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

Abstract

The coding sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 slr0095 gene was cloned and functionally expressed in Escherichia coli. The corresponding enzyme was classified as a cation-and S-adenosyl-L-methionine-dependent O-methyltransferase (SynOMT), consistent with considerable amino acid sequence identities to eukaryotic O-methyltransferases (OMTs). The substrate specificity of SynOMT was similar with those of plant and mammalian CCoAOMT-like proteins accepting a variety of hydroxycinnamic acids and flavonoids as substrates. In contrast to the known mammalian and plant enzymes, which exclusively methylate the meta-hydroxyl position of aromatic di-and trihydroxy systems, Syn-OMT also methylates the para-position of hydroxycinnamic acids like 5-hydroxyferulic and 3,4,5-trihydroxycinnamic acid, resulting in the formation of novel compounds. The x-ray structure of SynOMT indicates that the active site allows for two alternative orientations of the hydroxylated substrates in comparison to the active sites of animal and plant enzymes, consistent with the observed preferred para-methylation and position promiscuity. Lys 3 close to the N terminus of the recombinant protein appears to play a key role in the activity of the enzyme. The possible implications of these results with respect to modifications of precursors of polymers like lignin are discussed.2 (EC 2.1.1) is a common modification in secondary product biosynthesis (1). Sitespecific O-methylation modulates the physiological properties and the chemical reactivity of phenolic compounds and renders them more hydrophobic. 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). They are therefore referred to as catechol OMTs (COMT) and have been investigated as potential targets to cure degenerate brain diseases (5). In plants, caffeoyl-coenzyme A O-methyltransferases (CCoAOMTs), named after their preferred in vitro substrate, in conjunction with a second group of cation-independent caffeic acid OMTs, are crucial for determining the structural integrity of lignin in plant vascular tissues (6, 7). Specific subtypes of CCoAOMT-like proteins also methylate, besides caffeoyl-CoA, other phenylpropanoids, preferentially flavonoids, with vicinal dihydroxy groups (2). The threedimensional structures of eukaryotic animal and plant OMTs known so far are quite similar despite their otherwise low sequence identities, irrespective of the involvement of bivalent cations and substrates. Structural data obtained so far reveal a conserved AdoMet-binding site in all OMTs from the animal and plant kingdom (8 -10). The methyl transfer mechanism proceeds via an S n 2-like transition state and a cation-facilitated deprotonation of one of the two hydroxyl groups.Atomic structures of the cation-dependent catechol OMT from rat ...

Novel Flavonol Glycosides from the Aerial Parts of Lentil (Lens culinaris)

“…In the case of the 3,4,5-trihydroxycinnamic acid, present in the compound 4, an analytical standard was not available and the compound was tentatively identified on the base of its molecular mass. We could not find any precise literature data about its UV maxima, but the UV spectrum we obtained (see Supplementary Figure S196) was similar to one presented in the work of Kopycki et al [16].…”

“…In this study we presented the purification and structure elucidation of 18 acylated and non-acylated glycosides of kaempferol and quercetin, including 16 (11). Kaempferol and quercetin are known as potent antioxidants with diverse biological activity and health-promoting properties.…”

Two O-methyltransferases from Picea abies: characterization and molecular basis of different reactivity

“…The molecular weights of the OMTs in this group range from 23 to 27 kDa. A subgroup within the CCoAOMT-like enzymes is found in plants and microbes (Ibdah et al 2003;Lee et al 2006;Kopycki et al 2008b). They have similar molecular weights compared to classical CCoAOMTs but they utilize both Xavonoids and caVeoyl-CoA.…”

Structural Modeling and Biochemical Characterization of Flavonoid O-Methyltransferase from Rice

“…The recombination proteins were expressed and purified via the method developed by Kim et al 18 The O-methyltransferase assay was conducted as previously described. 11 Reaction mixture contained 60 µM of flavonoid, 500 µM of SAM and 3 µg of the purified wild type ROMT9 or 90 µg of D275L or 65 µg of H328R in 500 µL of 50 mM Tris/HCl (pH 7.5). The reaction mixture was incubated at 37 o C for 30 min.…”

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