Biochemical characterization of a putative wheat caffeic acid O-methyltransferase

Plant Physiology and Biochemistry 47 (2009) 322–326

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Biochemical characterization of a putative wheat caffeic acid O-methyltransferase Jian-Min Zhou a, Yong Weon Seo b, Ragai K. Ibrahim a, * a b

Plant Biochemistry Laboratory, Concordia University, Montre´al, Que´bec, Canada H4B 1R6 Laboratory of Plant Molecular Breeding, Korea University, Seoul 136-701, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 October 2008 Accepted 26 November 2008 Available online 7 December 2008

A wheat (Triticum aestivum L., near isogenic line of Hamlet) O-methyltransferase (OMT) was previously reported as a putative caffeic acid OMT (TaCOMT1), involved in lignin biosynthesis, based on its high sequence similarity with a number of graminaceous COMTs. The fact that the putative TaCOMT1 exhibits a significantly high sequence homology to another recently characterized wheat flavone-specific OMT (TaOMT2), and that molecular modeling studies indicated several conserved amino acid residues involved in substrate binding and catalysis of both proteins, prompted an investigation of its appropriate substrate specificity. We report here that TaCOMT1 exhibits highest preference for the flavone tricetin, and lowest activity with the lignin precursors, caffeic acid/5-hydroxyferulic acid as the methyl acceptor molecules, indicating that it is not involved in lignin biosynthesis. We recommend its reannotation to a flavone-specific TaOMT1 that is distinct from TaOMT2. Crown Copyright Ó 2008 Published by Elsevier Masson SAS. All rights reserved.

Keywords: Flavone-specific O-methyltransferase Biochemical characterization Reannotation Tricetin Triticum aestivum L. Wheat

1. Introduction Flavonoid compounds play important roles in plant growth and development, and act as signaling molecules in UV protection, reproduction, pathogenesis and symbiosis, to mention only a few ([16] and references therein). Enzymatic O-methylation is catalyzed by an extensive family of O-methyltransferases (OMTs, EC 2.1.1.6-) and involves the transfer of the methyl group of S-adenosyl-Lmethionine (AdoMet) to a specific hydroxyl group of an acceptor molecule with the concomitant formation of its methyl ether derivative and S-adenosyl-L-homocysteine (AdoHcy), as products [8]. In addition to its essential role in the biosynthesis of lignin precursors [11], O-methylation of phenolic compounds reduces the reactivity of their phenolic hydroxyl groups and hence, their carcinogenicity [22] and increases their lipophilicity and antimicrobial activity [5]. Of the >200 plant OMT entries in the databases only a few have been biochemically characterized and annotated [10]. However, several of these were reported as putative caffeic acid OMTs (COMTs) solely on the basis of their amino acid sequence homology to previously reported, although not necessarily well characterized, OMTs (e.g. [3]). It Abbreviations: AdoMet, S-adenosyl-L-methionine; OMT, O-methyltransferase; CA, caffeic acid; 5HFA, 5-hydroxyferulic acid; HPLC, high-performance liquid chromatography; ORF, open reading frame; TLC, thin-layer chromatography. * Corresponding author. Department of Biology, Concordia University, 7141 Sherbrooke Street West, Montre´al, Que´bec, Canada H4B 1R6. Tel.: þ1 514 848 2424x3399; fax: þ1 514 848 2881. E-mail address: [email protected] (R.K. Ibrahim).

is well known that a high sequence homology among genes may not necessarily imply similarity of function [4], since a difference of one or a few amino acids between two gene products may alter their substrate preference [6,7,17]. For example, the Arabidopsis thaliana OMT1 (accession no. U70424) was formerly reported to encode a putative caffeic acid/5-hydroxyferulic acid OMT (COMT) based on a high sequence similarity with other plant COMTs [19]. However, expression of this gene in a heterologous system and biochemical characterization of its gene product identified it as a flavonol 30 -OMT [13]. We describe here the biochemical characterization of wheat (Triticum aestivum) OMT that was previously reported as a putative caffeic acid OMT (TaCOMT1) based on its high sequence similarity with other graminaceous enzymes [9]. TaCOMT1 exhibits 96% similarity and 93% identity with another recently characterized TaOMT2. The latter catalyzes the sequential methylation of the flavone tricetin to its monomethyl(selgin), dimethyl-(tricin) and trimethyl derivatives [20]. The significantly high sequence identity (93%) between both proteins, and the similarity of the amino acid residues involved in binding and catalysis, prompted us to biochemically characterize and reannotate TaCOMT1 according to its preferred substrate. 2. Materials and methods 2.1. Chemicals Most of the flavonoid compounds used in this study were from our laboratory collection, except tricetin which was purchased from

0981-9428/$ – see front matter Crown Copyright Ó 2008 Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2008.11.011

J.-M. Zhou et al. / Plant Physiology and Biochemistry 47 (2009) 322–326

Indofine Chemical Company (Hillsborough, NJ). The methylated tricetin derivatives, which are not available commercially, were synthesized using, as starting chemicals, the appropriately substituted acetophenones and benzaldehydes corresponding to the B-rings of selgin, tricin and trimethyltricetin, followed by C-ring oxidation [2]. Their purity and identity were verified by HPLC analysis, and UV- and 1H NMR spectroscopic methods. [14C]AdoMet (55 mCi/mmol) and [3H]AdoMet (80 Ci/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO), and unlabeled AdoMet from Sigma (Oakville, ON). Protein quantification reagents and 40% acrylamide/bis solution (37.5:1) were purchased from Bio-Rad (Mississauga, ON). Unless otherwise specified, all other chemicals were of analytical grade. 2.2. Isolation of TaCOMT1 and its expression in Escherichia coli The open reading frame (ORF) sequence of TaCOMT1 cDNA [9] was confirmed in pBluescript SK phagemid, amplified and subcloned into the expression vector pET200/D-TOPO using the primers: TaCOMT1F (50 -CACCATG GGC TCC ACC GCA GCCGAC) and TaCOMT1R (50 -CTA CTT GGT GAA CTC GAT GGC). To reinforce the reliability of TaOMT1 cDNA ORF sequence, AccuPrimePfx DNA Polymerase (Invitrogen, Carlsbad, CA) was used in the PCR reaction following the manufacturer’s instructions. The recombinant protein was expressed in E. coli BL21 (DE3) cells (EMD, Darmstadt, Germany) and purified to near homogeneity by affinity chromatography on an Ni-NTA column (Qiagen, Mississauga, ON). SDS-PAGE was used to monitor purity of the protein preparations, and the highly purified fraction was desalted on PD-10 columns and stored at 4  C until used. 2.3. OMT assay and identification of reaction products Enzyme assays were performed as previously described [20] using 50 mM of the phenolic substrate, 50 mM AdoMet containing 25 nCi of the 14C-label, and 0.1–2.0 mg of the affinity-purified recombinant protein. The reaction products from several radiolabeled enzyme assays were combined, lyophilized, and re-dissolved in 20 mL of MeOH for TLC on Silica gel 60 F254 plates (Merck, Darmstadt) in toluene–dioxane–acetic acid (18:5:1, v/v/v) as the solvent system. Control assays were performed in absence of the phenolic substrate or with boiled enzyme for background correction, and all assays were conducted in triplicates. Identity of the methylated reaction products was confirmed by co-chromatography with reference compounds, visualization under UV light, and autoradiography on X-ray film. Semi-preparative enzyme assay incubations were also carried out using nonlabeled AdoMet, and the methylated reaction products were analyzed by HPLC with a Millennium HPLC System (Waters, Milford, MA), using a Waters YMC-PackPro C18 column (150  4.6 mm I.D., S-5mM), and a linear gradient consisting of 40–90% MeOH in 1% acetic acid for 30 min at a flow rate of 1.0 mL/ min. This gradient was maintained for a further 10 min before returning to the initial conditions. Identity of the reaction products was confirmed by comparison of their retention times (Rt) and UV-absorption maxima with those of reference compounds. Kinetic analyses were performed using 0.1 mg of the affinitypurified OMT proteins with a saturating concentration of AdoMet (50 mM), containing 25 nCi of the [3H] label, and varied concentrations (5–100 mM) of the flavonoid substrate. Assays were performed in triplicates, and Lineweaver–Burk plots [14] were applied for the determination of Km and Vmax values. 2.4. Homology modeling In order to gain an insight on the degree of structural similarity between TaCOMT1 and TaOMT2, molecular modeling of the latter

323

protein was carried out using the Medicago sativa caffeic acid/5hydroxyferulic acid 3/5-O-methyltransferase (MsCOMT; pdb, 1kyz) as a template [23], as was previously described [18]. 3. Results and discussion 3.1. Characterization of recombinant TaCOMT1 The full-length of TaCOMT1 cDNA is 1434 bp, and contains an ORF of 1080 bp that encodes a 360 amino acid polypeptide, with a calculated molecular mass of 38.9 kDa and a theoretical isoelectric point of 5.75. The TaCOMT1 protein harboring an His-tag at its N-terminal was expressed in E. coli BL21 (DE3) cells, and the recombinant protein purified to near homogeneity on an Ni-NTA affinity chromatography, as verified by SDS-PAGE (Fig. 1). Its apparent molecular mass corresponds to the combined Mr of the protein subunit (38.9 kDa) and the His-tag (3.0 kDa). The putative molecular mass obtained and the lack of requirement for Mg2þ for enzymatic activity are representative of Class II OMT proteins. The purified recombinant protein is quite stable, with almost no loss of its catalytic activity when stored at 4  C for 3–4 weeks. 3.2. Substrate specificity of TaCOMT1 In order to study the substrate preference of TaCOMT1, the affinity-purified recombinant protein was assayed, in triplicate, with a variety of potential phenolic and flavonoid substrates. The enzyme exhibits the highest (100%) relative activity with the flavone tricetin followed, in a decreasing order, by luteolin (72%), quercetin (51%) and eriodictyol (35%), as substrates (see structures in Fig. 2). The fact that CA and 5HFA exhibited low methylating activity (ca. 10% and 15%, respectively, of the control) rules out the involvement of TaCOMT1 in lignin biosynthesis. Furthermore, it exhibited relatively low (<20%) enzyme activity with other flavonoid substrates tested, such as taxifolin, quercetagetin, gossypetin and myricetin (the flavonol analog of tricetin). TaCOMT1 exhibits a Km value of 4.07 mM and a Vmax of 11.64 pkat mg1 for tricetin methylation. The enzyme reaction products were characterized by their retention times and UV-absorption maxima on HPLC (Fig. 3A), as well as co-chromatography with authentic samples and autoradiography on TLC (Fig. 3B). This indicates that TaCOMT1 is a flavone-specific OMT that exhibits a preference for flavones over flavonols as substrates, and is therefore re-annotated to TaOMT1.

Fig. 1. SDS-PAGE profiles of the recombinant TaOMT1 (TaCOMT1) protein during affinity purification on an Ni-NTA column. Lanes: 1, Mr ladder of standard proteins; 2, E. coli lysate; 3, flow-through; 4, buffer wash; 5, Ni-eluate; 6, PD-10-desalted protein. SDS-PAGE was performed using 4% acrylamide in the stacking gel and 12% acrylamide in the running gel.

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OH R1 3' 3

OH 4 5

HO R2 6

8

O

A

C

OH

O

B

OH 4'

2 3

OH

O Phenylpropanoids R1, OH; R2, H = Caffeic acid R1, OMe, R2, OH = 5-OH-ferulic acid

Flavonols :Quercetin (as above) 2,3-Dihydro- = Taxifolin 6-Hydroxy- = Quercetagetin 8-Hydroxy- = Gossypetin 3',4',5'-TriOH- = Myricetin OH 3'

A

C

OH 4'

B 5'

O

HO

2

R

3

OH

(unpublished data), coupled with the difficulty in introducing a bulky methyl group to a 40 -hydroxy-30 ,50 -dimethoxy-substituted B-ring may explain the low relative enzyme activity with tricin as substrate.

O

Flavones R, H = Luteolin R, H; 2,3-Dihydro- = Eriodictyol R, OH = Tricetin Fig. 2. Structural formulae of the phenolic substrates used in this study.

As was recently shown with wheat TaOMT2 [20] and rice OsOMT1 [21], TaOMT1 (TaCOMT1) catalyzes the sequential methylation of tricetin to its monomethyl-(selgin), dimethyl-(tricin) and trimethyl derivatives, although tricin appears to be the major enzyme reaction product (Fig. 3A and B). These results are consistent with the fact that the TaCOMT1 recombinant protein utilizes both tricetin (100% relative activity) and selgin (87.4%), as compared with tricin (3.6%), as methyl acceptors. In fact, trimethyltricetin acts as a competitive inhibitor of the enzyme reaction

3.3. Structure–function relationship In spite of the significantly high sequence homology (96% similarity, 93% identity) between TaCOMT1 and TaOMT2, they are obviously two distinct enzymes with discernible features. First, their amino acid sequence alignment (Fig. 4) shows that TaCOMT1 contains four extra amino acid residues at positions 4–7, as well as eight conservative and nine nonconservative amino acid replacements. Second, although they share their highest preference for the flavone tricetin as the methyl acceptor, their behaviour towards CA/ 5HFA as well as other substrates is evidently different. TaCOMT1 exhibits lowest enzyme activity with phenylpropanoids, whereas TaOMT2 accepts 5HFA, but with a nearly 4-fold turnover value lower than that for tricetin, as substrates [20]. Third, unlike TaCOMT1, TaOMT2 exhibits higher enzyme activities (relative to tricetin) with luteolin (89%), quercetin (80%), eriodictyol (75%), quercetagetin (67%), taxifolin (64%), gossypetin (54%) and myricetin (25%) as substrates [20], indicating that they are two distinct OMTs. However, both the newly annotated TaOMT1 (formerly TaCOMT1 [9]) and TaOMT2 may be considered ‘homologs’ considering the hexaploid nature of wheat and the fact that they are derived from two different wheat cultivars. Homology-based modeling of TaOMT2, using the structurally determined MsCOMT (pdb, 1kyz) as a template [23], indicates the involvement of several amino acid residues in binding of the ligands: tricetin (Asn124, Trp259, Asp263, Glu290 and Glu322,), selgin (Asn124, Trp259, Asp263 and Gly305) and tricin (Asn124, Asp263 and Gly305), as indicated by several hydrogen bonds within 1.60–2.61 Å between these ligands and their neighboring residues (Yoongho Lim, personal communication). An example of the residues involved in binding of tricetin is shown in Fig. 5. The fact that these amino acid residues as well as those involved in

Fig. 3. Characterization of a 30-min enzyme reaction product of recombinant TaCOMT1 assayed with tricetin as the substrate using: (A) HPLC and (B) autoradiography following cochromatography of the reaction products with reference compounds on TLC. HPLC peaks and radioactive spots were characterized as described in Section 2 as (1) tricetin 30 monomethyl- (selgin); (2) 30 ,50 -dimethyl-(tricin); (3) 30 ,40 ,50 -trimethyl ether, derivatives.

J.-M. Zhou et al. / Plant Physiology and Biochemistry 47 (2009) 322–326

325

TaOMT2 TaCOMT1 MsCOMT

MGS-------IAAGADEDACMYALQLVSSSILPMTLKNAIELGLLETLMAAG-GKFLTPA 52 MGST---AADMAASADEEACMYALQLVSSSILPMTLKNAIELGLLETLVAAG-GKLLTPA 56 MGSTGETQITPTHISDEEANLFAMQLASASVLPMILKSALELDLLEIIAKAGPGAQISPI 60

TaOMT2 TaCOMT1 MsCOMT

EVAAKLPSAANPEAPDMVDRMLRLLASYNVVSCRTEDGKDGRLSRRYGAAPVCKYLTPNE 112 EVAAKLPSTANPAAADMVDRMLRLLASYNVVSCTMEEGKDGRLSRRYRAAPVCKFLTPNE 116 EIASQLP-TTNPDAPVMLDRMLRLLACYIILTCSVRTQQDGKVQRLYGLATVAKYLVKNE 119

TaOMT2 TaCOMT1 MsCOMT

DGVSMSALALMNQDKVLMESWYYLKDAVLDGGIPFNKAYGMSAFEYHGTDPRFNRVFNEG DGVSMAALALMNQDKVLMESWYYLKDAVLDGGIPFNKAYGMSAFEYHGTDPRFNRVFNEG DGVSISALNLMNQDKVLMESWYHLKDAVLDGGIPFNKAYGMTAFEYHGTDPRFNKVFNKG * MKNHSIIITKKLLESYKGFEGLGTLVDVGGGVGATVAAITAHYPTIKGINFDLPHVISEA MKNHSIIITKKLLEVYKGFEGLGTIVDVGGGVGATVGAITAAYPAIKGINFDLPHVISEA MSDHSTITMKKILETYTGFEGLKSLVDVGGGTGAVINTIVSKYPTIKGINFDLPHVIEDA

TaOMT2 TaCOMT1 MsCOMT TaOMT2 TaCOMT1 MsCOMT TaOMT2 TaCOMT1 MsCOMT TaOMT2 TaCOMT1 MsCOMT

PPFPGVTHVGGDMFQKVPSGDAILMKWILHDWSDEHCATLLKNCYDALPAHGKVVLVECI QPFPGVTHVGGDMFQKVPSGDAILMKWILHDWSDEHCATLLKNCYDALPAHGKVVLVECI PSYPGVEHVGGDMFVSIPKADAVFMKWICHDWSDEHCLKFLKNCYEALPDNGKVIVAECI * * * LPVNPEATPKAQGVFHVDMIMLAHNPGGRERYEREFEALAKGAGFAAMKTTYXYANAWAI LPVNPEATPKAQGVFHVDMIMLAHNPGGRERYEREFEALAKGAGFKAIKTTYIYANAFAI LPVAPDSSLATKGVVHIDVIMLAHNPGGKERTQKEFEDLAKGAGFQGFKVHCNAFNTYIM * EFTK-- 356 EFTK-- 360 EFLKKV 365

172 176 179 232 236 239 292 296 299 352 356 359

Fig. 4. Amino acid sequence alignment of TaCOMT1 (accession no. AAP23942), TaOMT2 (accession no. DQ223971) and MsCOMT (accession no. AAB46623). Amino acid residues proposed to be involved in substrate binding of TaOMT2 (Y. Lim, personal communication) are shown in red; those involved in catalysis of MsCOMT [23] are shown in blue. Stars in green refer to residues involved in both binding and catalysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

catalysis [23] are also conserved among TaCOMT1 and TaOMT2 sequences (Fig. 4) indicates the close similarity in structure and function of the two latter proteins. In contrast with the spacious active site of MsCOMT that has been related to its promiscuity [23], that of TaOMT2, as would also be expected for TaOMT1 (TaCOMT1), is quite compact (data not shown) that would allow the sequential methylation of tricetin within the same pocket, and without the need for channeling of the methylated intermediates. Several OMTs have been reported in the literature that were either incompletely characterized, or erroneously annotated based solely on amino acid sequence comparisons. For example, the characterization of the two COMT sequences from sorghum [1] and sugarcane [15] was based solely on sequence comparisons with the

incompletely characterized maize COMT [3]. The ryegrass COMT1 [12] was tested only with CA, to the exclusion of any of the flavonoid compounds. In fact, the ryegrass, sorghum and sugarcane OMTs exhibit significantly high identity (78–88%) and similarity (85–93%) to both TaCOMT1 and TaOMT2. This emphasizes the need to conduct proper biochemical characterization of OMT gene products for their preferred methyl acceptor molecules, especially those encoding the methylation of phenylpropanoid and/or flavonoid compounds, before proper annotation can be assigned. The distinction between phenylpropanoid and flavonoid OMTs represents a real dilemma to plant biochemists as to their correct annotation. This is partly because of the structural similarity between the phenylpropanoid moiety and the B-ring and 3-C side chain of flavonoids (Fig. 2) and the high sequence homologies among COMTs and flavonoid OMTs (Fig. 4). It has also been shown that a difference of as little as one or a few amino acids near the active site can modify the substrate preferences of OMTs [6,7,17]. It is imperative, therefore, that previously characterized proteins as putative COMTs be re-examined for their substrate preferences, coupled with a rigorous characterization of the enzyme reaction products, if erroneous annotation of new OMT genes is to be avoided. 4. Conclusions TaCOMT1, previously reported as a putative caffeic acid OMT [9], is being re-annotated as a flavone-specific TaOMT1 with highest preference for the flavone, tricetin as the methyl acceptor molecule that is quite distinct from its homolog TaOMT2. Its reannotation was based on substrate specificity studies and homology-based molecular modeling using MsCOMT [23] as the template protein. This emphasizes the need to biochemically characterize OMTs before assigning their appropriate functions.

Fig. 5. Stereoview of the modeled TaOMT2 structure showing the amino acid residues involved in binding of tricetin (right) in presence of the cosubstrate, AdoMet (left). Hydrogen bonds are shown in yellow-dotted lines. Add 2 for the real numbers of the indicated amino acid residues (Y. Lim, personal communication). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Acknowledgements This work was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada to R.K.I., and partially supported by the Technology Development Program for

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Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea to Y.W.S. We wish to thank Dr. Y. Fukushi, Hokkaido University, Japan, for the synthesis of the tricetin methyl ether derivatives, and Professor Yoongho Lim, Dept. of Bioscience and Biotechnology, Konkuk University, S. Korea, for molecular modeling of TaOMT2 and production of Fig. 5. References [1] S. Bout, W. Vermerris, A candidate-gene approach to clone the sorghum Brown midrib gene encoding caffeic acid O-methyltransferase, Mol. Genet. Genomics 269 (2003) 205–214. [2] W.L. Chan, Y.C. Lin, W.H. Zhang, P.L. Tang, Y.S. Szeto, One-step synthesis of polyhydroxyflavanones from hydroxyacetophenones and hydroxybenzaldehydes, Heterocycles 43 (1996) 551–554. [3] P. Collazo, L. Montoliu, P. Puigdome`nech, J. Rigau, Structure and expression of the lignin O-methyltransferase gene of Zea mays L. Plant Mol. Biol. 20 (1992) 857–867. [4] J.A. Eisen, M. Wu, Phylogenetics analysis and gene functional predictions: phylogenomics in action, Theor. Popul. Biol. 61 (2002) 481–487. [5] C.J. French, M. Elder, F. Leggett, R.K. Ibrahim, G.H.N. Towers, Flavonoids inhibit mosaic virus infectivity, Can. J. Plant. Pathol. 13 (1991) 1–6. [6] S. Frick, T.M. Kutchan, Molecular cloning and functional expression of Omethyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis, Plant J. 17 (1999) 329–339. [7] A. Gauthier, P.J. Gulick, R.K. Ibrahim, Characterization of two cDNA clones which encode O-methyltransferases for the methylation of both flavonoid and phenylpropanoids compounds, Arch. Biochem. Biophys. 351 (1998) 243–249. [8] R.K. Ibrahim, D. Anzellotti, The enzymatic basis of flavonoid biodiversity, in: J.T. Romeo (Ed.), Integrative Phytochemistry: From Ethnobotany to Molecular Ecology, Pergamon, New York, 2003, pp. 1–36. [9] C.S. Jang, J.W. Johnson, Y.W. Seo, Differential expression of TaLTP3 and TaCOMT1 induced by Hessian fly larval infestation in a wheat line possessing H21 resistance gene, Plant Sci. 168 (2005) 1319–1326.

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