Two forms of O-methyltransferase in tobacco cell suspension culture

An S-adenosyl-l-methionine: o-dihydric phenol O-methyltransferase was isolated from tobacco cell suspension culture and was partially purified by (NH₄)₂SO₄ precipitation and successive chromatography on DEAE-Sepharose, Sephacryl S-200 and hydroxyapatite columns. It catalysed the O-methylation of 3 cinnamic acids, two coumarins and two flavonoids, but to different extents. Results obtained from polyacrylamide gel electrophoresis, m-/p-methylation ratios and mixed substrate experiments indicated the existence of two forms of the enzyme which were resolved by chromatography on DEAE-cellulose. One form (MW 74000, pI 6.1, opt. pH 7.3) catalysed the meta-methylation of caffeic acid, while the other (MW 70000, pI 6.3, opt. pH 8.3) mediated the para-methylation of quercetin, though each form exhibited some activity against other substrates.     

Glial metabolism of quercetin reduces its neurotoxic potential

The neuroprotective effects of flavonoids will ultimately depend on their interaction with both neuronal and glial cells. In this study, we show that the potential neurotoxic effects of quercetin are modified by glial cell interactions. Specifically, quercetin is rapidly conjugated to glutathione within glial cells to yield 2′-glutathionyl-quercetin, which is exported from cells but has significantly reduced neurotoxicity. In addition, quercetin underwent intracellular O-methylation to yield 3′-O-methyl-quercetin and 4′-O-methyl-quercetin, although these were not exported from glia at the same rate as the glutathionyl adduct. The neurotoxic potential of both quercetin and 2′-glutathionyl-quercetin paralleled their ability to modulate the pro-survival Akt/PKB and extracellular signal-regulated kinase (ERK) signalling pathways. These data were supported by co-culture investigation, where the neurotoxic effects of quercetin were significantly reduced when they were cultured alongside glial cells. We propose that glial cells act to protect neurons against the neurotoxic effects of quercetin and that 2′-glutathionyl-quercetin represents a novel quercetin metabolite.     

Regioselective synthesis of flavonoid bisglycosides using Escherichia coli harboring two glycosyltransferases

Regioselective glycosylation of flavonoids cannot be easily achieved due to the presence of several hydroxyl groups in flavonoids. This hurdle could be overcome by employing uridine diphosphate-dependent glycosyltransferases (UGTs), which use nucleotide sugars as sugar donors and diverse compounds including flavonoids as sugar acceptors. Quercetin rhamnosides contain antiviral activity. Two quercetin diglycosides, quercetin 3-O-glucoside-7-O-rhamnoside and quercetin 3,7-O-bisrhamnoside, were synthesized using Escherichia coli expressing two UGTs. For the synthesis of quercetin 3-O-glucoside-7-O-rhamnoside, AtUGT78D2, which transfers glucose from UDP-glucose to the 3-hydroxyl group of quercetin, and AtUGT89C1, which transfers rhamnose from UDP-rhamnose to the 7-hydroxyl group of quercetin 3-O-glucoside, were transformed into E. coli. Using this approach, 67 mg/L of quercetin 3-O-glucoside-7-O-rhamnoside was synthesized. For the synthesis of quercetin 3,7-O-bisrhamnoside, AtUGT78D1, which transfers rhamnose to the 3-hydroxy group of quercetin, and AtUGT89C1 were used. The RHM2 gene from Arabidopsis thaliana was coexpressed to supply the sugar donor, UDP-rhamnose. E. coli expressing AtUGT78D1, AtUGT89C1, and RHM2 was used to obtain 67.4 mg/L of quercetin 3,7-O-bisrhamnoside.     

Flavonoide aus salix alba. Die struktur des Terniflorins und eines weiteren acylflavonoides

Eight flavonoids were isolated from the leaves of Salix alba. One, apigenin 7-O-(4-p-coumarylglucoside), is a new natural compound; another, terniflorin, the 6-isomer, is an artefact. The others are quercetin 3-O-glucoside, quercetin 3-O-rutinoside, isorhamnetin 3-O-glucoside, isorhamnetin 3-O-rutinoside and quercetin 7,′3-dimethylether 3-O-glucoside.     

Molecular mechanism of RNase R substrate sensitivity for RNA ribose methylation

RNA 2′-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3′-5′ exoribonuclease and is particularly sensitive to RNA 2′-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2′-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR’s ribonuclease activity and significant sensitivity to RNA 2′-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2′-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2′-O-methylations research in biological and clinical samples.     

Temporal lag between gene expression and metabolite accumulation in flavonol biosynthesis of Arabidopsis roots

The temporal lag between gene expression and metabolite accumulation has been estimated in flavonol biosynthesis, but the time difference between these events is unclear. In the present study, we investigated the expression of flavonol biosynthetic genes ELONGATED HYPOCOTYL5, MYELOBLASTOSIS PROYEIN12/PRODUCTION OF FLAVONOL GLYCOSYDES1, CHALCONE SYNTHASE, CHALCONE ISOMERASE, FLAVANONE 3-HYDROXYLASE, and FLAVONOL SYNTHASE1, and the accumulation of flavonol glycosides (kaempferol and quercetin glycosides) in time-series samples of Arabidopsis thaliana roots. All genes started to be expressed within 3 h after sequential light irradiation (HAS) and reached their maximum expression levels at 12 HAS, and the accumulation of the flavonol glycosides started at 6 HAS. Metabolome analysis using liquid chromatography-mass spectrometry showed that the accumulation of kaempferol 3-O-glucoside-7-O-rhamnoside and kaempferol 3-O-rhamnosyl (1 → 2) glucoside-7-O-rhamnoside reached their maximum levels at 48 HAS, whereas other flavonol glycosides, such as kaempferol/quercetin 3-O-rhamnoside-7-O-rhamnoside, quercetin 3-O-glucoside-7-O-rhamnoside and quercetin 3-O-rhamnosyl (1 → 2) glucoside-7-O-rhamnoside, increased gradually until 96 HAS. These results show that the expression of the flavonol genes is an early response against light exposure, and that the accumulation of the flavonol glycosides is a late response.     

Nitrogen-containing derivatives of O-tetramethylquercetin: Synthesis and biological profiles in prostate cancer cell models

Forty-eight nitrogen-containing quercetin derivatives were synthesized from readily available rutin or quercetin for the in vitro evaluation of their biological profiles. The WST-1 cell proliferation assay data indicate that thirty-nine out of the forty-eight derivatives possess significantly improved antiproliferative potency as compared with quercetin and fisetin, as well as the parent 3,3′,4′,7-O-tetramethylquercetin toward both androgen-sensitive (LNCaP) and androgen-insensitive (PC-3 and DU145) human prostate cancer cell lines. 5-O-Aminoalkyl-3,3′,4′,7-O-tetramethylquercetins were established as a better scaffold for further development as anti-prostate cancer agents. Among them, 5-O-(N,N-dibutylamino)propyl-3,3′,4′,7-O-tetramethylquercetin (44) was identified as the optimal derivative with IC₅₀ values of 0.55–2.82 µM, being over 35–182 times more potent than quercetin. The flow cytometry-based assays further demonstrate that 44 effectively activates PC-3 cell apoptosis.     

Biologically-active flavonoids from Gossypium arboreum

A new flavonol glycoside, gossypetin 8-O-rhamnoside, was isolated from flower petals of Gossypium arboreum along with quercetin 7-O-glucoside, quercetin 3-O-glucoside and quercetin 3′-O-glucoside. These compounds showed antibacterial activity against Pseudomonas maltophilia and Enterobacter cloacae.     

Flavonoids of Mayaca fluviatilis (Mayacaceae)

Mayaca is an aquatic monocot of the monogeneric family Mayacaceae. The flavonol glycosides quercetin 3-O-glucoside, quercetin 3-O-rutinoside, and kaempferol 3-O-glucoside, and the flavone luteolin 5-O-glucoside were found in methanolic leaf extracts. The presence of flavonol and flavone O-glycosides sets the Mayacaceae apart from the Commelinaceae, which accumulates predominantly flavone C-glycosides.     

Synthesis of Flavonoid O-Pentosides by Escherichia coli through Engineering of Nucleotide Sugar Pathways and Glycosyltransferase

Plants produce two flavonoid O-pentoses, flavonoid O-xyloside and flavonoid O-arabinoside. However, analyzing their biological properties is difficult because flavonoids are not naturally produced in sufficient quantities. In this study, Escherichia coli was used to synthesize the plant-specific flavonoid O-pentosides quercetin 3-O-xyloside and quercetin 3-O-arabinoside. Two strategies were used. First, E. coli was engineered to express components of the biosynthetic pathways for UDP-xylose and UDP-arabinose. For UDP-xylose biosynthesis, two genes, UXS (UDP-xylose synthase) from Arabidopsis thaliana and ugd (UDP-glucose dehydrogenase) from E. coli, were overexpressed. In addition, the gene encoding ArnA (UDP-l-Ara4N formyltransferase/UDP-GlcA C-4″-decarboxylase), which competes with UXS for UDP-glucuronic acid, was deleted. For UDP-arabinose biosynthesis, UXE (UDP-xylose epimerase) was overexpressed. Next, we engineered UDP-dependent glycosyltransferases (UGTs) to ensure specificity for UDP-xylose and UDP-arabinose. The E. coli strains thus obtained synthesized approximately 160 mg/liter of quercetin 3-O-xyloside and quercetin 3-O-arabinoside.     

The flavonoids of ferns in the isolated genera Loxsoma and Loxsomopsis

Kaempferol and quercetin 3-O-glucosides and 3-O-rhamnoglucosides are common to both Loxsoma cunninghamii and Loxsomopsis costaricensis, but in the former the new flavonoid glycosides, kaempferol and quercetin 3-O-glucoside-7-O-arabinoside have also been identified. The data are consistent with the proposed taxonomic relationship between these geographically isolated genera. Comparative flavonoid chemistry indicates that the Loxsomaceae may be a primitive family, not closely related to the Hymenophyllaceae or the Cyatheaceae.     

Characterization of three distinct flavonol O-methyltransferases from Chrysosplenium americanum

The partially purified O-methyltransferase (OMT) system of Chrysosplenium americanum was found to catalyse the stepwise O-methylation of quercetin to its mono-, di- and trimethyl derivatives. It also utilized the partially methylated flavonol intermediates to form the next higher order of O-methylated products; thus indicating the involvement of several OMTs. The latter were resolved by chromatofocusing into three distinct peaks of enzyme activity which focused at pI values 4.8, 5.4 and 5.7. The former enzyme O-methylated quercetin at the 3-position, whereas the latter two O-methylated 3, 7-di-O-methyl quercetagetin at the 3′- and 6-positions, respectively. None of the focused enzymes accepted caffeic acid, or other flavonoids such as kaempferol or luteolin, as substrates; thus indicating specificity towards flavonols with 3′, 4′- substitution. The three OMTs had similar MWs and the K_m values for their substrates were of the same order of magnitude. The biochemical role of these novel enzymes is discussed in relation to the biosynthesis of polymethylated flavonols in this tissue.     

Flavonoids of Heterogaura (Onagraceae)

Heterogaura is a monotypic genus of the tribe Onagreae of the Onagraceae. It is endemic to south western Oregon and California. Four flavonol glycosides, kaempferol 3-O-rhamnoside, quercetin 3-O-glucoside, quercetin 3-O-rhamnoglucoside and myricetin 3-O-glucoside, were found to occur in methanolic leaf extracts of each of the populations sampled. The presence of only flavonols is consistent with flavonoid analyses from other genera of the Onagreae, including Clarkia, the closest relative of Heterogaura.     

Control by bacteriophage T4 of two sequential phosphorylations of the alpha subunit of Escherichia coli RNA polymerase

The effect of 2′-O-methylation upon the base-stacking properties of dinucleoside monophosphates has been studied by circular dichroism measurements over the temperature range from ?20 °C to +80 °C at high and at low salt concentration of 13 2′-O-methyl derivatives in neutral aqueous solution. It is found that 2′-O methylation generally enhances the stacking propensity of dinucleoside monophosphates except for the dimers with adenine in the 3′-linked nucleoside, where the converse trend is observed. The influence of 2′-O-methylation upon the base-stacking property of a dimer correlates in part with the effect of a reduction in salt concentration, suggesting that the 2′-O-methyl group effects the stacking by displacing ions from the immediate environment of the dimer as well as by intramolecular steric effects. The dimers which exhibit an enhanced stacking due to the 2′-O-methylation are found in a larger than statistical abundance in yeast transfer RNA, whereas those showing a reduced stacking occur in minor abundance. These observations are discussed in relation to some current views on the role of modified nucleosides in the conformation of ribonucleic acids.     

Flavonoid glycosides from the leaves of Aphananthe aspera (Thunb.) Planch. (Cannabaceae) and their chemotaxonomic significance

From the leaves of Aphananthe aspera (Thunb.) Planch. (Family: Cannabaceae), six flavonol glycosides, such as quercetin 3-O-β-glucopyranoside (1), kaempferol 3-O-β-glucopyranoside (2), quercetin 3-O-rutinoside (3), kaempferol 3-O-rutinoside (4), quercetin 3-O-neohesperidoside (5) and kaempferol 3-O-neohesperidoside (6) were isolated and identified. Structure elucidation of these compounds was performed on the basis of NMR spectral data. All these compounds were isolated for the first time from the genus Aphananthe. Chemotaxonomic significance and distribution of these flavonoid derivatives among the genera of Cannabaceae are explained in detail.     

Flavonoids of Balsamorhiza deltoidea and Wyethia mollis

Eleven O-methylated derivatives of kaempferol, quercetin and quercetagetin were isolated from the dichloromethane leaf-wash of Balsamorhiza deltoidea. Four of these compounds represent new reports from either Balsamorhiza or Wyethia: 6-hydroxykaempferol 7-O-methyl ether, quercetin 3′,4′-O-dimethylether, quercetagetin 7-O-methyl ether, and quercetagetin 3,6,7-O-trimethyl ether. We also confirmed the presence of two isoflavones, santal and orobol 3′-O-methyl ether, in W. mollis. The 8-C-prenylated derivatives of naringenin, eriodictyol, and dihydroisorhamnetin were also identified as constituents of W. mollis. The vacuolar flavonoid fraction of Balsamorhiza deltoidea and Wyethia helenioides was shown to consist of simple mono and diglycosides of kaempferol and quercetin.     

Flavonoids in the leaves of Hillebrandia and Begonia species (Begoniaceae)

The fresh leaves of Hillebrandia sandwicensis and 126 Begonia taxa were chemotaxonomically surveyed for flavonoids. Of their taxa, H. sandwicensis and 119 species, one variety and three hybrids were analyzed for flavonoids for the first time. Ten flavonols and eleven C-glycosylflavones were isolated and characterized as quercetin 3-O-rutinoside (1), kaempferol 3-O-rutinoside (2), isorhamnetin 3-O-rutinoside (3), quercetin 3-O-glucoside (4), quercetin 3-methyl ether 7-O-rhamnosylglucoside (5), quercetin 3,3'-dimethyl ether 7-O-rhamnosylglucoside (6), quercetin glycoside (13), quercetin glycoside (acylated) (14), kaempferol glycoside (17) and quercetin 3-O-rhamnoside (18) as flavonols, and isovitexin (7), vitexin (8), isoorientin (9), orientin (10), luteolin 6-C-pentoside (11), luteolin 8-C-pentoside (12), schaftoside (15), isoschaftoside (16), chrysoeriol 6,8-di-C-pentoside (19), apigenin 6,8-di-C-arabinoside (20) and isovitexin 2''-O-glucoside (21) as C-glycosylflavones. Quercetin 3-O-rutinoside (1) alone was isolated from H. sandwicensis endemic to Hawaii. Major flavonoids of almost Begonia species was also 1. Begonia species were divided into two chemotypes, i.e. flavonol containing type and C-glycosylflavone containing type. Of 14 section of the Begonia, almost species of many section, i.e. sect. Augustia, Coelocentrum, Doratometra, Leprosae, Loasibegonia, Monopteron and Ruizoperonia, were flavonol types. On the other hand, C-glycosyflavone type was comparatively most in sect. Platycentrum.     

Flavonoid glycosides of Kalanchoe spathulata

A new glycoside, patuletin 3,7-di-O-rhamnoside, together with patuletin, quercetin, quercetin 3-O-glucoside-7-O-rhamnoside, kaempferol and kaempferol 3-O-rhamnoside were identified from leaves and flowers of Kalanchoe spathulata.     

Flavonol tetraglycosides from the leaves of Prunus cerasus and P. Avium

Four new flavonol glycosides have been identified from fresh leaves and fruits of sweet and sour cherries (Prunus avium and P. cerasus) as minor flavonoids: quercetin 3-O-rutinosyl-7,3′-O-bisglucoside; two quercetin 3-O-rutinosyl-4′-di-O-glucosides; kaempferol 3-O-rutinosyl-4′-di-O-glucoside.     

Flavonol glycoside gallates from Tellima grandiflora

Quercetin 3-O-(6″-O-galloyl)-β-d-glucoside has been identified as a constituent of Tellima grandiflora (Saxifragaceae). In all, twelve gallates were encountered: two isomeric gallates of quercetin 3-O-glucoside and two of quercetin 3-O-galactoside, a similar set involving kaempferol, and a similar set involving myricetin.