Molecular evolution of flavonoid dioxygenases in the family Apiaceae

Plant species of the family Apiaceae are known to accumulate flavonoids mainly in the form of flavones and flavonols. Three 2-oxoglutarate-dependent dioxygenases, flavone synthase or flavanone 3 beta-hydroxylase and flavonol synthase are involved in the biosynthesis of these secondary metabolites. The corresponding genes were cloned recently from parsley (Petroselinum crispum) leaves. Flavone synthase I appears to be confined to the Apiaceae, and the unique occurrence as well as its high sequence similarity to flavanone 3beta-hydroxylase laid the basis for evolutionary studies. In order to examine the relationship of these two enzymes throughout the Apiaceae, RT-PCR based cloning and functional identification of flavone synthases I or flavanone 3beta-hydroxylases were accomplished from Ammi majus, Anethum graveolens, Apium graveolens, Pimpinella anisum, Conium maculatum and Daucus carota, yielding three additional synthase and three additional hydroxylase cDNAs. Molecular and phylogenetic analyses of these sequences were compatible with the phylogeny based on morphological characteristics and suggested that flavone synthase I most likely resulted from gene duplication of flavanone 3beta-hydroxylase, and functional diversification at some point during the development of the apiaceae subfamilies. Furthermore, the genomic sequences from Petroselinum crispum and Daucus carota revealed two introns in each of the synthases and a lack of introns in the hydroxylases. These results might be explained by intron losses from the hydroxylases occurring at a later stage of evolution.     

Interaction between the aryl hydrocarbon receptor and its antagonists, flavonoids

Flavonoids have been reported to be dietary antagonists of an aryl hydrocarbon receptor (AhR). However, little is known about the molecular mechanism on their antagonistic effects. In this study, the inhibitory effect of flavonoids on ligand binding to the AhR and interaction between flavonoids and the AhR complex (AhRc) were investigated in each flavonoid subclass. Flavone, flavonol, and flavanone but not catechin inhibited the specific binding between the AhR and 3-methylcholanthrene dose-dependently, indicating that the former three subclasses possibly act as competitive antagonists of the AhR. However, catechin in addition to the former three subclasses directly interacted with the AhRc by surface plasmon resonance analysis. The dissociation constant values showed an inverse correlation with the suppressive effect on the DNA binding activity. These results suggest that flavone, flavonol, and flavanone act as competitive antagonists of the AhR, while catechin associates with the AhRc and indirectly exhibits its antagonistic effects.     

Flavanone-7-O-glucosyltransferase activity from Petunia hybrida

Citrus spp. are known for the accumulation of flavanone glycosides (e.g., naringin comprises up to 70% of the dry weight of very young grapefruit). In contrast, petunia utilizes relatively more naringenin for production of flavonol glycosides and anthocyanins. This investigation addressed whether or not petunia is capable of glucosylation of naringenin and if so, what are the characteristics of this flavanone glucosylating enzyme. Petunia leaf tissue contains some flavanone-7-O-glucosyltransferase (E.C. 2.4.1.185) activity, although at 90-fold lower levels than grapefruit leaves. This activity was partially purified 89-fold via ammonium sulfate fractionation followed by FPLC on Superose 12 and Mono Q yielding three chromatographically separate peaks of activity. The enzymes in the peak fractions glucosylated flavanone, flavonol, and flavone substrates. Enzymes in Mono Q peaks I and II were relatively more specific toward flavanone substrates and peak I was significantly more active. Enzyme activity was not effected by Ca2+, Mg2+, AMP, ADP, or ATP. The petunia enzyme was over 10,000 times more sensitive to UDP inhibition (Ki 0.89 microM) than the flavanone-specific 7GT in grapefruit. These and other results suggest that different flavanoid accumulation patterns in these two plants may be partially due to the different relative levels and biochemical properties of their flavanone glucosylating (7GT) enzymes.     

Isolation and identification of flavonoids, including flavone rotamers, from the herbal drug 'Crataegi folium cum flore' (hawthorn)

Twelve flavonoids, including seven flavones, four flavonols and one flavanone, were isolated from methanolic extract of the herbal drug 'Crataegi folium cum flore' (hawthorn leaves and flowers) by a combination of CC (over Amberlite XAD-7 and Sephadex LH-20) and preparative HPLC. Their structures, including that of the novel flavonol 8-methoxykaempferol 3-O-(6"-malonyl-beta-glucopyranoside), were elucidated by homo- and heteronuclear NMR and electrospray/MS. The 1H- and 13C-NMR of all compounds, including rotameric pairs of five flavone C-glycosides, were assigned. The presence and relative proportion of each rotamer was shown by various NMR experiments, including two-dimensional nuclear Overhauser and exchange spectroscopy, to depend on solvent, linkage position and structure of the C-glycosyl substituent.     

Three 2-oxoglutarate-dependent dioxygenase activities of Equisetum arvense L. forming flavone and flavonol from (2S)-naringenin

Equisetum arvense L. (Equisetaceae-horsetail) accumulates various flavones and flavonols in infertile shoot. Enzyme assays conducted with crude extracts of the green tissue revealed chalcone synthase activity and also three further activities assigned to flavonoid biosynthesis and identified as flavone synthase I, flavanone 3β-hydroxylase and flavonol synthase. The latter three activities were characterized as soluble, 2-oxoglutarate-dependent dioxygenases by their typical cofactor requirements and peculiar inhibition. Notably, this is the first report of flavone synthase I which had been considered to be restricted solely to species of the Apiaceae from a distant plant taxon.     

Regioselectivity of 7-O-methyltransferase of poplar to flavones

POMT-7, an O-methyltransferase from poplar (Populus deltoids) was used to modify a variety of flavonoid compounds. POMT-7 was able to transfer a methyl group to several flavonoids containing a C-7 hydroxyl group. However, POMT-7 showed a higher affinity toward flavonol and flavone such as apigenin, kaempferol, luteolin, and quercetin than flavanone and isoflavone. Based on comparison of HPLC retention times with authentic compounds and corresponding nuclear magnetic resonance spectroscopy data, the methylation position of the reaction products was determined to be at the hydroxyl group of C-7. Biotransformation kinetics indicated that the enzyme converted more than 80% of the apigenin, kaempferol, luteolin and quercetin substrates, which were added at concentration of 70 microM, into corresponding 7-methoxy compounds within 24 h.     

Molecular characterization of flavonoid malonyltransferase from Oryza sativa

In this study, a flavonoid malonyltransferase (OsMaT-2) was cloned from Oryza sativa, and the recombinant protein OsMaT-2 was purified via affinity chromatography. OsMaT-2 utilized a variety of flavonoid glucosides, including flavanone glucosides, flavone glucosides, flavonol glucosides, and isoflavone glucosides as substrates, but did not utilize anthocyanin. As an acyl donor, OsMaT-2 utilized only malonyl-CoA. Based on reactions with various quercetin 3-O-sugars, we identified the probable position of malonylation as the 6″-hydroxyl group of the sugar. This is the first report, to the best of our knowledge, of the cloning of a flavonoid malonyltransferase from O. sativa.     

Heterologous Expression of Two Gentian Cytochrome P450 Genes can Modulate the Intensity of Flower Pigmentation in Transgenic Tobacco Plants

Two different heterologous expression systems, microsomal fractions of Saccharomyces cerevisiae and transgenic tobacco plants, were used to investigate the enzymatic activities of flavonoid 3′-hydroxylase (GtF3′H) and flavone synthase II (GtFSII) homologues isolated from gentian petals. Recombinant GtF3′H expressed in yeast showed hydroxylation activities in the 3′ position with several flavonoid substrates, while recombinant GtFSII was able to produce flavone from flavanone. GtF3′ H-expressing transgenic tobacco plants showed a slight increase in anthocyanin content and flower color intensity, and conversion of the flavonol quercetin from kaempferol. On the other hand, GtFSII-expressing plants showed a remarkable reduction in anthocyanin content and flower color intensity, and additional accumulation of flavone, especially luteolin derivatives. We demonstrated that two cytochrome P450s from gentian petals have F3′H and FSII enzymatic activities both in vitro and in vivo, and might therefore be useful in modification of flower color using genetic engineering.     

Purification and antigenicity of flavone synthase I from irradiated parsley cells

Flavone synthase I, a soluble 2-oxoglutarate-dependent dioxygenase catalyzing the oxidation of flavanones to flavones in several Apiaceae species, was induced in parsley cell cultures by continuous irradiation with ultraviolet/blue light for 20 h. The enzyme was extracted from these cells and purified by a revised purification protocol including the fractionation on hydroxyapatite, Fractogel EMD DEAE, and Mono Q anion exchangers, which resulted in an apparently homogeneous flavone synthase at approximately 10-fold higher yield as compared to the previous report. The homogeneous enzyme was employed to raise an antiserum in rabbit for partial immunological characterization. The specificity of the polyclonal antibodies was demonstrated by immunotitration and Western blotting of the crude ammonium sulfate-fractionated enzyme as well as of the enzyme at various stages of the purification. High titer cross-reactivity was observed toward flavone synthase I, showing two bands in the crude extract corresponding to molecular weights of 44 and 41 kDa, respectively, while only the 41 kDa was detected on further purification. The polyclonal antiserum did not cross-react with recombinantly expressed flavanone 3beta-hydroxylase from Petunia hybrida or flavonol synthase from Citrus unshiu, two related 2-oxoglutarate-dependent dioxygenases involved in the flavonoid pathway.     

Phenolic Glycosides with antiproteasomal activity from Centaurea urvillei DC. subsp. urvillei

A new flavanone glycoside, naringenin-7-O-β-d-glucuronopyranoside, and a new flavonol glycoside, 6-hydroxykaempferol-7-O-β-d-glucuronopyranoside were isolated together with 12 known compounds, 5 flavone glycoside; hispidulin-7-O-β-d-glucuronopyranoside, apigenin-7-O-β-d-methylglucuronopyranoside, hispidulin-7-O-β-d-methylglucuronopyranoside, hispidulin-7-O-β-d-glucopyranoside, apigenin-7-O-β-d-glucopyranoside, a flavonol; kaempferol, two flavone; apigenin, and luteolin, a flavanone glycoside; eriodictyol-7-O-β-d-glucuronopyranoside, and three phenol glycoside; arbutin, salidroside, and 3,5-dihydroxyphenethyl alcohol-3-O-β-d-glucopyranoside from Centaurea urvillei subsp. urvillei. The structure elucidation of the new compounds was achieved by a combination of one- (¹H and ¹³C) and two-dimensional NMR techniques (G-COSY, G-HMQC, and G-HMBC) and LC-ESI-MS. The isolated compounds were tested for their antiproteasomal activity. The results indicated that kaempferol, a well known and widely distributed flavonoid in the plant kingdom, was the most active antiproteasomal agent, followed by apigenin, eriodictyol-7-O-β-d-glucuronopyranoside, 3,5-dihydroxyphenethyl alcohol-3-O-β-d-glucopyranoside, and salidroside, respectively.     

Aromatase inhibition by flavonoids

Several synthetic flavones were found to inhibit the aromatization of androstenedione to estrone catalyzed by human placental microsomes. Twenty-one compounds were tested and the IC50 of the most active were: flavone, 10 microM; 7-hydroxyflavone, 0.5 microM; 7,4'-dihydroxyflavone, 2.0 microM; flavanone, 8.0 microM; and 4'-hydroxyflavanone, 10 microM. Most of the others had IC50 values ranging from 80 to greater than 200 microM. These findings show that 4'-hydroxylation results in either no change or very little change in IC50 for flavanone, isoflavone and isoflavanone as well as other ring A hydroxylated flavones. Derivatives of flavone with a hydroxyl substituent at position 5, 6 and 7 were also screened. 7-Hydroxyflavone (11) was the most effective competitive inhibitor (IC50 = 0.5 microM) with an apparent Ki value of 0.25 microM. Compound 11 also induced a change in the absorption spectrum of the aromatase cytochrome P-450 which is indicative of substrate displacement. The relative binding affinities of the flavonoid analogs were determined and only ring A adn ring B dihydroxylated analogs were found to bind to the estrogen receptor.     

Biosynthesis of plant-specific flavones and flavonols in Streptomyces venezuelae

Recently, recombinant Streptomyces venezuelae has been established as a heterologous host for microbial production of flavanones and stilbenes, a class of plant-specific polyketides. In the present work, we expanded the applicability of the S. venezuelae system to the production of more diverse plant polyketides including flavones and flavonols. A plasmid with the synthetic codon-optimized flavone synthase I gene from Petroselium crispum was introduced to S. venezuelae DHS2001 bearing a deletion of the native pikromycin polyketide synthase gene, and the resulting strain generated flavones from exogenously fed flavanones. In addition, a recombinant S. venezuelae mutant expressing a codon-optimized flavanone 3beta-hydroxylase gene from Citrus siensis and a flavonol synthase gene from Citrus unshius also successfully produced flavonols.     

Solution structure and function of YndB,an AHSA1 protein from Bacillus subtilis

The solution structure of the Bacillus subtilis protein YndB has been solved using NMR to investigate proposed biological functions. The YndB structure exhibits the helix‐grip fold, which consists of a β‐sheet with two small and one long α‐helix, forming a hydrophobic cavity that preferentially binds lipid‐like molecules. Sequence and structure comparisons with proteins from eukaryotes, prokaryotes, and archaea suggest that YndB is very similar to the eukaryote protein Aha1, which binds to the middle domain of Hsp90 and induces ATPase activity. On the basis of these similarities, YndB has been classified as a member of the activator of Hsp90 ATPase homolog 1‐like protein (AHSA1) family with a function that appears to be related to stress response. An in silico screen of a compound library of ～18,500 lipids was used to identify classes of lipids that preferentially bind YndB. The in silico screen identified, in order of affinity, the chalcone/hydroxychalcone, flavanone, and flavone/flavonol classes of lipids, which was further verified by 2D ¹H‐¹⁵N HSQC NMR titration experiments with trans‐chalcone, flavanone, flavone, and flavonol. All of these compounds are typically found in plants as precursors to various flavonoid antibiotics and signaling molecules. The sum of the data suggests an involvement of YndB with the stress response of B. subtilis to chalcone‐like flavonoids released by plants due to a pathogen infection. The observed binding of chalcone‐like molecules by YndB is likely related to thesymbiotic relationship between B. subtilis and plants. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Mass spectrometry of silylated flavone and flavanone glycosides

The electron impact mass spectra of 14 trimethylsilylated flavone and flavanone mono- and diglycosides are reported for the first time. All spectra show well defined molecular ion peaks and those of O-glycosides additionally give evidence of the aglycone and the sugar(s), the sugar attachment, the sugar sequence and the interglycosidic linkage (of flavone and flavanone biosides).     

Mechanisms involved in the chemoprevention of flavonoids

Flavonoids, widespread in edible plants, have been studied extensively for their anticarcinogenic properties. However, only few studies have been done with these constituents being administered by the dietary route. In our research, the effects of feeding rats with flavone, flavanone, tangeretin, and quercetin were investigated on two steps of aflatoxin B1 (AFB1)-induced hepatocarcinogenesis (initiation and promotion). Nonpolar flavonoids such as flavone, flavanone and tangeretin administered through the initiation period, decreased the number of -gamma-glutamyl transpeptidase-preneoplastic foci. In the same conditions of administration, quercetin, a polyhydroxylated flavonoid, showed no protective effect. Moreover, feeding rats with flavanone during the phenobarbital-induced promotion step significantly reduced the areas of placental glutathione S-transferase preneoplastic foci. Quercetin, flavone, and tangeretin, administered in the same conditions, caused no significant effect. Therefore flavanone act as an anti-initiator as well as an anti-promotor. Several mechanisms were involved in the anti-initiating effects of flavone, flavanone, and tangeretin: enhancement of enzymes involved in the detoxication of AFB1 (glutathione S-transferase, UDP-glucuronyl transferase), increase of the formation of AFB1-glutathione conjugates and inhibition of the binding of AFB1 to DNA. Although the relevance of these data to the human situation remains to be demonstrated, they confirm that several flavonoids administered by the dietary route possess promising chemoprotective effects.     

Changes in citrus leaf flavonoid concentrations resulting from blight-induced zinc-deficiency

Increased flavonoid concentrations were found to correlate with the elevated levels of leaf phenolic compounds occurring in blight-induced zinc-deficient citrus. In orange (Citrus sinensis L.) leaves, the increases occurred primarily in hesperidin and diosmin, whereas in grapefruit (C. paradisi Macf.) the largest increases occurred in naringin and rhoifolin. Zinc-deficiency occurring in the blighted citrus leaves appeared to be the important contributing factor to the increased flavonoid content. Although the leaves from trees with blight were typically smaller than leaves from unaffected trees, the increased flavonoid content was not significantly due to a concentration effect. Large differences occurred in the percent increases in concentrations of certain citrus leaf flavonoids. While large increases occurred for a number of flavanone and flavone glycosides, much smaller percent increases occurred for other minor flavone glycosides, and the polymethoxyflavone aglycones. The parallel increases occurring in the concentrations of certain flavone glycosides and their flavanone analogs provide a further indication that flavanone glycosides are precursors in the biosynthesis of flavone glycosides in citrus.     

Total synthesis of three naturally occurring 6,8-di-C-glycosylflavonoids: phloretin, naringenin, and apigenin bis-C-beta-D-glucosides

Three naturally occurring di-C-glycosylflavonoids, phloretin (dihydrochalcone), naringenin (flavanone), and apigenin (flavone) bis-6,8-C-beta-D-glucopyranosides (4, 5, and 6), were synthesized in total yields of 52.3%, 53.5%, and 36.4%, respectively, starting from the key compound, di-C-beta-D-glucopyranosylphloroacetophenone (1). Benzyl protection of the phenolic hydroxyls in 1 and a subsequent aldol condensation with benzyloxybenzaldehyde led to the production of chalcone 3, which, after hydrogenolysis or acid hydrolysis and deprotection, gave 4 and 5, respectively. The acetylation of 5, followed by DDQ oxidation and deprotection, gave 6.     

Biochemical and molecular characterization of flavonoid 7-sulfotransferase from Arabidopsis thaliana

Flavonoid compounds play important roles as flower pigments, stress metabolites formed in response to UV, during pollen germination and for polar auxin transport (Trends Plant Sci. 1 (1996) 377). Flavonoid sulfate esters are common in plants, especially the Asteraceae; however, due to the lack of information regarding the factors that regulate their accumulation, their exact role remains to be elucidated. The biosynthesis of flavonol sulfate esters is catalyzed by a number of position specific flavonol sulfotransferases (STs). An Arabidopsis thaliana database search has allowed us to identify and classify 18 putative ST coding sequences. We report here the cloning and characterization of the AtST3a member of this family that is expressed at early stages of seedling development and in the inflorescence stem and siliques of mature plants. The recombinant AtST3a protein exhibits strict specificity for position 7 of flavonoids. In contrast to previously characterized flavonol 7-ST from Flaveria bidentis that sulfonates only flavonol disulfates, AtST3a was found to accept as substrates a number of flavonols and flavone aglycones, as well as their monosulfate esters. The discovery of a flavonol ST from A. thaliana suggests that flavonol sulfates are more widely distributed than originally believed and this model plant could be used to study their biological significance.     

Four glucosyltransferases from rice: cDNA cloning, expression, and characterization

Four UDP-dependent glucosyltransferase (UGT) genes, UGT706C1, UGT706D1, UGT707A3, and UGT709A4 were cloned from rice, expressed in Escherichia coli, and purified to homogeneity. In order to find out whether these enzymes could use flavonoids as glucose acceptors, apigenin, daidzein, genistein, kaempferol, luteolin, naringenin, and quercetin were used as potential glucose acceptors. UGT706C1 and UGT707A3 could use kaempferol and quercetin as glucose acceptors and the major glycosylation position was the hydroxyl group of carbon 3 based on the comparison of HPLC retention times, UV spectra, and NMR spectra with those of corresponding authentic flavonoid 3-O-glucosides. On the other hand, UGT709A4 only used the isoflavonoids genistein and daidzein and transferred glucose onto 7-hydroxyl group. In addition, UGT706D1 used a broad range of flavonoids including flavone, flavanone, flavonol, and isoflavone, and produced at least two products with glycosylation at different hydroxyl groups. Based on their substrate preferences and the flavonoids present in rice, the in vivo function of UGT706C1, UGT706D1, and UGT707A3 is most likely the biosynthesis of kaempferol and quercetin glucosides.