UDP-glucuronic Acid:Anthocyanin Glucuronosyltransferase from Red Daisy (Bellis perennis) Flowers

Abstract

In contrast to the wealth of biochemical and genetic information on vertebrate glucuronosyltransferases (UGATs), only limited information is available on the role and phylogenetics of plant UGATs. Here we report on the purification, characterization, and cDNA cloning of a novel UGAT involved in the biosynthesis of flower pigments in the red daisy (Bellis perennis). The purified enzyme, BpUGAT, was a soluble monomeric enzyme with a molecular mass of 54 kDa and catalyzed the regiospecific transfer of a glucuronosyl unit from UDP-glucuronate to the 2″-hydroxyl group of the 3-glucosyl moiety of cyanidin 3-O-6″-O-malonylglucoside with a kcat value of 34 s–1 at pH 7.0 and 30 °C. BpUGAT was highlyspecific for cyanidin 3-O-glucosides (e.g. Km for cyanidin 3-O-6″-O-malonylglucoside, 19 μm) and UDP-glucuronate (Km, 476 μm). The BpUGAT cDNA was isolated on the basis of the amino acid sequence of the purified enzyme. Quantitative PCR analysis showed that transcripts of BpUGAT could be specifically detected in red petals, consistent with the temporal and spatial distributions of enzyme activity in the plant and also consistent with the role of the enzyme in pigment biosynthesis. A sequence analysis revealed that BpUGAT is related to the glycosyltransferase 1 (GT1) family of the glycosyltransferase superfamily (according to the Carbohydrate-Active Enzymes (CAZy) data base). Among GT1 family members that encompass vertebrate UGATs and plant secondary product glycosyltransferases, the highest sequence similarity was found with flavonoid rhamnosyltransferases of plants (28–40% identity). Although the biological role (pigment biosynthesis) and enzymatic properties of BpUGAT are significantly different from those of vertebrate UGATs, both of these UGATs share a similarity in that the products produced by these enzymes are more water-soluble, thus facilitating their accumulation in vacuoles (in BpUGAT) or their excretion from cells (in vertebrate UGATs), corroborating the proposed general significance of GT1 family members in the metabolism of small lipophilic molecules. In contrast to the wealth of biochemical and genetic information on vertebrate glucuronosyltransferases (UGATs), only limited information is available on the role and phylogenetics of plant UGATs. Here we report on the purification, characterization, and cDNA cloning of a novel UGAT involved in the biosynthesis of flower pigments in the red daisy (Bellis perennis). The purified enzyme, BpUGAT, was a soluble monomeric enzyme with a molecular mass of 54 kDa and catalyzed the regiospecific transfer of a glucuronosyl unit from UDP-glucuronate to the 2″-hydroxyl group of the 3-glucosyl moiety of cyanidin 3-O-6″-O-malonylglucoside with a kcat value of 34 s–1 at pH 7.0 and 30 °C. BpUGAT was highlyspecific for cyanidin 3-O-glucosides (e.g. Km for cyanidin 3-O-6″-O-malonylglucoside, 19 μm) and UDP-glucuronate (Km, 476 μm). The BpUGAT cDNA was isolated on the basis of the amino acid sequence of the purified enzyme. Quantitative PCR analysis showed that transcripts of BpUGAT could be specifically detected in red petals, consistent with the temporal and spatial distributions of enzyme activity in the plant and also consistent with the role of the enzyme in pigment biosynthesis. A sequence analysis revealed that BpUGAT is related to the glycosyltransferase 1 (GT1) family of the glycosyltransferase superfamily (according to the Carbohydrate-Active Enzymes (CAZy) data base). Among GT1 family members that encompass vertebrate UGATs and plant secondary product glycosyltransferases, the highest sequence similarity was found with flavonoid rhamnosyltransferases of plants (28–40% identity). Although the biological role (pigment biosynthesis) and enzymatic properties of BpUGAT are significantly different from those of vertebrate UGATs, both of these UGATs share a similarity in that the products produced by these enzymes are more water-soluble, thus facilitating their accumulation in vacuoles (in BpUGAT) or their excretion from cells (in vertebrate UGATs), corroborating the proposed general significance of GT1 family members in the metabolism of small lipophilic molecules. UDP-glucuronosyltransferases (UGATs) 1The abbreviations used are: UGAT, UDP-glucuronosyltransferase; GT, glycosyltransferase; BpUGAT, B. perennis UGAT; Cy3MG, cyanidin 3-O-6″-O-malonylglucoside; HPLC, high performance liquid chromatography; PSPG, plant secondary product glycosyltransferase; UDP-glucuronate, uridine 5′-diphosphoglucuronic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; UBGAT, UDP-glucuronate:baicalein 7-UGAT.1The abbreviations used are: UGAT, UDP-glucuronosyltransferase; GT, glycosyltransferase; BpUGAT, B. perennis UGAT; Cy3MG, cyanidin 3-O-6″-O-malonylglucoside; HPLC, high performance liquid chromatography; PSPG, plant secondary product glycosyltransferase; UDP-glucuronate, uridine 5′-diphosphoglucuronic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; UBGAT, UDP-glucuronate:baicalein 7-UGAT. catalyze the transfer of a glucuronosyl group from uridine 5′-diphosphoglucuronic acid (UDP-glucuronate) to an acceptor substrate. The known UGATs have all been shown to be members of the glycosyltransferase superfamily. This superfamily is subclassified, based on their phylogenetics, into 74 families (GT1–GT75; according to the Carbohydrate-Active Enzymes (CAZy) data base (1Campbell J.A. Davies G.J. Bulone V. Henrissat B. Biochem. J. 1997; 326: 929-942Crossref PubMed Scopus (625) Google Scholar); afmb.cnrs-mrs.fr/CAZY/index.html) where UGAT activities have been identified in four separate families (GT1, GT43, GT47, and GT70). In vertebrates, UGATs are expressed in the endoplasmic reticulum of cells of the liver and other organs and are involved in the glucuronide conjugation of xenobiotics (e.g. drugs and dietary phytoalexins) and endobiotics (e.g. bilirubin and steroid hormones) (2Brierley C.H. Burchell B. BioEssays. 1993; 15: 749-754Crossref PubMed Scopus (50) Google Scholar, 3Bock K.W. Biochem. Pharmacol. 2003; 66: 691-696Crossref PubMed Scopus (120) Google Scholar). These UGATs confer water solubility on these lipophilic compounds and facilitate their excretion from cells, thus playing a very important role in their detoxification and elimination. Extensive biochemical and genetic studies of these UGATs have shown that they are metal-activated, membrane-bound, oligomeric enzymes and are related to the GT1 family of the superfamily (4Radominska-Pandya A. Czernik P.J. Little J.M. Battaglia E. Mackenzie P.I. Drug Metab. Rev. 1999; 31: 817-899Crossref PubMed Scopus (433) Google Scholar, 5Lim E-K. Bowles D.J. EMBO J. 2004; 23: 2915-2922Crossref PubMed Scopus (190) Google Scholar). In animals, another type of UGAT, galactosylgalactosylxylosylprotein 3-β-UGATs, a family GT43 enzyme, is involved in the biosynthesis of proteoglycans (6Shimoda Y. Tajima Y. Nagase T. Harii K. Osumi N. Sanai Y. J. Biol. Chem. 1999; 274: 17115-17122Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 7Seiki T. Oka S. Terayama K. Imiya K. Kawasaki T. Biochem. Biophys. Res. Commun. 1999; 255: 182-187Crossref PubMed Scopus (70) Google Scholar). In plants, UGATs or UGAT-related genes have been proposed to play important roles in growth and metabolism, and these roles appear to be distinct from those of the animal enzymes mentioned above. For example, a UGAT-like gene (NpGUT1), belonging to the GT47 family of the superfamily, presumably codes for pectin UGAT, which is involved in pectin biosynthesis. Genetic investigations indicate that this enzyme is essential for intercellular attachment in meristems of Nicotiana plumbaginifolia (8Iwai H. Masaoka N. Ishii T. Satoh S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16319-16324Crossref PubMed Scopus (202) Google Scholar). A gene (PsUGT1) showing a sequence similarity to human UGAT has also been identified in Pisum sativum through genetic approaches and has been reported to be essential for normal plant growth and development (9Woo H-H. Orbach M.J. Hirsch A.M. Hawes M.C. Plant Cell. 1999; 11: 2303-2315Crossref PubMed Scopus (67) Google Scholar). However, the specificity, catalytic properties, and biochemical properties of these UGAT-like proteins remain to be clarified. UGATs are also involved in plant secondary metabolism of biomedical and agricultural relevance, such as the biosynthesis of baicalin (5,6-dihydroxy-7-O-glucuronosylflavone), a major flavonoid of the dried roots of Scutellaria baicalensis, that shows antiallergic, anti-HIV, and antitumor activities (10Nagashima S. Hirotani M. Yoshikawa T. Phytochemistry. 2000; 53: 533-538Crossref PubMed Scopus (57) Google Scholar). Some of these UGATs (11Schulz M. Weissenbock G. Phytochemistry. 1988; 27: 1261-1267Crossref Scopus (23) Google Scholar, 12Kurosawa Y. Takahara H. Shiraiwa M. Planta. 2002; 215: 620-629Crossref PubMed Scopus (43) Google Scholar), including UDP-glucuronate:baicalein 7-UGAT (UBGAT) of S. baicalensis (10Nagashima S. Hirotani M. Yoshikawa T. Phytochemistry. 2000; 53: 533-538Crossref PubMed Scopus (57) Google Scholar), have been purified and characterized. However, the genetic aspects of these UGATs have yet to be studied, although the primary structure of UBGAT has recently been submitted to data bases (GenBank™/DDBJ/EBI accession number BAC98300). To further enhance our knowledge of the enzymology, phylogenetics, and physiological importance of plant UGATs and shed light on structure/function relationship of these enzymes, we initiated a study of UGATs that are involved in the biosynthesis of cyanidin 3-O-2″-O-β-glucuronosyl-6″-O-malonylglucoside, an anthocyanin responsible for the red coloration of daisy (Bellis perennis) flowers (13Saito N. Toki K. Honda T. Kawase K. Phytochemistry. 1988; 27: 2963-2966Crossref Scopus (48) Google Scholar, 14Toki K. Saito N. Honda T. Phytochemistry. 1991; 30: 3769-3771Crossref Scopus (25) Google Scholar). It is generally thought that the glucuronosylation of anthocyanin should enhance its solubility and the stability of the pigment and is important in maintaining the coloration and hues of the flowers (14Toki K. Saito N. Honda T. Phytochemistry. 1991; 30: 3769-3771Crossref Scopus (25) Google Scholar, 15Brouillard R. Dangles O. Harbone J.B. The Flavonoids. Chapman & Hall/CRC, Washington, D. C.1994: 565-599Google Scholar, 16Strack D. Wray V. Harbone J.B. The Flavonoids. Chapman & Hall/CRC, Washington, D. C.1994: 1-22Google Scholar). We describe here the isolation, gene cloning, and functional characterization of a novel enzyme, UDP-glucuronic acid:anthocyanidin 3-O-glucoside 2″-O-β-glucuronosyltransferase, which we hereafter refer to as BpUGAT. Red daisy (B. perennis; Sakata, Yokohama, Japan) was purchased from a local market in Sendai, Japan, and used throughout this study. Recently opened flowers (less than 15 mm in diameter) were collected and stored at –80 °C until used. Anthocyanins were isolated and purified at Minami-Kyushu University, Miyazaki, Japan, and were used as authentic samples after of their by UDP-glucuronate and were purchased from CHAPS, and were products of The of mm pH anthocyanin UDP-glucuronate and the enzyme. For cyanidin 3-O-6″-O-malonylglucoside was used as an anthocyanin substrate. The in the of enzyme, was at 30 and the was by the of the enzyme. at 30 °C for the was by the of of Anthocyanins in the were by high performance liquid on a mm a as H. T. K. Y. N. M. Y. T. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (57) Google Scholar). and their were by the data to the by of a analysis Biochem. Sci. 15: Full Text PDF PubMed Scopus Google Scholar). was by the of Biochem. PubMed Scopus Google a with as the were at °C. and opened red flowers of B. perennis were in of an mm mm and mm and were for 30 in a by at for The was further through A of of red daisy flowers was in the as to a of of which was to The of the was in of was to the enzyme to a of the for the was by The was mm pH enzyme was to a of with mm pH the with the the enzyme was with mm pH and The were mm pH enzyme was to a of with mm pH and the with the the enzyme activity was with a of pH in the The were and on an was to the enzyme to a of of enzyme was to an with with mm pH at and The was with the The enzyme were by mm pH and by enzyme was to an with with mm pH The enzyme activity was by the of the from mm in mm for The were by and mm pH and enzyme was to a with the at a of a liquid The was with the The enzyme activity was with a of mm in in the at a of The were collected and by enzyme was to a of with mm pH CHAPS, and and at a of The were collected and by a enzyme was to a with mm pH at and The was with the The enzyme were collected and a A of BpUGAT was at this was according to the of PubMed Scopus Google Scholar). The proteins on the were by with For of proteins in the were to an The were by reported H. M. Biochem. PubMed Scopus Google Scholar, M. H. S. T. 1991; PubMed Scopus Google a Japan) the To amino acid of BpUGAT purified from red daisy purified was with a from and the were by as H. Y. K. M. M. Y. T. T. Plant J. PubMed Google Scholar, T. K. T. S. Y. M. T. M. Y. T. T. 2000; PubMed Scopus Google Scholar). acid of of these were by sequence and on the amino acid as were based on sequence based on sequence and based on sequence where and and indicate and was isolated from the B. perennis flowers by a The was a with the and and The PCR was the as a and and The which was in was the PCR A cDNA of B. perennis flowers was a cDNA the the as a the cDNA of B. perennis was as H. T. K. Y. N. M. Y. T. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The cDNA of was into the and to a with a analysis The BpUGAT cDNA was by PCR the and to and the and into the and of the The was into a a PCR cloning for to the of PCR The was with and and the was with a that been with and to the which BpUGAT. was used as a for the of BpUGAT. cells were at 30 °C in of an with for until the were by were at °C. were in of mm pH mm mm and and were with by a Japan) by was to the to a of the for the was by of the was as above. was from the organs of a B. perennis plant the plant The BpUGAT in of from the organs was by PCR with the and PCR was the and the were °C for and °C for 15 by of °C for 15 °C for and °C for A the BpUGAT cDNA was used as a for the The of daisy flowers are as mm in mm mm mm mm studies showed that BpUGAT was a soluble enzyme, and a high activity was found in recently opened flowers (less than 15 mm in a of such flowers was used as the for the enzyme. the enzyme to be to and the of to the was essential for the enzyme The of UGAT activity on and with those of a that this was responsible for the UGAT the enzyme could be purified to with an activity of after The BpUGAT in the was for the of on the enzyme. The molecular mass of the purified BpUGAT was to be kDa by on that the enzyme was of purified BpUGAT. The enzyme enzyme purified from B. perennis enzyme purified from S. and proteins were the of PubMed Scopus Google and with of BpUGAT from red daisy in a of the purified BpUGAT with a which with the major anthocyanin of red daisy as by mass the product showed a molecular at and the mass of showed a at These the of a glucuronosyl moiety in the product of the product revealed that was cyanidin N. Toki K. Honda T. Kawase K. Phytochemistry. 1988; 27: 2963-2966Crossref Scopus (48) Google and 14Toki K. Saito N. Honda T. Phytochemistry. 1991; 30: 3769-3771Crossref Scopus (25) Google Scholar, also for the the purified enzyme the regiospecific transfer of the glucuronosyl group with of its to the 2″-hydroxyl group of the 3-glucosyl moiety of the anthocyanin and is as a UDP-glucuronic acid:anthocyanidin 3-O-glucoside The kcat value for the glucuronosyl transfer from UDP-glucuronate to was 34 and the Km for and UDP-glucuronate were 19 and 476 The purified enzyme was a pH of with a activity at pH 30 and was at pH °C for and molecular properties of kcat Km for Km for UDP-glucuronate for for UDP-glucuronate Km for cyanidin of these are shown in activity of these are shown in of these are shown in of these are shown in of these are shown in properties pH for pH stability to to The of these are shown in in a of BpUGAT was with UDP-glucuronate, and as a The activities for and were than of the activity for UDP-glucuronate, that the enzyme was for glucuronosyl acceptor of BpUGAT was a of including and In to Cy3MG, the enzyme also showed activities cyanidin 3-O-glucoside for and 3-O-glucoside However, the enzyme showed or only glucuronosyl transfer activity the cyanidin of and 3-O-6″-O-malonylglucoside, and and of and and flavonoid and was also as a substrate. We also the of BpUGAT to catalyze the transfer of glucuronosyl to other lipophilic compounds and T. Biochem. PubMed Scopus Google Scholar), and the were with those the for a UGAT that is involved in glucuronide conjugation in and metabolism in (2Brierley C.H. Burchell B. BioEssays. 1993; 15: 749-754Crossref PubMed Scopus (50) Google Scholar, 3Bock K.W. Biochem. Pharmacol. 2003; 66: 691-696Crossref PubMed Scopus (120) Google The showed that all of these were for as for BpUGAT. The of and a on BpUGAT activity were the The enzyme activity was by mm and mm and by mm mm and mm It should be that the enzyme by and be to their on the enzyme such as these are known to J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). mm and and mm on the catalytic activity in of The of compounds that are related to the and products on enzyme activity were also the The enzyme was by 1 mm and 1 mm and by 1 mm was by 1 mm uridine 1 mm and 1 mm The enzyme was in the of at pH 7.0 and °C for activity after with 1 mm the We the PCR on the basis of the amino acid with the purified BpUGAT 1 and for and a PCR of B. perennis flowers as a The was further to a PCR product of this as a the cDNA was high to The cDNA from these was The amino acid sequence of the a (GenBank™/DDBJ/EBI accession number of amino with a molecular mass of The amino acid for the purified enzyme and were at and We were also to a sequence plant secondary product glycosyltransferase that has been identified the of other family members and vertebrate UGATs, for of the glycosyltransferase superfamily and proposed to the Y. S. E-K. Bowles D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Plant Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Planta. PubMed Scopus Google Scholar, K. N. Plant Biol. PubMed Scopus Google Scholar). In an for similarity of the amino acid sequence in data bases and the the highest was found with the of M. E. R. J. J. Biol. Chem. 1991; Full Text PDF PubMed Google and the of J. E. A. Y. J. R. Plant J. PubMed Scopus Google Scholar). A sequence similarity was found with anthocyanin from plant M. M. Y. Y. T. Saito K. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the similarity in specificity, BpUGAT showed only a very sequence similarity to vertebrate UGATs and all known plant UGATs (8Iwai H. Masaoka N. Ishii T. Satoh S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16319-16324Crossref PubMed Scopus (202) Google Scholar), (9Woo H-H. Orbach M.J. Hirsch A.M. Hawes M.C. Plant Cell. 1999; 11: 2303-2315Crossref PubMed Scopus (67) Google Scholar), and UBGAT (GenBank™/DDBJ/EBI accession number The BpUGAT cDNA was expressed the of the in the S. cells as a at a of of The could be purified to from of cells that were the as those used for the of the BpUGAT from daisy The showed a BpUGAT catalytic and molecular properties of the enzyme were to those of the BpUGAT thus that the cDNA codes for BpUGAT. To the functional significance of BpUGAT in the coloration of red daisy the and in BpUGAT in the daisy plant were by PCR development was into different The BpUGAT was identified in of red and the highest of the was at of flower The was identified in the of or only a of the could be found in the and temporal and spatial of BpUGAT are consistent with the for BpUGAT activity and the in the B. perennis plant the of BpUGAT is flower as is the for the genes responsible for anthocyanin and is consistent with the temporal and spatial distributions of enzyme activity and These are consistent with the role of the enzyme in the of that are the of the red coloration of daisy flowers (13Saito N. Toki K. Honda T. Kawase K. Phytochemistry. 1988; 27: 2963-2966Crossref Scopus (48) Google Scholar, 14Toki K. Saito N. Honda T. Phytochemistry. 1991; 30: 3769-3771Crossref Scopus (25) Google Scholar). In this primary structure and phylogenetics of a novel UGAT that is involved in plant secondary metabolism pigment biosynthesis) were with its molecular properties and studies revealed that BpUGAT to the glycosyltransferase superfamily (1Campbell J.A. Davies G.J. Bulone V. Henrissat B. Biochem. J. 1997; 326: 929-942Crossref PubMed Scopus (625) Google and is related to the GT1 which of vertebrate UGATs that are involved in glucuronide conjugation and T. Plant Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). It showed only sequence similarity to vertebrate UGATs its similarity in and to to the of T. Plant Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). It is very important to here that play roles in the metabolism of endobiotics and xenobiotics in plants T. Planta. PubMed Scopus Google Scholar, T. J. H. K. J. Chem. 2002; PubMed Scopus Google Scholar), and their are with those of vertebrate UGATs E-K. Bowles D.J. EMBO J. 2004; 23: 2915-2922Crossref PubMed Scopus (190) Google Scholar). plant secondary and also be to a of The accumulation of such compounds in plant cells with the physiological of the plants, the compounds are or to a the solubility of these compounds and their thus maintaining the of plants E-K. Bowles D.J. EMBO J. 2004; 23: 2915-2922Crossref PubMed Scopus (190) Google Scholar, T. Planta. PubMed Scopus Google Scholar). the enzymatic by these family GT1 members water solubility on the facilitating product accumulation in vacuoles (in and BpUGAT) or the product excretion from cells (in the of vertebrate The analysis of the UBGAT sequence (GenBank™/DDBJ/EBI accession number revealed that this plant UGAT should also be a of consistent with its role (e.g. of the in baicalin could be that BpUGAT and UBGAT are of that have been to an to the role of the GT1 family in plant with a relationship of BpUGAT with BpUGAT is more in its enzymatic properties to other than to vertebrate UGATs. For example, BpUGAT is a soluble enzyme that for its catalytic BpUGAT is to be a enzyme, from the of in the enzyme, its pH for activity and pH stability and the of a analysis of the BpUGAT sequence For a vertebrate enzyme that is involved in glucuronide is an endoplasmic membrane-bound, enzyme that shows an of for activity (4Radominska-Pandya A. Czernik P.J. Little J.M. Battaglia E. Mackenzie P.I. Drug Metab. Rev. 1999; 31: 817-899Crossref PubMed Scopus (433) Google Scholar). BpUGAT shows a high activity s–1 at 30 more than than those of vertebrate UGATs. BpUGAT is for shows a lipophilic compounds for It should be that that are involved in metabolism also acceptor T. Planta. PubMed Scopus Google Scholar, T. J. H. K. J. Chem. 2002; PubMed Scopus Google Scholar), and the acceptor of BpUGAT should be related to its role in the biosynthesis of flower It should also be that the enzymatic properties of UGAT of Y. Takahara H. Shiraiwa M. Planta. 2002; 215: 620-629Crossref PubMed Scopus (43) Google significantly from those of BpUGAT and UBGAT are to those of vertebrate UGATs. For example, UGAT to be a enzyme and for catalytic activity Y. Takahara H. Shiraiwa M. Planta. 2002; 215: 620-629Crossref PubMed Scopus (43) Google Scholar). This be related to a in their of the primary structure of UGAT is with of have shown that in the with members high sequence and of acceptor with other (e.g. and T. Planta. PubMed Scopus Google Scholar). BpUGAT and UBGAT to of these known BpUGAT is related to UBGAT and to be more related to flavonoid rhamnosyltransferases M. E. R. J. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, J. E. A. Y. J. R. Plant J. PubMed Scopus Google the of BpUGAT and these rhamnosyltransferases are distinct from members of the known and and UBGAT they catalyze the transfer of a unit to an moiety catalyze the transfer to a moiety of flavonoid BpUGAT and these rhamnosyltransferases appear to another of enzymes that a of the family GT1 of other and their be to this The to 3-O-glucosides have been and are in plant the other the of including (e.g. and further (e.g. with plant The of BpUGAT important information to the of anthocyanin in daisy and cyanidin 3-O-glucoside were for BpUGAT that these as physiological glucuronosyl in for more than of the in the daisy flowers used in this cyanidin was These that the anthocyanin of daisy flowers should be to cyanidin as as cyanidin and the of biosynthesis of this flower pigment a M. M. Y. Y. T. Saito K. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google where or A has been proposed for anthocyanin and in red M. M. Y. Y. T. Saito K. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). We for and with