Cloning and functional characterisation of two regioselective flavonoid glucosyltransferases from Beta vulgaris

Two full-length cDNAs encoding flavonoid-specific glucosyltransferases, UGT73A4 and UGT71F1, were isolated from a cDNA library of Beta vulgaris (Amaranthaceae) cell suspension cultures. They displayed high identity to position-specific betanidin and flavonoid glucosyltransferases from Dorotheanthus bellidiformis (Aizoaceae) and to enzymes with similar substrate specificities from various plant families. The open reading frame of the sequences encode proteins of 476 (UGT73A4) and 492 (UGT71F1) amino acids with calculated molecular masses of 54.07kDa and 54.39kDa, and isoelectric points of 5.8 and 5.6, respectively. Both enzymes were functionally expressed in Escherichia coli as His- and GST-tagged proteins, respectively. They exhibited a broad substrate specificity, but a distinct regioselectivity, glucosylating a variety of flavonols, flavones, flavanones, and coumarins. UGT73A4 showed a preference for the 4'- and 7-OH position in the flavonoids, whereas UGT71F1 preferentially glucosylated the 3- or the 7-OH position. Glucosylation of betanidin, the aglycone of the major betacyanin, betanin, in B. vulgaris was also observed to a low extent by both enzymes. Several O-glycosylated vitexin derivatives isolated from leaves of young B. vulgaris plants and rutin obtained from B. vulgaris tissue culture are discussed as potential endogenous products of UGT73A4 and UGT71F1. The results are analyzed with regard to evolution and specificity of plant natural product glucosyltransferases.     

Mining of UDP-glucosyltrfansferases in licorice for controllable glycosylation of pentacyclic triterpenoids

Pentacyclic triterpenoids have wide applications in the pharmaceutical industry. The precise glucosylation at C-3 OH of pentacyclic triterpenoids mediated by uridine 5'-diphospho-glucosyltransferase (UDP-glucosyltransferase [UGT]) is an important way to produce valuable derivatives with various improved functions. However, most reported UGTs suffer from low regiospecificity toward the OH and COOH groups of pentacyclic triterpenoids, which significantly decreases the reaction efficiency. Here, two new UGTs (UGT73C33 and UGT73F24) were discovered in Glycyrrhiza uralensis. UGT73C33 showed high activity but poor regioselectivity toward the C-3 OH and C-30 COOH of pentacyclic triterpenoid, producing three glucosides. UGT73F24 showed rigid regioselectivity toward C-3 OH of typical pentacyclic triterpenoids producing only C-3 O-glucosylated derivatives. In addition, UGT73C33 and UGT73F24 showed a broad substrate scope toward typical flavonoids with various sugar donors. Next, the substrate recognition mechanism of UGT73F24 toward glycyrrhetinic acid (GA) and UDP-glucose was investigated. Two key residues, I23 and L84, were identified to determine activity, and site-directed mutagenesis of UGT73F24-I23G/L84N increased the activity by 4.1-fold. Furthermore, three in vitro GA glycosylation systems with UDP-recycling were constructed, and high yields of GA-3-O-Glc (1.25 mM), GA-30-O-Glc (0.61 mM), and GA-di-Glc (0.26 mM) were obtained. The de novo biosynthesis of GA-3-O-glucose (26.31 mg/L) was also obtained in engineered yeast.     

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.     

UGT73C6 and UGT78D1, glycosyltransferases involved in flavonol glycoside biosynthesis in Arabidopsis thaliana

Flavonol glycosides constitute one of the most prominent plant natural product classes that accumulate in the model plant Arabidopsis thaliana. To date there are no reports of functionally characterized flavonoid glycosyltransferases in Arabidopsis, despite intensive research efforts aimed at both flavonoids and Arabidopsis. In this study, flavonol glycosyltransferases were considered in a functional genomics approach aimed at revealing genes involved in determining the flavonol-glycoside profile. Candidate glycosyltransferase-encoding genes were selected based on homology to other known flavonoid glycosyltransferases and two T-DNA knockout lines lacking flavonol-3-O-rhamnoside-7-O-rhamnosides (ugt78D1) and quercetin-3-O-rhamnoside-7-O-glucoside (ugt73C6 and ugt78D1) were identified. To confirm the in planta results, cDNAs encoding both UGT78D1 and UGT73C6 were expressed in vitro and analyzed for their qualitative substrate specificity. UGT78D1 catalyzed the transfer of rhamnose from UDP-rhamnose to the 3-OH position of quercetin and kaempferol, whereas UGT73C6 catalyzed the transfer of glucose from UDP-glucose to the 7-OH position of kaempferol-3-O-rhamnoside and quercetin-3-O-rhamnoside, respectively. The present results suggest that UGT78D1 and UGT73C6 should be classified as UDP-rhamnose:flavonol-3-Orhamnosyltransferase and UDP-glucose:flavonol-3-O-glycoside-7-O-glucosyltransferase, respectively.     

Regioselective synthesis of plant (iso)flavone glycosides in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The flavonoids genistein, biochanin A, luteolin, quercetin, and kaempferol are plant natural products with potentially useful pharmacological and nutraceutical activities. These natural products usually exist in plants as glycosides, and their glycosylation has a remarkable influence on their pharmacokinetic properties. The glycosyltransferases UGT71G1 and UGT73C8 from Medicago truncatula are excellent reagents for the regioselective glycosylation of (iso)flavonoids in Escherichia coli grown in Terrific broth. Ten to 20 mg/L of either genistein or biochanin A 7-O-glucoside was produced after feeding genistein or biochanin A to E. coli expressing UGT71G1, and similar levels of luteolin 4'-O- and 7-O-glucosides were produced after feeding luteolin to cultures expressing UGT73C8. For the production of kaempferol 3-O-glucoside or quercetin 3-O-glucoside, the Phe148Val or Tyr202Ala mutants of UGT71G1 were employed. Ten to 16 mg/L of either kaempferol 3-O- or quercetin 3-O-glucosides were produced on feeding kaempferol or quercetin to E. coli expressing these enzymes. More than 90% of the glucoside products were released to the medium, facilitating their isolation.     

Characterization of flavonoid 7-O-glucosyltransferase from Arabidopsis thaliana

Most flavonoids found in plants exist as glycosides, and glycosylation status has a wide range of effects on flavonoid solubility, stability, and bioavailability. Glycosylation of flavonoids is mediated by Family 1 glycosyltransferases (UGTs), which use UDP-sugars, such as UDP-glucose, as the glycosyl donor. AtGT-2, a UGT from Arabidopsis thaliana, was cloned and expressed in Escherichia coli as a gluthatione S-transferase fusion protein. Several compounds, including flavonoids, were tested as potential substrates. HPLC analysis of the reaction products indicated that AtGT-2 transfers a glucose molecule into several different kinds of flavonoids, eriodictyol being the most effective substrate, followed by luteolin, kaempferol, and quercetin. Based on comparison of HPLC retention times with authentic flavonoid 7-O-glucosides and nuclear magnetic resonance spectroscopy, the glycosylation position in the reacted flavonoids was determined to be the C-7 hydroxyl group. These results indicate that AtGT-2 encodes a flavonoid 7-O-glucosyltransferase.     

cDNA cloning and expression of isoflavonoid-specific glucosyltransferase from<Emphasis Type="Italic"> Glycyrrhiza echinata</Emphasis> cell-suspension cultures

A cDNA encoding UDP-glucose: formononetin 7-O-glucosyltransferase, designated UGT73F1, was cloned from yeast extract-treated Glycyrrhiza echinata L. cell-suspension cultures using probes from Scutellaria baicalensis UDP-glucose: flavonoid 7-O-glucosyltransferase. The open reading frame of the UGT73F1 cDNA encodes a 441-amino-acid protein with a predicted molecular mass of 48.7 kDa. The deduced amino acid sequence showed that the protein is related to the stress-inducible glucosyltransferases. UGT73F1 mRNA was not detected in untreated G. echinata cultures but was transiently induced by treatment with yeast extract. Recombinant UGT73F1 was expressed as a histidine-tag fusion protein in Escherichia coli and purified to near homogeneity by nickel chelate chromatography. The purified recombinant enzyme was selective for isoflavonoid, formononetin and daidzein as substrates, while flavonoids and various tested non-flavonoid compounds were poor substrates.Abbreviations GT UDP-glycosyltransferase - rUGT73F1 recombinant UGT73F1 - UBGT: UDP-glucose: baicalein 7-O-glucosyltransferase The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB098614.     

Molecular genetic analysis of tandemly located glycosyltransferase genes,UGT73B1,UGT73B2, andUGT73B3, inArabidopsis thaliana

In theArabidopsis genome, approximately 120 UDP-glycosyltransferases (UGTs) have been annotated. They generally catalyze the transfer of sugars to various acceptor molecules, including flavonoids. To better understand their physiological roles, we analyzed a tandemly located putative flavonoid UGT cluster comprisingUGT73B1, UGT73B2, andUGT73B3 on Chromosome IV. We then isolated four loss-of-function mutations —ugt73b1- 1, ugt73b2- 1, ugt73b3- 1, andugt73b3- 2. In our expression analysis, the closely related UCTs exhibited tissue-specific patterns of expression that were severely altered in their respective mutant plants. For example,UGT73B2 was up-regulated inugt73b1- 1, whereasUGT73B7 was highly expressed inugt73b2- 1, ugt73b3- t, andugt73b3- 2. Interestingly, each recessive mutant was resistant to methyl viologen (paraquat), an herbicide thought to cause oxidative stress. Our results suggest thatUGTs play an important role in the glycosylation pathways when responding to oxidative stress.     

Molecular and biochemical characterisation of a serine acetyltransferase of onion, Allium cepa (L.)

We have previously cloned a cDNA, designated SAT1, corresponding to a gene coding for a serine acetyltransferase (SAT) from onion (Allium cepa L.). The SAT1 locus was mapped to chromosome 7 of onion using a single-stranded conformation polymorphism (SSCP) in the 3' UTR of the gene. Northern analysis has demonstrated that expression of the SAT1 gene is induced in leaf tissue in response to low S-supply. Phylogenetic analysis has placed SAT1 in a strongly supported group (100% bootstrap) that comprises sequences that have been characterised biochemically, including Allium tuberosum, Spinacea oleracea, Glycine max, Citrullus vulgaris, and SAT5 (AT5g56760) of Arabidopsis thaliana. This group can be divided further with the SAT1 of A. cepa sequence grouping strongly with the A. tuberosum sequence. Translation of SAT1 from onion generates a protein of 289 amino acids with a calculated molecular mass of 30,573 Da and pI of 6.52. The conserved G277 and H282 residues that have been identified as critical for L-cysteine inhibition are observed at G272 and H277. SAT1 has been cloned into the pGEX plasmid, expressed in E. coli and SAT activity of the recombinant enzyme has been measured as acetyl-CoA hydrolysis detected at 232 nm. A Km of 0.72 mM was determined for l-serine as substrate, a Km of 92 microM was calculated with acetyl-CoA as substrate, and an inhibition curve for L-cysteine generated an IC50 value of 3.1 microM. Antibodies raised against the recombinant SAT1 protein recognised a protein of ca. 33 kDa in whole leaf onion extracts. These properties of the SAT1 enzyme from onion are compared with other SAT enzymes characterised from closely related species.     

A kinetic analysis of regiospecific glucosylation by two glycosyltransferases of Arabidopsis thaliana: domain swapping to introduce new activities

Plant Family 1 glycosyltransferases (GTs) recognize a wide range of natural and non-natural scaffolds and have considerable potential as biocatalysts for the synthesis of small molecule glycosides. Regiospecificity of glycosylation is an important property, given that many acceptors have multiple potential glycosylation sites. This study has used a domain-swapping approach to explore the determinants of regiospecific glycosylation of two GTs of Arabidopsis thaliana, UGT74F1 and UGT74F2. The flavonoid quercetin was used as a model acceptor, providing five potential sites for O-glycosylation by the two GTs. As is commonly found for many plant GTs, both of these enzymes produce distinct multiple glycosides of quercetin. A high performance liquid chromatography method has been established to perform detailed steady-state kinetic analyses of these concurrent reactions. These data show the influence of each parameter in determining a GT product formation profile toward quercetin. Interestingly, construction and kinetic analyses of a series of UGT74F1/F2 chimeras have revealed that mutating a single amino acid distal to the active site, Asn-142, can lead to the development of a new GT with a more constrained regiospecificity. This ability to form the 4 '-O-glucoside of quercetin is transferable to other flavonoid scaffolds and provides a basis for preparative scale production of flavonoid 4 '-O-glucosides through the use of whole-cell biocatalysis.     

Local Differentiation of Sugar Donor Specificity of Flavonoid Glycosyltransferase in Lamiales

Flavonoids are most commonly conjugated with various sugar moieties by UDP-sugar:glycosyltransferases (UGTs) in a lineage-specific manner. Generally, the phylogenetics and regiospecificity of flavonoid UGTs are correlated, indicating that the regiospecificity of UGT differentiated prior to speciation. By contrast, it is unclear how the sugar donor specificity of UGTs evolved. Here, we report the biochemical, homology-modeled, and phylogenetic characterization of flavonoid 7-O-glucuronosyltransferases (F7GAT), which is responsible for producing specialized metabolites in Lamiales plants. All of the Lamiales F7GATs were found to be members of the UGT88-related cluster and specifically used UDP-glucuronic acid (UDPGA). We identified an Arg residue that is specifically conserved in the PSPG box in the Lamiales F7GATs. Substitution of this Arg with Trp was sufficient to convert the sugar donor specificity of the Lamiales F7GATs from UDPGA to UDP-glucose. Homology modeling of the Lamiales F7GAT suggested that the Arg residue plays a critical role in the specific recognition of anionic carboxylate of the glucuronic acid moiety of UDPGA with its cationic guanidinium moiety. These results support the hypothesis that differentiation of sugar donor specificity of UGTs occurred locally, in specific plant lineages, after establishment of general regiospecificity for the sugar acceptor. Thus, the plasticity of sugar donor specificity explains, in part, the extraordinary structural diversification of phytochemicals.     

Molecular cloning, expression and characterization of a glycosyltransferase from rice

Secondary plant metabolites undergo several modification reactions, including glycosylation. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, we cloned one of the glycosyltransferases from rice, RUGT-5 resulting in 40–42% sequence homology with UGTs from other plants. RUGT-5 was functionally expressed as a glutathione S-transferase fusion protein in Escherichia coli and was then purified. Eight different flavonoids were used as tentative substrates. HPLC profiling of reaction products displayed at least two peaks. Glycosylation positions were located at the hydroxyl groups at C-3, C-7 or C-4′ flavonoid positions. The most efficient substrate was kaempferol, followed by apigenin, genistein and luteolin, in that order. According to in vitro results and the composition of rice flavonoids the in vivo substrate of RUGT-5 was predicted to be kaempferol or apigenin. To our knowledge, this is the first time that the function of a rice UGT has been characterized.     

Co-pigmentation and flavonoid glycosyltransferases in blue Veronica persica flowers

Glycosylation is one of the key modification steps for plants to produce a broad spectrum of flavonoids with various structures and colors. A survey of flavonoids in the blue flowers of Veronica persica Poiret (Lamiales, Scrophulariaceae), which is native of Eurasia and now widespread worldwide, led to the identification of highly glycosylated flavonoids, namely delphinidin 3-O-(2-O-(6-O-p-coumaroyl-glucosyl)-6-O-p-coumaroyl-glucoside)-5-O-glucoside (1) and apigenin 7-O-(2-O-glucuronosyl)-glucuronide (2), as two of its main flavonoids. Interestingly, the latter flavone glucuronide (2) caused a bathochromic shift on the anthocyanin (1) toward a blue hue in a dose-dependent manner, showing an intermolecular co-pigment effect. In order to understand the molecular basis for the biosynthesis of this glucuronide, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT88D8), based on the structural similarity to flavonoid 7-O-glucuronosyltransferases (F7GAT) from Lamiales plants. Enzyme assays showed that the recombinant UGT88D8 protein catalyzes the 7-O-glucuronosylation of apigenin and its related flavonoids with preference to UDP-glucuronic acid as a sugar donor. Furthermore, we identified and functionally characterized a cDNA encoding another UGT, UGT94F1, as the anthocyanin 3-O-glucoside-2″-O-glucosyltransferase (A3Glc2″GlcT), according to the structural similarity to sugar-sugar glycosyltransferases classified to the cluster IV of flavonoid UGTs. Preferential expression of UGT88D8 and UGT94F1 genes in the petals supports the idea that these UGTs play an important role in the biosynthesis of key flavonoids responsible for the development of the blue color of V. persica flowers.     

Responses of the flavonoid pathway to UV-B radiation stress and the correlation with the lipid antioxidant characteristics in the desert plant Caryopteris mongolica

Caryopteris mongolica is a dwarf shrub mainly found in grassland and desert areas of north-west China, and which can survive severe environmental stress. This study aimed to assess the responses of the flavonoid pathway to UV-B radiation treatments and its correlation to the lipid peroxide and antioxidant systems in C. mongolica. In UV-B radiation experiments, plants were exposed to UV-B radiation treatments with a intensity of 30 J/s for 1, 4 and 24 h, respectively. A control group without UV-B radiation treatment was also used. The chlorophyll fluorescence parameters, contents of chlorophyll and carotenoid, levels of lipid peroxidation, activities of antioxidant system enzymes, accumulations of total flavonoids and anthocyanins, and activities of phenylalanine ammonialyase (PAL) and chalcone isomerase (CHI) under different UV-B radiation treatments were investigated. The correlations between products and key enzymes in the flavonoid pathway and the lipid peroxide and antioxidant systems were also analyzed. The results showed that chlorophyll fluorescence parameters decreased within 24 h of treatment. The chlorophyll contents decreased within 4 h and remained stable after 24 h. Carotenoid content significantly increased. The level of MDA, the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POD) and the contents of total flavonoids and anthocyanidins increased, while catalase (CAT) activity decreased under UV-B stress. The activities of PAL and CHI also increased with the increased content of total flavonoids. The flavonoid products anthocyanidins had a significant positive correlation with MDA level, as well as the activities of antioxidant enzyme SOD. In conclusion, UV-B radiation induced the degradation of photosynthetic pigments and decreased photochemical efficiency of Photosystem II; increased the contents of MDA, total flavonoids and anthocyanidins; and also enhanced activities of antioxidant enzymes (SOD, APX and POD) and key enzymes (PAL and CHI) in the flavonoid pathway in C. mongolica. Thus, we speculate that the flavonoid pathway were involved in the regulation of stress resistance in C. mongolica.     

Complete assignment of structural genes involved in flavonoid biosynthesis influencing bulb color to individual chromosomes of the shallot (Allium cepa L.)

We analyzed Japanese bunching onion (Allium fistulosum L.) - shallot (Allium cepa L. Aggregatum group) alien chromosome addition lines in order to assign the genes involved in the flavonoid biosynthesis pathway to chromosomes of the shallot. Two complete sets of alien monosomic additions (2n = 2x + 1 = 17) were used for determining the chromosomal locations of several partial sequences of candidate genes, CHS, CHI, F3H, DFR, and ANS via analyses of PCR-based markers. The results of DNA marker analyses showed that the CHS-A, CHS-B, CHI, F3H, DFR, and ANS genes should be assigned to chromosomes 2A, 4A, 3A, 3A, 7A, and 4A, respectively. HPLC analyses of 14 A. fistulosum - shallot multiple alien additions (2n = 2x + 2 - 2x + 7 = 18 - 23) were conducted to identify the anthocyanin compounds produced in the scaly leaves. A direct comparison between the genomic constitution and the anthocyanin compositions of the multiple additions revealed that a 3GT gene for glucosylation of anthocyanidin was located on 4A. Thus, we were able to assign all structural genes involved in flavonoid biosynthesis influencing bulb color to individual chromosomes of A. cepa.     

Substrate specificities of family 1 UGTs gained by domain swapping

Family 1 glycosyltransferases are a group of enzymes known to embrace a large range of different substrates. This study devises a method to enhance the range of substrates even further by combining domains from different glycosyltransferases to gain improved substrate specificity and catalytic efficiency. Chimeric glycosyltransferases were made by combining domains from seven different family 1 glycosyltransferases, UGT71C1, UGT71C2, UGT71E1, UGT85C1, UGT85B1, UGT88B1 and UGT94B1. Twenty different chimeric glycosyltransferases were formed of which twelve were shown to be catalytically active. The chimeric enzymes of Arabidopsis thaliana UGT71C1 and UGT71C2 showed major changes in acceptor substrate specificity and were able to glycosylate etoposide significantly better than the parental UGT71C1 and UGT71C2 enzymes, with K_cat and efficiency coefficients 3.0 and 2.6 times higher, respectively. Chimeric glycosyltransferases of UGT71C1 combined with Stevia rebaudiana UGT71E1, also afforded enzymes with high catalytic efficiency, even though the two enzymes only display 38% amino acid sequence identity. These chimeras show a significantly altered regiospecificity towards especially trans-resveratrol, enabling the production of trans-resveratrol-β-4′-O-glucoside (resveratroloside). The study demonstrates that it is possible to obtain improved catalytic properties by combining domains from both closely as well as more distantly related glycosyltransferases. The substrate specificity gained by the chimeras is difficult to predict because factors determining the acceptor specificity reside in the N- terminal as well as the C-terminal domains.