Flavonoid Substrates Research Articles

Role of Serine 286 in cosubstrate binding and catalysis of a flavonolO-methyltransferase

O-Methyltransferases catalyze the transfer of the methyl groups of S-adenosyl-L-methionine to specific hydroxyl groups of several classes of flavonoid compounds. Of the several cDNA clones isolated from a Chrysosplenium americanum library, FOMT3' encodes the 3'/5'-O-methylation of partially methylated flavonols. The recombinant protein of another clone, FOMTx which differs from FOMT3' by a single amino acid residue (Ser286Arg) exhibits no enzymatic activity towards any of the flavonoid substrates tested. Replacement of Ser 286 in FOMT3' with either Ala, Leu, Lys or Thr, almost abolished O-methyltransferase activity. In contrast with FOMT3', no photoaffinity labeling could be achieved using [(14)CH(3)]AdoMet with the mutant recombinant proteins indicating that Ser 286 is also required for cosubstrate binding. These results are corroborated by isothermal titration microcalorimetry measurements. Circular dichroism spectra ruled out any significant conformational differences in the secondary structures of both FOMT3' and Ser286Arg. Modeling FOMT3' on the structure of chalcone methyltransferase indicates that serine 286 is greater than 10 A from any of the residues of the active site or the AdoMet binding site of FOMT3'. At the same time, residues 282 to 290 are conserved in most of the Chrysosplenium americanum OMTs. These residues form a large part of the subunit interface, and at least five of these residues are within 4 A of the opposing subunit. It would appear, therefore, that mutations in Ser286 exert their influence by altering the contacts between the subunits and that these contacts are necessary for maintaining the integrety of the AdoMet binding site and active site of this group of enzymes.

In vitro flavon-3-ol oxidation mediated by a B ring hydroxylation pattern.

Flavonols are potent naturally occurring antioxidants. Chemical oxidation reactions in combination with modern spectroscopic techniques have been employed to identify oxidized flavonoid products. Although many oxidized derivatives have been generated from commercially available starting materials, few studies have developed a sequence of flavonoid substrates with a particular hydroxylation pattern to probe the mechanism of flavonoid oxidation. Here, we use AIBN (2,2'-azobisisobutyronitrile) in combination with a series of hydroxylated flavonols to probe the mechanism of flavonoid dimer formation and the role of singly or doubly oxidized species in generating and promoting oxidized flavonoid products. 3-Methoxyquercetin (3-hydroxyl group blocked) and luteolin (lack of 3-hydroxyl) were reacted with AIBN alone or with a second flavonol to serve as a C-3 hydroxyl donor to examine the mechanism of dimer formation. 3-Hydroxyflavones with increasing hydroxyl substitutions in the B ring were also reacted with AIBN in the presence or absence of an external nucleophile to examine the role of various hydroxyls in the formation of a carbocation intermediate via a doubly oxidized species. The presence of a free C-3 hydroxyl, coupled with a B ring ortho hydroxy unit, appears essential for dimer formation. An increase in the number of hydroxyls in the B ring facilitates products generated from a doubly oxidized species.

Mechanistic Studies on Three 2-Oxoglutarate-dependent Oxygenases of Flavonoid Biosynthesis: ANTHOCYANIDIN SYNTHASE, FLAVONOL SYNTHASE, AND FLAVANONE 3β-HYDROXYLASE

Anthocyanidin synthase (ANS), flavonol synthase (FLS), and flavanone 3beta-hydroxylase (FHT) are involved in the biosynthesis of flavonoids in plants and are all members of the family of 2-oxoglutarate- and ferrous iron-dependent oxygenases. ANS, FLS, and FHT are closely related by sequence and catalyze oxidation of the flavonoid "C ring"; they have been shown to have overlapping substrate and product selectivities. In the initial steps of catalysis, 2-oxoglutarate and dioxygen are thought to react at the ferrous iron center producing succinate, carbon dioxide, and a reactive ferryl intermediate, the latter of which can then affect oxidation of the flavonoid substrate. Here we describe work on ANS, FLS, and FHT utilizing several different substrates carried out in 18O2/16OH2, 16O2/18OH2, and 18O2/18OH2 atmospheres. In the 18O2/16OH2 atmosphere close to complete incorporation of a single 18O label was observed in the dihydroflavonol products (e.g. (2R,3R)-trans-dihydrokaempferol) from incubations of flavanones (e.g. (2S)naringenin) with FHT, ANS, and FLS. This and other evidence supports the intermediacy of a reactive oxidizing species, the oxygen of which does not exchange with that of water. In the case of products formed by oxidation of flavonoid substrates with a C-3 hydroxyl group (e.g. (2R,3R)-trans-dihydroquercetin), the results imply that oxygen exchange can occur at a stage subsequent to initial oxidation of the C-ring, probably via an enzyme-bound C-3 ketone/3,3-gem-diol intermediate.

Bio-fermentation of modified flavonoids: an example of in vivo diversification of secondary metabolites

A bio-fermentation technique was used for the in vivo diversification of flavonoid structures based on expression in Escherichia coli of six O-methyltransferases (OMTs) from Mentha x piperita and one O-glucosyltransferase (GT) each from Arabidopsis thaliana and Allium cepa. Enzymes were shown to be regio-specific in in vitro experiments and modified a broad range of flavonoid substrates at various positions. Using the flavonol quercetin as a model substrate, we show that the product spectrum produced with the in vivo approach is identical to that found in vitro. Additionally, using mixed cultures of E. coli expressing different classes of modifying genes (OMTs and GTs), the production of polymethylated flavonoid glucosides was observed. This report demonstrates the potential to increase the structural diversity of plant secondary metabolites using a multi-enzyme, bio-fermentation approach.

Cloning and regiospecificity studies of two flavonoid glucosyltransferases from Allium cepa

Two UDP-glucose-dependent flavonoid glucosyltransferases (EC 2.4.1.-) isolated from the epidermal layer of yellow onion ( Allium cepa) were functionally expressed in Escherichia coli and their substrate specificity investigated. The two enzymes exhibited different substrate- and regio-specificity profiles. A. cepa UGT73G1 used a wide range of different flavonoid substrates including flavonoids not naturally occurring in onion. Regiospecificity was indicated for hydroxyl-groups of the C-3, C-7 and C-4′ positions of the flavan backbone structure to yield flavonoid mono- and diglucosides. In contrast, A. cepa UGT73J1 showed activity only with the flavonoid mono-glucoside isoquercitrin and the isoflavone aglycone genistein, with regiospecificity for the C-7 position. The regiospecificity for both enzymes included positions that are glucosylated in flavonoids of onion bulbs, indicating their involvement in flavonoid biosynthesis in A. cepa.

Regioselectivity of glucosylation of caffeic acid by a UDP-glucose:glucosyltransferase is maintained in planta.

Caffeic acid is a phenylpropanoid playing an important role in the pathways leading to lignin synthesis and the production of a wide variety of secondary metabolites. The compound is also an antioxidant and has potential utility as a general protectant against free radicals. Three glucosylated forms of caffeic acid are known to exist: the 3- O - and 4- O -glucosides and the glucose ester. This study describes for the first time a glucosyltransferase [UDP-glucose:glucosyltransferase (UGT)] that is specific for the 3-hydroxyl, and not the 4-hydroxyl, position of caffeic acid. The UGT sequence of Arabidopsis, UGT71C1, has been expressed as a recombinant fusion protein in Escherichia coli, purified and assayed against a range of substrates in vitro. The assay confirmed that caffeic acid as the preferred substrate when compared with other hydroxycinnamates, although UGT71C1 also exhibited substantial activity towards flavonoid substrates, known to have structural features that can be recognized by many different UGTs. The expression of UGT71C1 in transgenic Arabidopsis was driven by the constitutive cauliflower mosaic virus 35 S (CaMV35S) promoter. Nine independent transgenic lines were taken to homozygosity and characterized by Northern-blot analysis, assay of enzyme activity in leaf extracts and HPLC analysis of the glucosides. The level of expression of UGT71C1 was enhanced considerably in several lines, leading to a higher level of the corresponding enzyme activity and a higher level of caffeoyl-3- O -glucoside. The data are discussed in the context of the utility of UGTs for natural product biotransformations.

Daphnetin Methylation by a Novel O-Methyltransferase Is Associated with Cold Acclimation and Photosystem II Excitation Pressure in Rye

In plants, O-methylation of phenolic compounds plays an important role in such processes as lignin synthesis, flower pigmentation, chemical defense, and signaling. However, apart from phenylpropanoids and flavonoids, very few enzymes involved in coumarin biosynthesis have been identified. We report here the molecular and biochemical characterization of a gene encoding a novel O-methyltransferase that catalyzes the methylation of 7,8-dihydroxycoumarin, daphnetin. The recombinant protein displayed an exclusive methylation of position 8 of daphnetin. The identity of the methylated product was unambiguously identified as 7-hydroxy-8-methoxycoumarin by co-chromatography on cellulose TLC and coelution from high performance liquid chromatography, with authentic synthetic samples, as well as by UV, mass spectroscopy, (1)H NMR spectral analysis, and NOE correlation signals of the relevant protons. Northern blot analysis and enzyme activity assays revealed that the transcript and corresponding enzyme activity are up-regulated by both low temperature and photosystem II excitation pressure. Using various phenylpropanoid and flavonoid substrates, we demonstrate that cold acclimation of rye leaves increases O-methyltransferase activity not only for daphnetin but also for the lignin precursors, caffeic acid, and 5-hydroxyferulic acid. The significance of this novel enzyme and daphnetin O-methylation is discussed in relation to its putative role in modulating cold acclimation and photosystem II excitation pressure.

AN9, a petunia glutathione S-transferase required for anthocyanin sequestration, is a flavonoid-binding protein.

AN9 is a glutathione S-transferase from petunia (Petunia hybrida) required for efficient anthocyanin export from the site of synthesis in the cytoplasm into permanent storage in the vacuole. For many xenobiotics it is well established that a covalent glutathione (GSH) tag mediates recognition of molecules destined for vacuolar sequestration by a tonoplast-localized ATP-binding cassette pump. Here we inquired whether AN9 catalyzes the formation of GSH conjugates with flavonoid substrates. Using high-performance liquid chromatography analysis of reaction mixtures containing enzyme, GSH, and flavonoids, including anthocyanins, we could detect neither conjugates nor a decrease in the free thiol concentration. These results suggest that no conjugate is formed in vitro. However, AN9 was shown to bind flavonoids using three assays: inhibition of the glutathione S-transferase activity of AN9 toward the common substrate 1-chloro 2,4-dinitrobenzene, equilibrium dialysis, and tryptophan quenching. We conclude that AN9 is a flavonoid-binding protein, and propose that in vivo it serves as a cytoplasmic flavonoid carrier protein.

Characterization of Two cDNA Clones Which EncodeO-Methyltransferases for the Methylation of both Flavonoid and Phenylpropanoid Compounds

EnzymaticO-methylation of phenylpropanoid and flavonoid compounds is believed to be catalyzed by distinct classes ofO-methyltransferases [EC 2.1.1.6x]. The O-methylated derivatives of phenylpropanoids and flavonoids play an important role in lignification and as antimicrobial compounds, respectively. Two cDNA clones,OMT1andOMT2,which differ in three amino acid residues were isolated and characterized from the semiaquatic freshwater weedChrysosplenium americanum(Saxifragaceae). These two novel cDNA clones encode enzymes which catalyze the 3′-O-methylation of the flavonoid aglycones luteolin and quercetin, although they also catalyze the efficient 3/5-O-methylation of the phenylpropanoids caffeic and 5-hydroxyferulic acids, respectively. Both recombinant proteins were partially purified from anEscherichia coliexpression system and their kinetic parameters were compared using two flavonoids and two phenylpropanoids as substrates. Although both gene products methylate caffeic acid and 5-hydroxyferulic acid to a similar extent, they exhibit a threefold higher affinity for and a four- to sixfold increase in turnover of flavonoid compounds. The gene product ofOMT1accepts the flavonoid substrates luteolin and quercetin for methylation at a higher rate than that ofOMT2,as indicated by a two- to threefold increase in itsVmaxvalues and turnover ratios. The fact thatC. americanumaccumulates a variety of highly methylated flavonols and exhibits little lignification suggests that these two flavonoid OMT clones have retained their ability toO-methylate phenylpropanoids as well. These results are discussed in relation to differences in the amino acid sequences of these two clones, as well as with otherO-methyltransferases, and the evolutionary divergence of these genes in plants.

β-Glucosylation of plant phenolics by phenol β-glucosyltransferase in larval tissues of the tobacco hornworm, Manduca sexta (L.)

Activity of phenol β-glucosyltransferase (PGT) in labial gland and fat body tissue preparations from feeding 5th stadium larval tobacco hornworms, Manduca sexta, towards a variety of plant phenolics was determined by HPLC analysis of the β-glucoside product formed. The enzymes in the 15,000 g fractions of tissue homogenates with uridine 5′-diphosphoglucose as a cofactor showed a wide range of activity in conjugating simple mono-, di-, and triphenols; phenolic acids, aldehydes, and alcohols; and coumarins and flavonoids. Highest specific activity of both labial glan and fat body enzyme preparations occured with diphenol substrates such as catechol, hydroquinone, and vanillin, whereas lowest activities generally were observed with flavonoid substrates. The labial gland PGT had higher specific activity for the simple phenolic substrates than did the fat body enzyme. However, the coumarins and flavonoids were glucosylated at nearly the same rates by both enzyme preparations. Although the functions of PGT in labial glands and fat body are unknown, evidence from this study suggests that they could be important in conjugating a number of potentially toxic plant phenols occuring in the larval diet.

Purification and characterization of S-adenosyl- l-methionine: Caffeic acid 3- O-methyltransferase from suspension cultures of alfalfa ( Medicago sativa L.)

Caffeic acid O-methyltransferase (COMT) is one of a group of proteins present in alfalfa cell cultures which can be photoaffinity labeled with S-adenosyl- l-[ methyl- 3H]methionine. The enzyme was purified to homogeneity from elicitor-treated suspension cultures and shown to exist as an active monomer of subunit M r 41,000. COMT could be separated into two forms on the basis of their isoelectric points and relative affinities for S-adenosylmethionine and S-adenosylhomocysteine. Both forms had equal affinities for caffeic acid, were highly specific for the 3-hydroxyl group of substituted cinnamic acids, and exhibited negligible activity toward flavonoid substrates. An antiserum raised against COMT from aspen immunoprecipitated alfalfa COMT activity. Peptide mapping studies indicated that the two forms of COMT and an isoflavone O-methyltransferase from alfalfa are closely related proteins. The extractable activity of COMT doubled over a 48-h period following exposure of alfalfa cell suspensions to a yeast elicitor preparation, and this was associated with a small change in the relative proportions of the two forms of the enzyme.

Enzymatic synthesis of polymethylated flavonols in Chrysosplenium americanum. II. Substrate interaction and product inhibition studies of flavonol 3-, 6-, and 4′- O-methyltransferases

The steady-state kinetic behavior of three position-specific O-methyltransferases (3-, 4′-, and 6-OMTs) was compared with reference to substrate inhibition patterns in Chrysosplenium americanum. The 6-OMT was severely inhibited by the flavonoid substrate at concentrations close to K m , whereas the other two enzymes were less affected by their respective flavonoid substrates. Substrate interaction kinetics for the 6-OMT gave converging lines consistent with a sequential binding mechanism, whereas the data generated for the 3- and 4′-OMTs could be fitted to the equation for a ping-pong mechanism or to that of a sequential binding mechanism where K ia was much smaller K a . More information on the mechanism of reaction was obtained from product inhibition studies. The three enzymes exhibited competitive inhibition patterns between S-adenosyl- l-methionine (SAM) and S-adenosyl- l-homocysteine (SAH), whereas other patterns were either noncompetitive or uncompetitive. The steadystate kinetic properties of the 3-, 4′-, and 6-OMTs were consistent with a sequential ordered reaction mechanism, in which SAM and SAH were leading reaction partners and included an abortive EQB complex. Product inhibition constants were sufficiently low to suggest that these may be important in regulating the pathway of polymethylated flavonoid synthesis. It was suggested that due to their greater sensitivity to inhibition by SAH, the OMTs involved in earlier steps of the methylation sequence may regulate the rate of synthesis of final products in Chrysosplenium.

O-methylation of flavonoids by cell-free extracts of calamondin orange

Cell-free extracts of calamondin orange ( Citrus mitis) catalysed the O-methylation of almost all hydroxyls of a number of flavonoids, indicating the existence in citrus tissues of ortho, meta, para and 3- O-methyltransferases. The latter, hitherto unreported enzyme, catalysed the formation of 3- O-methyl ethers of galangin and quercetin. The stepwise O-methylation of a number of compounds, especially quercetin and quercetagetin, tends to suggest a coordinated sequence of O-methylations on the surface of a multienzyme complex. The methyl acceptor abilities of the flavonoid substrates used are discussed in relation to their hydroxyl substitution patterns and their negative electron density distribution.

O-methylation of flavonoid substrates by a partially purified enzyme from soybean cell suspension cultures

A methyltransferase, which catalyzes the methylation of luteolin ( K m , 16 μM) using S-adenosyl- l-methionine as the methyl donor, has been purified about 38-fold from cell suspension cultures of soybean ( Glycine max L., var. Mandarin). The following 3,4-dihydroxy phenolic compounds were also methylated: luteolin 7- O-glucoside ( K m , 28 μ m), quercetin ( K m , 35 μ m), eriodictyol ( K m , 75 μ m), 5-hydroxyferulic acid ( K m , 227 μ m), dihydroquercetin ( K m , 435 μ m), and caffeic acid ( K m , 770 μ m). Rutin and quercetin 3- O-glucoside were poor substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of p-coumaric acid, m-coumaric acid, ferulic acid, isoferulic acid, sinapic acid, apigenin, or naringenin. While the isoflavones biochanin A and daidzein did not serve as substrates, texasin (6,7-dihydroxy-3′-methoxyisoflavone) was methylated ( K m , 35 μ m). The methylation of caffeic acid and quercetin showed a pH optimum of 8.6–8.9. The enzyme required Mg 2+ ions for maximum activity (approximately 1 m m) and could be totally inhibited by EDTA (10 m m). The K m for S-adenosyl- l-methionine was 11 μ m. S-Adenosyl- l-homocysteine inhibited the methylation of luteolin by S-adenosyl- l-methionine.