O-Methyltransferases from Arabidopsis thaliana
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Abstract:
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.Keywords:
Natural product
O-methyltransferase
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Isoeugenol
O-methyltransferase
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Summary In plants, type I and II S ‐adenosyl‐ l ‐methionine‐dependent O ‐methyltransferases (OMTs) catalyze most hydroxyl group methylations of small molecules. A homology‐based RT‐PCR strategy using Catharanthus roseus (Madagascar periwinkle) RNA previously identified six new type I plant OMT family members. We now describe the molecular and biochemical characterization of a seventh protein. It shares 56–58% identity with caffeic acid OMTs (COMTs), but it failed to methylate COMT substrates, and had no activity with flavonoids. However, the in vitro incubations revealed unusually high background levels without added substrates. A search for the responsible component revealed that the enzyme methylated dithiothreitol (DTT), the reducing agent added for enzyme stabilization. Unexpectedly, product analysis revealed that the methylation occurred on a sulfhydryl moiety, not on a hydroxyl group. Analysis of 34 compounds indicated a broad substrate range, with a preference for small hydrophobic molecules. Benzene thiol ( K m 220 μ m ) and furfuryl thiol ( K m 60 μ m ) were the best substrates (6–7‐fold better than DTT). Small isosteric hydrophobic substrates with hydroxyl groups, like phenol and guaiacol, were also methylated, but the activities were at least 5‐fold lower than with thiols. The enzyme was named C. roseus S ‐methyltransferase 1 (CrSMT1). Models based on the COMT crystal structure suggest that S ‐methylation is mechanistically identical to O ‐methylation. CrSMT1 so far is the only recognized example of an S ‐methyltransferase in this protein family. Its properties indicate that a few changes in key residues are sufficient to convert an OMT into a S ‐methyltransferase (SMT). Future functional investigations of plant methyltransferases should consider the possibility that the enzymes may direct methylation at sulfhydryl groups.
O-methyltransferase
Thiol
Transferase
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Abstract For Abstract see ChemInform Abstract in Full Text.
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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.
Tricin
Transferase
Docking (animal)
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Catharanthus roseus
O-methyltransferase
Flavonols
Flavones
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Abstract Salicylic acid (SA) is a signaling molecule utilized by plants in response to various stresses. Through conjugation with small organic molecules such as glucose, an inactive form of SA is generated which can be transported into and stored in plant vacuoles. In the model organism Arabidopsis thaliana, SA glucose conjugates are formed by two homologous enzymes (UGT74F1 and UGT74F2) that transfer glucose from UDP-glucose to SA. Despite being 77% identical and with conserved active site residues, these enzymes catalyze the formation of different products: UGT74F1 forms salicylic acid glucoside (SAG), while UGT74F2 forms primarily salicylic acid glucose ester (SGE). The position of the glucose on the aglycone determines how SA is stored, further metabolized, and contributes to a defense response. We determined the crystal structures of the UGT74F2 wild-type and T15S mutant enzymes, in different substrate/product complexes. On the basis of the crystal structures and the effect on enzyme activity of mutations in the SA binding site, we propose the catalytic mechanism of SGE and SAG formation and that SA binds to the active site in two conformations, with each enzyme selecting a certain binding mode of SA. Additionally, we show that two threonines are key determinants of product specificity.
Aglycone
Product inhibition
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Physcomitrella patens
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Medicago truncatula
Polyamine
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O-methyltransferase
Plant hormone
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The Preiss-Handler pathway, which salvages nicotinate (NA) for NAD synthesis, is an indispensable biochemical pathway in land plants. Various NA conjugations (mainly methylation and glycosylation) have been detected and have long been proposed for NA detoxification in plants. Previously, we demonstrated that NA O-glucosylation functions as a mobilizable storage form for NAD biosynthesis in the Brassicaceae. However, little is known about the functions of other NA conjugations in plants. In this study, we first found that N-methylnicotinate is a ubiquitous NA conjugation in land plants. Furthermore, we functionally identified a novel methyltransferase (At3g53140; NANMT), which is mainly responsible for N-methylnicotinate formation, from Arabidopsis (Arabidopsis thaliana). We also established that trigonelline is a detoxification form of endogenous NA in plants. Combined phylogenetic analysis and enzymatic assays revealed that NA N-methylation activity was likely derived from the duplication and subfunctionalization of an ancestral caffeic acid O-methyltransferase (COMT) gene in the course of land plant evolution. COMT enzymes, which function in S-lignin biosynthesis, also have weak NANMT activity. Our data suggest that NA detoxification conferred by NANMT and COMT might have facilitated the retention of the Preiss-Handler pathway in land plants.
O-methyltransferase
Detoxification
Metabolic pathway
Monolignol
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