Structure-function relationships of wheat flavone O-methyltransferase: Homology modeling, site-directed mutagenesis and regulation
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Wheat (Triticum aestivum) O-methyltransferase (TaOMT2) catalyzes the sequential methylation of the flavone,tricetin (5,7,3',4',5'-pentahydroxyflavone) to its 3'-methyl-(selgin), 3',5'-dimethyl-(tricin) and 3',4',5'-trimethyl ether derivatives, although tricin is the major product of this reaction. The novelty of TaOMT2 to perform three sequential methylations of tricetin as a substrate, the chemopreventive properties of its major product, tricin, and the compelling interest in the protein’s structure-function relationships, prompted us to further investigate this novel protein at the biochemical, molecular and structural levels. A 3-D model of this protein was constructed 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. Homology modeling experiments in which each of the substrates tricetin, selgin and tricin, was docked into the model revealed a number of amino acid residues putatively involved in substrate binding and catalysis. Results suggest that 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. A possible reaction mechanism is discussed.
The biological significance of this methylation reaction was also investigated by analyzing its expression, enzyme activity patterns at different wheat developmental stages, in response to cold acclimation and to different abiotic stresses such as salt and drought. Results show that TaOMT2 predominantly accumulates in wheat influorescences compared to leaves, coinciding with the increased methyltransferase activity in the influorescence tissues. The effect of abiotic stresses on wheat reveals that TaOMT2 accumulates in cold-acclimated winter wheat leaves. In contrast, TaOMT2 activity with tricetin as a substrate shows a tendency to decrease during cold acclimation. Other abiotic stresses, such as salt and drought have no effects on TaOMT2 accumulation in wheat leaves, but a slight decrease in activity. The importance of tricetin methylation during developmental stages and during abiotic stresses is discussed.Keywords:
Tricin
Transferase
Docking (animal)
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Abstract Chlorogenic acids (CGAs) are a group of phenolic secondary metabolites produced by certain plant species and an important component of coffee (Coffea spp.). The CGAs have been implicated in biotic and abiotic stress responses, while the related shikimate esters are key intermediates for lignin biosynthesis. Here, two hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyl transferases (HCT/HQT) from coffee were biochemically characterized. We show, to our knowledge for the first time, that in vitro, HCT is capable of synthesizing the 3,5-O-dicaffeoylquinic acid diester, a major constituent of the immature coffee grain. In order to further understand the substrate specificity and catalytic mechanism of the HCT/HQT, we performed structural and mutagenesis studies of HCT. The three-dimensional structure of a native HCT and a proteolytically stable lysine mutant enabled the identification of important residues involved in substrate specificity and catalysis. Site-directed mutagenesis confirmed the role of residues leucine-400 and phenylalanine-402 in substrate specificity and of histidine-153 and the valine-31 to proline-37 loop in catalysis. In addition, the histidine-154-asparagine mutant was observed to produce 4-fold more dichlorogenic acids compared with the native protein. These data provide, to our knowledge, the first structural characterization of a HCT and, in conjunction with the biochemical and mutagenesis studies presented here, delineate the underlying molecular-level determinants for substrate specificity and catalysis. This work has potential applications in fine-tuning the levels of shikimate and quinate esters (CGAs including dichlorogenic acids) in different plant species in order to generate reduced or elevated levels of the desired target compounds.
Shikimic acid
Shikimate pathway
Coffea
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O-methylation mediated by O-methyltransferases (OMTs) is a common modification in natural product biosynthesis and contributes to diversity of secondary metabolites. OMTs use phenylpropanoids, flavonoids, other phenolics and alkaloids as substrates, and share common domains for S-adenosyl-L-methionine (AdoMet) and substrate binding. We searched Arabiposis genome and found 17 OMTs genes (AtOMTs). AdoMet- and substrate-binding sites were predicted. AdoMet binding domain of AtOMTs is highly conserved, while substrate-binding domain is diverse, indicating use of different substrates. In addition, expressions of six AtOMT genes in response to UV and in different tissues were investigated using real-time quantitative reverse transcriptase-polymerase chain reaction. All the AtOMTs investigated were expressed under normal growth condition and most, except AtOMT10, were induced after UV illumination. AtOMT1 and AtOMT8 were expressed in all the tissues, whereas AtOMT10 showed flower-specific expression. Analysis of these AtOMT gene expressions could provide some clues on AtOMT involvement in the cellular processes.
Natural product
O-methyltransferase
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Antirrhinum majus
Benzoic acid
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Isoeugenol
O-methyltransferase
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Functional divergence
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Abstract The biosynthesis of monolignols can potentially occur via two parallel pathways involving free acids or their coenzyme A (CoA) esters. Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze functionally identical reactions in these two pathways, resulting in the formation of mono- or dimethoxylated lignin precursors. The activities of the two enzymes increase from the first to the sixth internode in stems of alfalfa (Medicago sativa L.), preceding the deposition of lignin. Alfalfa CCOMT is highly similar at the amino acid sequence level to the CCOMT from parsley, although it contains a six-amino acid insertion near the N terminus. Transcripts encoding both COMT and CCOMT are primarily localized to vascular tissue in alfalfa stems. Alfalfa CCOMT expressed in Escherichia coli catalyzesO-methylation of caffeoyl and 5-hydroxyferuloyl CoA, with preference for caffeoyl CoA. It has low activity against the free acids. COMT expressed in E. coli is active against both caffeic and 5-hydroxyferulic acids, with preference for the latter compound. Surprisingly, very little extractableO-methyltransferase activity versus 5-hydroxyferuloyl CoA is present in alfalfa stem internodes, in which relativeO-methyltransferase activity against 5-hy-droxyferulic acid increases with increasing maturity, correlating with increased lignin methoxyl content.
O-methyltransferase
Coenzyme A
Phenolic acid
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Cinnamyl alcohol dehydrogenase (CAD) is involved in the final step of the phenylpropanod pathway, catalyzing the NADPH-dependent reduction of hydroxy-cinnamaldehydes into the corresponding alcohols. The rice genome contains twelve CAD and CAD-like genes, collectively called OsCADs. To elucidate the biochemical function of the OsCADs, OsCAD1, 2, 6, and 7, which are highly expressed in rice, were cloned from rice tissues. The cloned OsCADs were heterologously expressed in Escherichia coli as His-tag fusion proteins. The activity assay of the recombinant OsCADs showed that OsCAD2, 6, and 7 have CAD activity toward hydroxycinnamaldehydes, but OsCAD1 has no detectable catalytic activity. The kinetic parameters of the enzyme reactions demonstrated that OsCAD2 has the highest catalytic activity among the examined enzymes. This result agrees well with the finding that the Zn binding and NADPH binding motifs and the residues constituting the substrate binding pocket in bona fide plant CADs were fully conserved in OsCAD2. Although they have large variations in the residue for the substrate binding pocket, OsCAD6 and 7 catalyzed the reduction of hydroxycinnamaldehydes with a similar efficiency. Alignment of amino acid sequences showed that OsCAD1 lacks the GxxxxP motif for NADPH binding and has mismatches in residues important in the reduction process, which could be responsible for the loss of catalytic activity. OsCAD2 belongs to CAD Class I with bona fide CADs from other plant species and is constitutively expressed throughout the developmental stages of rice, with preferential expression in actively lignifying tissues such as the root, stem, and panicle, suggesting that it is mainly involved in developmental lignification in rice. The expression of OsCAD2 was also induced by biotic and abiotic stresses such as Xanthomonas oryzae pv. oryzae (Xoo) infection and UV-irradiation, suggesting that it plays a role in the defense response of rice, in addition to a bona fide role in developmental lignification. OsCAD6 and 7 belong in CAD Class II. Their expression is relatively lower than that of OsCAD2 and is confined to certain tissues, such as the leaf sheath, stem, and panicle. The expression of OsCAD6 was stimulated by Xoo infection and UV-irradiation. Thus OsCAD6 appears to be an inducible OsCAD that is likely involved in the defense response of rice against biotic and abiotic stresses.
Cinnamyl-alcohol dehydrogenase
Cinnamyl alcohol
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Monolignol
O-methyltransferase
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O-methyltransferase
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Hydroxycinnamic acid
Agmatine
Acyltransferases
Hordeum
Acyltransferases
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