of TaOMT2.
Most phenolic compounds used in this study were from our laboratory collection. Tricetin was purchased from Indofine Chemical Company (Hillsborough, NJ). Its methylated derivatives were synthesized by the condensation of 2,4,6-trihydroxyacetophenone with a suitably substituted benzaldehyde to give rise to the corresponding flavanone, followed by dehydrogenation with iodine and NaOAc . Identity of the methylated products was verified by NMR and mass spectroscopy. [3H]SAM (80 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). Unless otherwise specified, all other reagents were of analytical grade.
Homology modeling and molecular docking
The 356 amino acid sequence of TaOMT2 was obtained from GenBank (Accession number ABB03907). For homology modeling, the 2.2 Å resolution X-ray structure  of MsCOMT (1KYZ.pdb) was used as the template. The sequences of the two proteins are 63% identical, their substrates are structurally analogous, and the reactions they catalyze are methylations of similar compounds mediated by SAM as the co-substrate. Based on the above considerations, there is a good reason to believe that MsCOMT is a good template for TaOMT2. There are three chains (A, E and C) that are readily seen in the unit cell of MsCOMT even though the protein in solution, like TaOMT2, is probably a dimer . The A and E chains form a tight dimer interface, whereas the dimeric complement to the C chain is not visible. It is essential to point out that even though the dimer of TaOMT2 is undoubtedly the functional unit in solution, monomers are also catalytically active. All three chains contain the reaction products S-adenosyl-L-homocysteine (SAH) and 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid.
The modeled three dimensional structure of TaOMT2 was built using SWISS-MODEL http://swissmodel.expasy.org. The template consisted of the 360 residues visible in the crystal structure of the MsCOMT E-chain; the polyhistidine tag and the first five residues are not visible. The monomeric structure of TaOMT2, 356 residues omitting the first seven residues, was constructed using the MsCOMT-E chain as a template. All molecular modeling experiments were done with SYBYL (Tripos, St. Louis, MO; http://www.tripos.com), except for calculations of the solvated protein (see below). The secondary structure of the modeled TaOMT2 was determined using the method of Kabsch and Sander provided in Sybyl. Sybyl calculations were carried out on an Intel Core 2 Quad Q6600 (2.4 GHz) Linux PC workstation. The initial structure obtained from SWISS-MODEL was subjected to energy minimization (EM) and molecular dynamics (MD) . Gasteiger-Huckel charge was used to determine the 3-D structures of the ligands: tricetin, selgin, and tricin, which were subjected to EM by the conjugate gradient algorithm using the Tripos Force Field; these are the most probable structures in solution but are probably distorted in the actual protein. For solvation, the protein was embedded in a 5Å shell of 10,942 water molecules, and the protein-water complex was transferred into Sybyl.
Since MsCOMT contains SAH as a co-factor, its location in TaOMT2 was determined by superimposing the TaOMT2 on MsCOMT. Tricetin, selgin, and tricin were docked manually into TaOMT2 using FlexX Single Receptor Module in Sybyl. Residues Met123, Asn124, Phe169, Met173, Val309, Ile312, Met313 and Asn317 were assigned as the binding site for docking as reported for MsCOMT . The selection radius for automatic docking was 6.5 Å, and the docking process was iterated 30 times per ligand. The final structure was chosen on the basis of the docking score and presumed to represent the spatially correct docking. The docking score for tricetin ranged between -19.03 and -12.48, and the docking model with a score of -15.71 was fitted to the binding site and used in this study.
The homodimeric structure of TaOMT2 was generated using the Biopolymer Module-Align Structure in the Sybyl program. 1KYZ.PDB contains no hydrogens; they were introduced into our modeled TaOMT2 using the H-bonds Module in Sybyl. The cutoff for H-bonds was a minimum of 3 Å between appropriate atoms. The size of the binding pocket was measured using the Docking Simulation Module in Sybyl. The secondary structures of TaOMT2 as well as the mutant proteins were determined using Biopolymer Display Module in Sybyl. The structural models generated in this study were viewed in PyMOL http://pymol.sourceforge.net or Sybyl.
TaOMT2 cDNA mutants were prepared using QuickChange site-directed mutagenesis kit (Stratagene, CA) and sequenced before subcloning into the expression vector. Since the MsCOMT cDNA clone was not available, the putative Medicago truncatula MtCOMT EST cDNA clone (NF035B09NR, GenBank Accession No. AW686202; Noble Foundation, Inc., Ardmore, OK), was used instead. MsCOMT and MtCOMT share 98% identity at the amino acid level. The wild type cDNAs in vector pET200/D-TOPO were used as templates for PCR.
Primers used for generating the mutants are shown in Additional file 2.
In vitro protein expression, enzyme assays and kinetic analyses
After sequencing, the mutant plasmids were transformed into E. coli BL21 (DE3) cells (EMD, Darmstadt, Germany) for protein expression. The recombinant proteins were purified to near homogeneity by affinity chromatography on a Ni-NTA column (Qiagen, Mississauga, ON). SDS-PAGE was used to check purity of the recombinant proteins, and the highly purified fractions were stored at 4°C until used.
The standard enzyme assays were performed as previously described  using 50 μM of the phenolic substrate, 50 μM SAM containing 25 nCi of radioactive label, and 0.1 to 2.0 μg of the affinity-purified recombinant protein.
Kinetic analyses were performed using 1.8 μg of the affinity-purified proteins with a saturating concentration of SAM, containing 25 nCi of radioactivity, and varied concentrations (5 μM to 50 μM) of the phenolic substrates. Assays were performed in triplicates and were repeated twice. Lineweaver-Burk plots were applied for the determination of K m , Vmax and Kcat values .
Structural model of TaOMT2
Substrate binding by TaOMT2
Modeling data for TaOMT2*
Distance from SAH-S(Å)
-Asp263γCOO- - 3'OH → 1.53
-His262δNH - 5'OH →2.08
-Glu322δCOO- - 5'OH → 2.11
-Asn124δNH2 -5O → 1.74
-Gly305CO - 7OH → 2.10
-Asn124δNH2 - 4O →1.87
-Asn124δO - 5OH → 2.0
-Trp259CO - 4'OH → 2.61
-Asp263γCOO- - 5'OH → 2.30
-Asp263γCOO- - 4'OH → 2.05
-Gly305CO-7OH → 1.92
-Asn124δNH2 -4O → 1.60
-Asn124δO-5OH → 2.13
-Asp263γCOO--4'OH → 1.76
Characterization of mutant proteins
Significance of the putative residues of TaOMT2 involved in binding ans/or catalysis and changes in the properties of their mutant proteins1
Properties of mutant proteins
Important residue for substrate binding; forms H-bonds with All OH groups of tricetin
Severe loss of activity is due to a conflict between the catalytic His262-imidazole group and Glu-CH2
Ile263 can not form a H-bond with 3'-OH group
Slight decrease in activity due to a decreased electronegativity of Asn-N compared to Asp-O, that affects charge transfer to tricetin-OH groups
H-bonds with tricetin 4'-OH; forms an H-bonding network with neighboring residues, esp. E290-COO- and H262-backbone-NH
Loss of activity is due to the fact that Ile can not form a H-bond with the 4'-OH of tricetin
This mutation results in a more extensive H-bonding that hinders charge transfer and affects B-ring flexibility
H-bonds with selgin 4'-OH and forms a H-bonding network with neighboring residues
Ala can maintain the H-bonding network between Trp259, Glu290 and His262, wheras Tyr cannot
H-bonds with tricetin 5'-OH.
Loss of charge or a change in the side chain affects H-bonding with the neighboring residues, especially His262
H-bonds with selgin 7-OH; important residue for substrate positioning
Change in polarity is less effective than chain length on catalytic activity.
Loss of activity due to loss of H-bonding with the amide group of the neighboring Asn348
H-bonds with O-4/O-5 of all substrates in order to orient them to the most favorable position
Resuled in a decreased substrate binding but not protein folding. Both mutations disrupt H-bonding with 5-OH group of tricetin
Putative catalytic base involved in deprotonation of tricetin hydroxyl groups
Resulted in almost complete loss of protein expression; all mutant proteins lack imidazole ring that is critical for proton flow among His262, Asp263 and the substrate
Replacement of Asp263 with either glutamic acid or isoleucine resulted in mutant proteins that exhibited severe loss of activity, indicating its critical role in substrate binding (Table 2). Likewise, substitution of His262 with arginine, leucine or phenylalanine abolished the catalytic activity of their mutant proteins. In fact, mutation of the His residue resulted in almost complete loss of protein expression and enzyme activity (Additional file 4A and Table 2). These results indicate the necessity of the imidazole ring for electron flow between Asp263 and His262. Kinetic analyses of mutant proteins with significant relative enzyme activity exhibited 2- to 3-fold lower affinity for tricetin, 70 to 90% reduced reaction velocity and 80 to 95% lower catalytic efficiency compared to the wild-type protein (Table 2). Taken together, these results indicate that changes in the H-bond network, charge transfer and/or size of the target residue have considerable effects on substrate binding and, consequently, the catalytic activity of the mutant proteins. However, it is interesting to note that HPLC analysis of the enzyme reaction products of those mutant proteins showed no significant differences in the product ratios between the wild-type and mutants, where tricin always constituted the predominant enzyme reaction product, with a trace of trimethyltricetin, but no selgin as would be expected (Additional Fig. 4B). Furthermore, mutant proteins with significant OMT activity can methylate selgin to tricin, and the latter to trace amounts of trimethyltricetin (data not shown), thus maintaining the sequential methylation characteristic of the wild-type protein.
Proposed reaction mechanism for TaOMT2
Substrate specificity of TaOMT2
Kinetic parameters of wild type TaOMT2 and mutant V309I for tricetin and 5HFA as substratesa
59.51 ± 0.79
110 ± 7.0
270.98 ± 11.51
210 ± 6.0
101.08 ± 9.68
35 ± 8.0
18.24 ± 1.72
59 ± 2.0
A 3-D structural model of wheat flavone OMT, TaOMT2, was generated based on the crystal structure of caffeic acid/5-hydroxyferulic acid MsCOMT . The high amino acid sequence identity, superposition of the backbones and the conservation of residues near the active sites (Additional file 3) provided the basis for a model to study the structure of this protein.
In contrast with other plant OMTs which mediate single methyl transfers, TaOMT2 catalyzes the sequential methylation of tricetin, by virtue of the unique architecture and disposition of its active sites (Fig. 3 and Figs. 4A to 4C). The fact that selgin does not accumulate neither in planta (unpublished data) nor in vitro enzyme assays [ and this work] indicates that selgin, the first methylated intermediate of tricetin methylation, does not leave the active site until completion of sequential methylation. Such a mechanism excludes channeling of the methylated intermediates from one active site to another, and is in agreement with a random enzyme reaction mechanism . The stepwise methylation of tricetin starts at the 3'-hydroxyl group which is the preferred (meta) position for methylation because of its highest negative electron density , as previously shown with the classical examples: rat liver catechol OMT  and lignin monomers COMT , followed by methylation at the 5'-position, through re-orientation of the first methylated intermediate, selgin. However, the significantly low level of methylation at the 4'-hydroxyl group of tricin (Additional file 4B) may be explained by (a) the weak binding affinity of the enzyme for tricin (data not shown), (b) the low negative electron density of this para hydroxyl group , (c) the steric hindrance caused by introduction of a bulky methyl group into the 4'-position of tricin, and/or (d) the competitive inhibition of the enzyme reaction by its final product, 3',4',5'-trimethyltricetin (ca < 5 μM) (unpublished data). In fact, the results of enzyme assays and HPLC analysis of the reaction products analysis support these assertions [4, 28].
The fact that a single amino acid residue, Val309 in TaOMT2 and Ile316 in MsCOMT, determines the preference for their respective substrates, tricetin and 5HFA (Table 3) is remarkable. Such single amino acid polymorphism [30–32] may define the evolutionary differences between the two closely related phenylpropanoid and flavonoid OMTs, which resulted in mis-annotation of several members of the latter OMT family . Several monocotyledonous flavone OMTs, as well as the Arabidopsis flavonol OMT1 (AtOMT1), share this common residue (valine), whereas it is replaced by either leucine or isoleucine in three COMTs: TaOMT4, MsCOMT and MtOMT1 (Additional file 1). Val is a small aliphatic residue and has one less methylene group than either leucine or isoleucine. It is located near the gate of the substrate binding pocket. Replacement of valine with isoleucine or leucine alters the volume of the binding pocket, thus changes the substrate preference of the enzyme. Furthermore, the natural occurrence of tricin in the forage crops, M. sativa  and M. truncatula , represents an example of the competition between both groups of OMTs involved in the methylation of lignin monomers  and the flavone, tricetin.
Tricin has been credited for its multifunctional properties and health promoting effects , including potent inhibition of expression and activity of cyclooxygenase enzymes, growth inhibition of human malignant breast tumor cells and colon cancer cells [37, 38] and reducing the numbers of intestinal adenomas , among others. The structure-function relationships of TaOMT2 reported here provide the basis for the enzymatic synthesis of tricin. Furthermore, the molecular model indicates that both Met313 and Asn317 lie within 3 to 4Å from the 4'-hydroxyl group of tricetin (data not shown), that may be involved in the 4'-O-methylation step. It will be interesting to investigate whether mutations of these two residues can alter the ratio of tricetin methylation products towards the metabolic engineering and optimization of tricin production in wheat .
TaOMT2 catalyzes the sequential methylation of the flavone tricetin. Substrate binding is mediated by an extensive H-bond network and van der Waals interactions which sequester both the substrate and co-substrate. Methylation is proposed to proceed by deprotonation of the hydroxyl groups via the His262-Asp263 pair, followed by the nucleophilic attack of the SAM-methyl group within the same active site. The sequence of methylation starts at the 3'-hydroxyl group, followed by the 5'-, then the 4'-hydroxyls through re-orientation of intermediates and possible conformational changes of the surrounding residues. Val309 defines the preference of TaOMT2 for its substrate, tricetin.
This work was supported by discovery grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada to JAK and RKI, and by grants KRF-2006-005-J03402 (KRF) and Biogreen 21 (RDA) to YL.
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