A Novel Mg2+-dependent O-Methyltransferase in the Phenylpropanoid Metabolism of Mesembryanthemum crystallinum

Abstract

Upon irradiation with elevated light intensities, the ice plant (Mesembryanthemum crystallinum) accumulates a complex pattern of methylated and glycosylated flavonol conjugates in the upper epidermal layer. Identification of a flavonol methylating activity, partial purification of the enzyme, and sequencing of the corresponding peptide fragments revealed a novel S-adenosyl-L-methionine-dependent O-methyltransferase that was specific for flavonoids and caffeoyl-CoA. Cloning and functional expression of the corresponding cDNA verified that the new methyltransferase is a multifunctional 26.6-kDa Mg 2؉ -dependent enzyme, which shows a significant sequence similarity to the cluster of caffeoyl coenzyme A-methylating enzymes. Functional analysis of highly homologous members from chickweed (Stellaria longipes), Arabidopsis thaliana, and tobacco (Nicotiana tabacum) demonstrated that the enzymes from the ice plant, chickweed, and A. thaliana possess a broader substrate specificity toward o-hydroquinone-like structures than previously anticipated for Mg 2؉ -dependent O-methyltransferases, and are distinctly different from the tobacco enzyme. Besides caffeoyl-CoA and flavonols, a high specificity was also observed for caffeoylglucose, a compound never before reported to be methylated by any plant O-methyltransferase. Based on phylogenetic analysis of the amino acid sequence and differences in acceptor specificities among both animal and plant Omethyltransferases, we propose that the enzymes from the Centrospermae, along with the predicted gene product from A. thaliana, form a novel subclass within the caffeoyl coenzyme A-dependent O-methyltransferases, with potential divergent functions not restricted to lignin monomer biosynthesis.Methylation by S-adenosyl-L-methionine (AdoMet) 1 dependent O-methyltransferases (OMTs) (EC 2.1.1) is a common modification in natural product biosynthesis. Site-specific O-methylation modulates the physiological properties of such compounds and reduces the chemical reactivity of phenolic hydroxyl groups (1). In plants, O-methylation is also required for monolignol biosynthesis and accounts for the structural differences and properties of lignin, which next to cellulose is the most prominent polymer on earth (2).Plant OMTs can be categorized into two major classes (3). Class I includes a group of low molecular weight (23,000 to 27,000) and Mg 2ϩ -dependent OMTs, whereas class II consists of higher molecular weight OMTs of about 38,000 to 43,000 that do not require Mg 2ϩ for catalytic activity. Prominent class II members include caffeic acid, flavonoid, coumarin, and alkaloid OMTs (4 -6). Within the class I OMTs, a group of small caffeoyl coenzyme A OMTs (CCoAOMTs) have been suggested to be key enzymes in the biosynthesis of monolignols, the precursors of gymnosperm and angiosperm lignins (7,8). In angiosperms, this task may also be performed by class II OMTs that are specific for caffeic acid, caffeyl aldehyde, or caffeyl alcohol (COMT) (9 -11). This apparent redundancy results in a cell-and tis...

Over‐expression of an S‐adenosylmethionine‐dependent methyltransferase leads to an extended lifespan of Podospora anserina without impairments in vital functions

“…1). However, a substrate specificity of some plant class I methyltransferases also for flavonoids was reported in a recent study (Ibdah et al, 2003;Kim et al, 2006). Moreover, rat COMT, another class I methyltransferase, is known to accept flavonoids as substrates (Zhu et al, 1994).…”

“…Mg 2+ ) as cofactors, and its size of 27 kDa, PaMTH1 belongs to the cation-dependent subclass (class I) of SAM-dependent methyltransferases (Joshi & Chiang, 1998;Ibdah et al ., 2003). In plants, animals and humans, members of this protein superfamily are involved in the biosynthesis of a variety of different secondary metabolites.…”

Functional and Structural Characterization of a Cation-dependent O-Methyltransferase from the Cyanobacterium Synechocystis sp. Strain PCC 6803

“…Cation-dependent OMTs constitute a small group of low molecular mass (23 to 27 kDa) enzymes (2). In mammals, these enzymes play important roles in the modification of catechol neurotransmitters in the brain or may inactivate potentially bioactive metabolites like quercetin in the liver and kidney (3,4).…”

Genetic Control and Evolution of Anthocyanin Methylation

“…The relationship between these proteins resembles that of the species, consistent with the idea that they are orthologous. Interestingly, the two most similar proteins from the Rosid Arabidopsis (At1g67980 and At1g67990) fall just outside the DIFe/ VvAOMT1 clade, while the next most similar protein (At4g26220) groups with methyltransferases from Mesembryanthemum crystallinum (Mg 2+ -dependent phenylpropanoid and flavonoid O-methyltransferase [PFOMT]) and Stellaria longipes (both Caryophyllales; Ibdah et al, 2003) and the Asterid cyclamen (Akita et al, 2011) in a distant clade. The Arabidopsis and M. crystalinum and S. longipes enzymes can in vitro methylate a broad but distinct range of substrates, which includes caffeoyl esters, flavones, and flavonols, also at hydroxyl groups other than those in the 3959 position (Ibdah et al, 2003;Wils et al, 2013).…”

“…flowers only express activities that can methylate anthocyanins, but not cinnamic acids, and do not contain ferulic or sinapic acids (Jonsson et al, 1982). In vitro assays of methyltransferases often employ substrate concentrations in the 0.1 to 1 mM range (Gang et al, 2002;Ibdah et al, 2003;Kim et al, 2006;Akita et al, 2011). Using similar anthocyanin substrate concentrations (0.4-1 mM), the AMTs in crude petal extracts, which originate from the three genes identified here, and recombinant MT2/DIFe1 protein could methylate anthocyanin 3-glucosides and 3-rutinosides in vitro (Jonsson et al, 1982;Fig.…”