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

ENZYMOLOGY AND PHYLOGENETICS OF A NOVEL GLUCURONOSYLTRANSFERASE INVOLVED IN FLOWER PIGMENT BIOSYNTHESIS*
  • Shin'ya Sawada
    Affiliations
    Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai 980-8579
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  • Hirokazu Suzuki
    Affiliations
    Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai 980-8579
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  • Fumiko Ichimaida
    Affiliations
    Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai 980-8579
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  • Masa-atsu Yamaguchi
    Affiliations
    Faculty of Horticulture, Minami-Kyushu University, Miyazaki 884-0003
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  • Takashi Iwashita
    Affiliations
    Suntory Institute for Bioorganic Research, Mishima-gun, Shimamoto-cho, Osaka 618-8503, Japan
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  • Yuko Fukui
    Affiliations
    Suntory Research Center, Mishima-gun, Shimamoto-cho, Osaka 618-8503, Japan
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  • Hisashi Hemmi
    Affiliations
    Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai 980-8579
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  • Tokuzo Nishino
    Affiliations
    Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai 980-8579
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  • Toru Nakayama
    Correspondence
    To whom correspondence should be addressed. Fax: 81-22-217-7293;
    Affiliations
    Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai 980-8579
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  • Author Footnotes
    * This work was supported in part by the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1S and 2S.The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank/EBI Data Bank with accession number(s) AB190262 (for BpUGAT).
Open AccessPublished:October 27, 2004DOI:https://doi.org/10.1074/jbc.M410537200
      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)
      The 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 (
      • Campbell J.A.
      • Davies G.J.
      • Bulone V.
      • Henrissat B.
      ); 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) (
      • Brierley C.H.
      • Burchell B.
      ,
      • Bock K.W.
      ). 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 (
      • Radominska-Pandya A.
      • Czernik P.J.
      • Little J.M.
      • Battaglia E.
      • Mackenzie P.I.
      ,
      • Lim E-K.
      • Bowles D.J.
      ). In animals, another type of UGAT, galactosylgalactosylxylosylprotein 3-β-UGATs, a family GT43 enzyme, is involved in the biosynthesis of proteoglycans (
      • Shimoda Y.
      • Tajima Y.
      • Nagase T.
      • Harii K.
      • Osumi N.
      • Sanai Y.
      ,
      • Seiki T.
      • Oka S.
      • Terayama K.
      • Imiya K.
      • Kawasaki T.
      ).
      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 (
      • Iwai H.
      • Masaoka N.
      • Ishii T.
      • Satoh S.
      ). 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 (
      • Woo H-H.
      • Orbach M.J.
      • Hirsch A.M.
      • Hawes M.C.
      ). 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 (
      • Nagashima S.
      • Hirotani M.
      • Yoshikawa T.
      ). Some of these UGATs (
      • Schulz M.
      • Weissenbock G.
      ,
      • Kurosawa Y.
      • Takahara H.
      • Shiraiwa M.
      ), including UDP-glucuronate:baicalein 7-UGAT (UBGAT) of S. baicalensis (
      • Nagashima S.
      • Hirotani M.
      • Yoshikawa T.
      ), 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 (
      • Saito N.
      • Toki K.
      • Honda T.
      • Kawase K.
      ,
      • Toki K.
      • Saito N.
      • Honda T.
      ). 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 (
      • Toki K.
      • Saito N.
      • Honda T.
      ,
      • Brouillard R.
      • Dangles O.
      ,
      • Strack D.
      • Wray V.
      ). 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.

      EXPERIMENTAL PROCEDURES

      Plant Materials, Anthocyanins, and Chemicals

      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 confirmation of their structures by instrumental analyses. UDP-glucuronate (trisodium salt), isoflavones, flavonols, and p-nitrophenyl β-d-glucoside were purchased from Sigma. CHAPS, phenylmethylsulfonyl fluoride, and polyvinylpolypyrrolidone were products of Nacalai Tesque, Kyoto, Japan.

      Enzyme and Protein Assays

      The standard reaction mixture (final volume, 100 μl) consisted of 20 mm potassium phosphate (Pi), pH 7.0, 120 μm anthocyanin substrate, 774 μm UDP-glucuronate (final concentrations), and the enzyme. For routine assays, cyanidin 3-O-6″-O-malonylglucoside (Cy3MG) was used as an anthocyanin substrate. The mixture, in the absence of enzyme, was preincubated at 30 °C, and the reaction was started by the addition of the enzyme. After incubation at 30 °C for 10 min, the reaction was stopped by the addition of 200 μl of ice-cold 0.5% (v/v) trifluoroacetic acid.
      Anthocyanins in the reaction mixture were routinely analyzed by reversed-phase high performance liquid chromatography (HPLC) on a Shodex Asahipak ODP-50 4E column (4.6 mm × 250 mm) using a Rainin Dynamax HPLC system (Rainin Instruments Co., Woburn, MA), as described previously (
      • Suzuki H.
      • Nakayama T.
      • Yonekura-Sakakibara K.
      • Fukui Y.
      • Nakamura N.
      • Nakao M.
      • Tanaka Y.
      • Yamaguchi M-A.
      • Kusumi T.
      • Nishino T.
      ). Kinetic parameters and their standard errors were estimated by fitting the initial velocity data to the Michaelis-Menten equation by means of a non-linear regression analysis (
      • Leatherbarrow R.J.
      ). Protein was quantified by the method of Bradford (
      • Bradford M.M.
      ) using a kit (Bio-Rad) with bovine serum albumin as the standard.

      Purification of BpUGAT from B. perennis Flowers

      All operations were performed at 0–4 °C.
      Step 1: Ammonium Sulfate Fractionation and Polyethyleneimine Treatment—Recently opened red flowers (0.5 kg) of B. perennis were suspended in 2,000 ml of an extraction buffer (100 mm potassium Pi containing 0.2% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, 5 mm EDTA, and 0.5 mm phenylmethylsulfonyl fluoride) containing 9% (w/v) polyvinylpolypyrrolidone and were disrupted for 30 s in a Waring blender followed by centrifugation at 8,000 × g for 20 min. The supernatant was further filtered through Whatman 114 filter paper. A total of 1.5 kg of red daisy flowers was treated in the same manner as above to give a total of 7,000 ml of crude extract, which was then subjected to ammonium sulfate fractionation. The precipitate of the 30–70% saturation fraction was dissolved in 2,500 ml of extraction buffer. Polyethyleneimine was then added to the enzyme solution to a final concentration of 0.3% (w/v). After stirring the mixture for 10 min, the precipitate was removed by centrifugation. The supernatant solution was extensively dialyzed against 40 mm potassium Pi, pH 7.5, containing 0.2% 2-mercaptoethanol.
      Step 2: First Q Sepharose—The enzyme solution was applied to a column (5 × 20 cm) of Q Sepharose Fast Flow (Amersham Biosciences) equilibrated with 40 mm potassium Pi, pH 7.5, containing 0.2% 2-mercaptoethanol. After washing the column with the equilibration buffer, the enzyme was eluted with 100 mm potassium Pi, pH 7.5, containing 0.2% 2-mercaptoethanol and 10% glycerol. The active fractions were dialyzed against 20 mm potassium Pi, pH 7.5, containing 0.2% 2-mercaptoethanol.
      Step 3: Second Q Sepharose—The enzyme solution (1,900 ml) was applied to a column (2 × 20 cm) of Q Sepharose Fast Flow equilibrated with 20 mm potassium Pi, pH 7.5, containing 0.2% 2-mercaptoethanol and 10% glycerol. After washing the column with the equilibration buffer, the enzyme activity was eluted with a linear gradient (20–200 mm) of potassium Pi, pH 7.5, in the buffer (total gradient volume, 1,000 ml). The active fractions were combined and concentrated on an Amicon 8200 ultrafiltration unit. Ammonium sulfate was added to the enzyme solution to a final concentration of 20% of saturation.
      Step 4: Phenyl-Sepharose—The enzyme solution (37.5 ml) was applied to an HR10/16 column (Amersham Biosciences) packed with phenyl-Sepharose 6 FF (low sub) (Amersham Biosciences) equilibrated with 100 mm potassium Pi, pH 7.5, containing ammonium sulfate at 20% saturation and 0.2% 2-mercaptoethanol. The column was washed with the same buffer. The flow-through fractions, containing enzyme activity, were collected, concentrated by ultrafiltration, dialyzed against 5 mm potassium Pi, pH 7.5, containing 0.2% 2-mercaptoethanol, and concentrated by ultrafiltration.
      Step 5: CHT Ceramic Hydroxyapatite—The enzyme solution (10 ml) was applied to an HR10/10 column (Amersham Biosciences) packed with CHT ceramic hydroxyapatite (Type I, Bio-Rad) equilibrated with 5 mm potassium Pi, pH 7.5, containing 0.2% 2-mercaptoethanol. The enzyme activity was eluted by increasing the potassium Pi concentration of the buffer from 10–100 mm in 10 mm steps (50 ml for each step). The active fractions were collected, concentrated by ultrafiltration, and dialyzed against 20 mm potassium Pi, pH 7.5, containing 0.2% 2-mercaptoethanol, 10% glycerol, and 0.1% (w/v) CHAPS.
      Step 6: Mono Q Fast Protein Liquid Chromatography—The enzyme solution (23 ml) was applied to a Mono Q HR10/10 column (Amersham Biosciences) equilibrated with the above buffer at a flow rate of 1.0 ml/min using a fast protein liquid chromatography system. The column was washed with the same buffer (50 ml). The enzyme activity was eluted with a linear gradient of potassium Pi (20–300 mm in 350 min) in the buffer at a flow rate of 1.0 ml/min. The active fractions were collected and concentrated by Centricon YM10 ultrafiltration.
      Step 7: Superdex 200 Gel Filtration—The enzyme solution (2.5 ml) was applied to a column of HiLoad 26/60 Superdex, 200 pg (Amersham Biosciences) equilibrated with 20 mm potassium Pi, pH 7.5, containing 0.15 m NaCl, 0.1% CHAPS, and 0.2% 2-mercaptoethanol and eluted at a flow rate of 0.9 ml/min. The active fractions were collected and concentrated by using a Centricon YM10 ultrafiltration device.
      Step 8: RESOURCE PHE—The enzyme solution (3 ml) was applied to a RESOURCE PHE column (6 ml, Amersham Biosciences) equilibrated with 100 mm potassium Pi, pH 7.5, containing ammonium sulfate at 20% saturation, 0.2% 2-mercaptoethanol, and 0.1% CHAPS. The column was washed with the same buffer. The flow-through fractions, containing enzyme activity, were collected and concentrated using a Centricon YM10. A homogeneous preparation of BpUGAT was obtained at this step.

      Protein Chemical Analyses

      SDS-PAGE was carried out according to the method of Laemmli (
      • Laemmli U.K.
      ). The proteins on the gels were visualized by staining with Coomassie Brilliant Blue R250. For sugar staining of protein bands, proteins in the SDS-polyacrylamide gel were transferred to an Immobilon-P membrane (Millipore). The glycoprotein blots were visualized by methods reported previously (
      • Bayer E.A.
      • Ben-Hur H.
      • Wilchek M.
      ,
      • Kondo M.
      • Harada H.
      • Sunada S.
      • Yamaguchi T.
      ) using a kit (G. P. Sensor, Seikagaku Corp., Tokyo, Japan) following the manufacturer's recommendations.
      To obtain internal amino acid sequences of BpUGAT purified from red daisy flowers, purified protein was digested with a lysylendopeptidase from Achromobacter lyticus M497-1 (Wako, Tokyo, Japan), and the resulting peptides were separated by reversed-phase HPLC as described previously (
      • Fujiwara H.
      • Tanaka Y.
      • Yonekura-Sakakibara K.
      • Fukuchi-Mizutani M.
      • Nakao M.
      • Fukui Y.
      • Yamaguchi M-A.
      • Ashikari T.
      • Kusumi T.
      ,
      • Nakayama T.
      • Yonekura-Sakakibara K.
      • Sato T.
      • Kikuchi S.
      • Fukui Y.
      • Fukuchi-Mizutani M.
      • Ueda T.
      • Nakao M.
      • Tanaka Y.
      • Kusumi T.
      • Nishino T.
      ). Amino acid sequences of some of these peptides were determined by automated Edman degradation: Ser-Gly-Pro-Asp-Phe-Glu-Thr-Ile-Leu-Ile-Lys (termed sequence 1), Arg-Glu-Glu-Ile-Ala-Ala-Val-Val-Arg-Lys (sequence 2), and Asn-Met-Glu-Ala-Glu-Val-Asp-Gly-Ile-Val (sequence 3).

      cDNA Cloning of BpUGAT

      Based on the amino acid sequences determined as above, degenerate oligonucleotide primers were synthesized: primer F1 (5′-GGICCIGAYTTYGARACIATHTTRATHAAR-3′, based on sequence 1; see above), primer R1 (5′-ARRTTYTCDATIACDATICCRTCIACYTC-3′, based on sequence 3), and primer R2 (5′-CCRTCIACYTCIFCYTCCATRTT-3′, based on sequence 3) where I indicates inosine and R, H, D, and Y indicate degenerate sites: R, A/G; H, A/C/T; D, A/G/T; and Y, C/T. Poly(A)+ RNA was isolated from the B. perennis flowers by using a kit (Straight A's mRNA isolation system, Novagen, Madison, WI). The reverse transcription-PCR was performed using a Qiagen One-Step reverse transcription-PCR kit (Qiagen, Hilden, Germany) with the primers F1 and R1 and poly(A)+ RNA. The nested PCR was completed using the amplified fragment as a template and primers F1 and R2. The amplified fragment, UGT1, which was ∼1 kbp in length, was digoxigenin-labeled using the PCR digoxigenin probe synthesis kit (Roche Diagnostics). A cDNA library of B. perennis flowers was constructed using a ZAP Express cDNA synthesis kit (Stratagene, Heidelberg, Germany) following the manufacturer's guidelines. With the digoxigenin-labeled fragment as a probe, the cDNA library of B. perennis flowers, ∼400,000 plaques, was screened as described previously (
      • Suzuki H.
      • Nakayama T.
      • Yonekura-Sakakibara K.
      • Fukui Y.
      • Nakamura N.
      • Nakao M.
      • Tanaka Y.
      • Yamaguchi M-A.
      • Kusumi T.
      • Nishino T.
      ). The cDNA of positive clones was subcloned into the pBluescript SK(–) phagemid vector (Stratagene) and subjected to DNA sequencing using a dye terminator cycle sequencing kit (Beckman Coulter) with a CEQ 2000XL DNA analysis system (Beckman Coulter).

      Expression and Purification of the Recombinant BpUGAT

      The full-length BpUGAT cDNA was amplified by PCR using the primers 5′-CTCGAGATGGATTCAAAAATCG-3′ and 5′-GGTACCTTAATTATTCATTTCAC-3′ to introduce XhoI and KpnI sites containing the translation initiation and stop codons into the 5′- and 3′-ends of the cDNA, respectively. The amplified fragment was cloned into a pCR4Blunt-TOPO vector using a kit (Zero Blunt TOPO PCR cloning kit for sequencing, Invitrogen) to confirm the absence of PCR errors. The plasmid was digested with XhoI and KpnI, and the resulting ∼1.4-kbp DNA fragment was ligated with a pESC-TRP vector (Stratagene) that had previously been digested with XhoI and KpnI to give the plasmid pESC-TRP-BpUGAT, which encodes BpUGAT. Saccharomyces cerevisiae YPH499 was used as a host for the expression of BpUGAT.
      Transformed cells were cultivated at 30 °C in 600 ml of an SG Trp dropout medium (Stratagene) with shaking for 7–10 days until the A600 reached 1.5. Cells were harvested by centrifugation. All subsequent operations were carried out at 0–4 °C. Cells (12.6 g, wet weight) were suspended in 26 ml of 100 mm potassium Pi, pH 7.5, containing 0.5% (w/v) 2-mercaptoethanol, 10% (w/v) glycerol, 5 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, and 0.1% (w/v) CHAPS and were disrupted with glass beads (0.5 mm) by using a Multibeads Shocker Model MBS200 (Yasui Co., Osaka, Japan) followed by centrifugation. Polyethyleneimine was then added to the supernatant to a final concentration of 0.3% (w/v). After stirring the mixture for 10 min, the precipitate was removed by centrifugation. Purification of the recombinant BpUGT was carried out essentially as described above.

      Quantitative Real Time PCR

      Total RNA was prepared from the individual organs of a B. perennis plant using the RNeasy plant minikit (Qiagen). The BpUGAT transcript in 200 ng of total RNA from the individual organs was quantified by quantitative real time PCR with the BpUGAT-specific primers (5′-GGATGCTCATCTCTACAC-3′ and 5′-GACCTTCTCATGCAATCAAC-3′). Real time PCR was carried out using the LightCycler Quick System Model 330 (Roche Diagnostics) and the QuantiTect SYBR Green reverse transcription-PCR kit (Qiagen). Thermal cycling conditions were 50 °C for 20 min and then 95 °C for 15 min followed by 35 cycles of 95 °C for 15 s, 55 °C for 20 s, and 72 °C for 10 s. A plasmid encoding the full-length BpUGAT cDNA was used as a template for the calibration. The developmental stages of daisy flowers are defined as follows: stage 1, 5–7 mm in diameter (no pigmentation); stage 2, 7–10 mm (slightly pigmented); stage 3, 10 mm (fully pigmented); stage 4, 10–15 mm (fully pigmented); stage 5, >15 mm (fully pigmented).

      RESULTS

      Purification and Molecular Properties of BpUGAT from B. perennis Flowers

      Preliminary studies showed that BpUGAT was a soluble enzyme, and a high specific activity was found in recently opened flowers (less than 15 mm in diameter); thus, a large amount of such flowers (1.5 kg, fresh weight) was used as the starting material for purifying the enzyme. Moreover the enzyme appeared to be susceptible to sulfhydryl oxidation, and the addition of 2-mercaptoethanol to the buffer was essential for purifying the enzyme efficiently. The elution profiles of UGAT activity during chromatographies on CHT ceramic hydroxyapatite, Mono Q, and Superdex 200 strictly coincided with those of a 54-kDa protein (Fig. 1), strongly suggesting that this 54-kDa protein was responsible for the UGAT activity. Finally the enzyme could be purified 540-fold to homogeneity (Fig. 1) with an activity yield of 2.1% after eight purification steps (Table I). The BpUGAT band in the SDS-polyacrylamide gels was negative for sugar staining (not shown), strongly suggesting the absence of sugar chains on the enzyme. The native molecular mass of the purified BpUGAT was estimated to be 49 kDa by gel filtration chromatography on Superdex 200, indicating that the enzyme was monomeric.
      Figure thumbnail gr2
      Scheme IBpUGAT-catalyzed transfer of a glucuronosyl group from UDP-glucuronate to anthocyanins. The glucuronosyl group is specifically transferred to the 2″-hydroxyl group of anthocyanin. Anthocyanin names are as follows: Cy3MG (R1 = OH, R2 = H, R3 = malonyl), pelargonidin 3-O-glucoside (R1 = (R2 = (R3 = H), cyanidin 3-O-glucoside (R1 = OH R2 = H R3 = H), and delphinidin 3-O-glucoside (R1 = R2 = OH, R3 = H). For the sake of convenience, structures of anthocyanins are shown as their flavylium forms.
      Figure thumbnail gr1
      Fig. 1SDS-PAGE of purified BpUGAT. The enzyme preparations (lane 1, native enzyme purified from B. perennis flowers; lane 2, recombinant enzyme purified from S. cerevisiae transformant cells) and marker proteins (lane 3) were simultaneously electrophoresed under the conditions of Laemmli (
      • Laemmli U.K.
      ) and stained with Coomassie Brilliant Blue R250.
      Table IPurification of BpUGAT from red daisy flowers
      StepTotal proteinTotal activitySpecific activityPurificationRecovery
      mgnanokatalspicokatals/mg protein-fold%
      Crude extract5,25047.09.01100
      Ammonium sulfate (Step 1)2,90035.0121.475
      Q Sepharose (Step 2)53021.4403.846
      Q Sepharose (Step 3)44019.7475.242
      Phenyl-Sepharose (Step 4)1176.9596.615
      Ceramic hydroxyapatite (Step 5)11.57.86807616
      Mono Q HR10/10 (Step 6)4.36.51,50016814
      Superdex 200 (Step 7)0.662.23,3003724.6
      RESOURCE PHE (Step 8)0.210.994,8005402.1

      Catalytic Properties

      Reaction of the purified BpUGAT with Cy3MG yielded a single product, which co-eluted with the major anthocyanin of red daisy flowers, as evidenced by analytical reversed-phase HPLC (not shown). By positive mode time-of-flight mass spectrometry, the product showed a molecular ion at m/z 711 [M]+, and the time-of-flight tandem mass spectrometry of m/z 711 showed a fragment ion peak at m/z 535 [M–176]+. These results suggest the presence of a glucuronosyl moiety in the product molecule. NMR analyses of the product revealed that it was cyanidin 3-O-β-2″-O-β-glucuronosyl-6″-O-malonylglucopyranoside (Refs.
      • Saito N.
      • Toki K.
      • Honda T.
      • Kawase K.
      and
      • Toki K.
      • Saito N.
      • Honda T.
      , see also Supplemental Fig. 1S for the complete NMR assignments). Thus, the purified enzyme catalyzes the regiospecific transfer of the glucuronosyl group with inversion of its anomeric configuration to the 2″-hydroxyl group of the 3-glucosyl moiety of the substrate anthocyanin (Scheme I) and is defined as a UDP-glucuronic acid:anthocyanidin 3-O-glucoside 2″-O-β-glucuronosyltransferase. The calculated kcat value for the BpUGAT-catalyzed glucuronosyl transfer from UDP-glucuronate to Cy3MG was 34 ± 7s–1, and the Km values for Cy3MG and UDP-glucuronate were 19 ± 7 and 476 ± 101 μm, respectively (Table II). The purified enzyme was active over a pH range of 6.0–8.5 with a maximum activity at pH 8.0 (at 30 °C) and was stable at pH 7.5 (at 20 °C for 17 h).
      Table IIKinetic and some molecular properties of BpUGAT
      Native BpUGATRecombinant BpUGAT
      Kinetic parameters
          kcat (s-1; donor, UDP-glucuronate; acceptor, Cy3MG)34 ± 735 ± 6
          Km for Cy3MG (μm; donor, UDP-glucuronate)19 ± 732 ± 7
          Km for UDP-glucuronate (μm; acceptor, Cy3MG)476 ± 101497 ± 91
          kcat/Km for Cy3MG (s-1 μm-1; donor, UDP-glucuronate)1.81.1
          kcat/Km for UDP-glucuronate (s-1 μm-1; acceptor, Cy3MG)0.070.07
          Km for cyanidin 3-O-glucoside
      The structures of these anthocyanins are shown in Scheme I.
      m; donor, UDP-glucuronate)
      85 ± 12211 ± 87
      Relative activity (%)
          Cy3MG
      The structures of these anthocyanins are shown in Scheme I.
      100100
          Cyanidin 3-O-glucoside
      The structures of these anthocyanins are shown in Scheme I.
      198238
          Delphinidin 3-O-glucoside
      The structures of these anthocyanins are shown in Scheme I.
      1421
          Pelargonin 3-O-glucoside
      The structures of these anthocyanins are shown in Scheme I.
      0.61.0
          Pelargonin 3,5-O-diglucoside0.11.2
      Other properties
          Optimum pH for activity8.08.0
          pH stability7.56.5
          Optimum temperature (°C)3530
          Temperature stability (°C)up to 35up to 35
      a The structures of these anthocyanins are shown in Scheme I.

      Substrate Specificity of BpUGAT

      Glycosyl Donor Specificity—The glycosyl donor specificity of BpUGAT was examined with UDP-glucuronate, UDP-glucose, and UDP-galactose using Cy3MG as a glycosyl acceptor. The relative activities for UDP-glucose and UDP-galactose were less than 0.1% of the activity for UDP-glucuronate, indicating that the enzyme was highly specific for UDP-glucuronate.
      Glucuronosyl Acceptor Specificity—The glucuronosyl acceptor specificity of BpUGAT was examined using a wide variety of phenolics including anthocyanins, flavonoids, and phenyl glucosides. In addition to Cy3MG, the enzyme also showed appreciable activities toward cyanidin 3-O-glucoside (relative activity, 198%; see Scheme I for structure) and delphinidin 3-O-glucoside (14%). However, the enzyme showed no or only negligible glucuronosyl transfer activity toward the following flavonoids (relative activity, <1%): pelargonidin 3-O-glucoside, cyanidin 3-O-3″,6″-O-dimalonylglucoside, 3,5-diglucosides of anthocyanidins (pelargonidin 3,5-O-diglucoside and pelargonidin 3-O-6″-O-malonylglucoside-5-O-glucoside), quercetin 3-O-glucoside, quercetin 3-O-6″-O-malonylglucoside, isoflavones (daidzin and genistin), isoflavone glucosides (daidzin, genistin, and 7-O-6″-O-malonylglucosides of daidzein and genistein), and flavonoid aglycons (cyanidin, quercetin, daidzein, and genistein). p-Nitrophenyl β-d-glucopyranoside was also inert as a substrate.
      We also examined the ability of BpUGAT to catalyze the transfer of glucuronosyl units to some other lipophilic compounds (β-estradiol, 17α-estradiol, 1-naphthol, 2-naphthol, 4-methylumbelliferone, and p-nitrophenol) using UDP-[U-14C]glucuronate (
      • Bansal S.K.
      • Gessner T.
      ), and the results were compared with those obtained under the same conditions for UGT1A1, a UGAT that is involved in glucuronide conjugation in xenobiotic and endobiotic metabolism in vertebrates (
      • Brierley C.H.
      • Burchell B.
      ,
      • Bock K.W.
      ) (Supplemental Fig. 2S). The results showed that all of these aglycons were good substrates for UGT1A1, whereas none served as substrates for BpUGAT.

      Effects of Metal Ions and Enzyme Inhibitors

      The effects of divalent metal ions (as chlorides) and a metal chelator (EDTA) on BpUGAT activity were examined under the standard assay conditions. The enzyme activity was completely inhibited by 0.1 mm Cu2+ and 0.1 mm Hg2+ ions and partially inhibited by 0.1 mm Ca2+ (residual activity, 57%), 0.1 mm Cd2+ (49%), and 0.1 mm Fe2+ ions (36%). It should be noted, however, that the observed enzyme inhibition by Cu2+ and Hg2+ may not solely be attributed to their effects on the enzyme itself because heavy metal ions such as these are known to destroy substrate anthocyanins (
      • Ford C.M.
      • Boss P.K.
      • Høj P.B.
      ). Other 0.1 mm metal ions (Co2+, Mg2+, Mn2+, Ni2+, Sn2+, and Zn2+) and 5 mm EDTA had negligible effects on the catalytic activity (residual activity, in excess of 70%). The effects of compounds that are structurally related to the substrates and products on enzyme activity were also examined under the standard assay conditions. The enzyme was strongly inhibited by 1 mm UDP (residual activity, 0%) and 1 mm UTP (9%) and somewhat by 1 mm UMP (36%) but was not inhibited by 1 mm uridine (98%), 1 mm sodium malonate (100%), and 1 mm glucose (100%). The enzyme was completely inactivated in the presence of 5 mmN-ethylmaleimide at pH 7.0 and 20 °C for 20 min; however, it retained full activity after incubation with 1 mm diethyl pyrocarbonate under the same conditions.

      cDNA Cloning, Sequencing, and Heterologous Expression

      We designed the PCR primers on the basis of the amino acid sequences determined with the purified BpUGAT (i.e. sequences 1 and 3, see “Experimental Procedures” for details) and executed a first round PCR using poly(A)+ RNAs of B. perennis flowers as a template. The amplified fragment was further subjected to nested PCR, yielding a PCR product of ∼1 kbp. Using this fragment as a probe, the cDNA library (∼400,000 plaques) was screened under high stringency conditions to give 80 positive clones. The longest cDNA from these clones was completely sequenced. The deduced amino acid sequence of the clone encoded a protein (GenBank™/DDBJ/EBI accession number AB190262) of 438 amino acids with a calculated molecular mass of 49,645 Da. The partial amino acid sequences determined for the purified enzyme (sequences 1, 2, and 3) were located at positions 102–112, 384–393, and 417–426, respectively. We were also able to identify a sequence (i.e. plant secondary product glycosyltransferase (PSPG) box) that has been ubiquitously identified among the sequences of some other family members (i.e. PSPGs and vertebrate UGATs, see “Discussion” for details) of the glycosyltransferase superfamily and proposed to constitute the nucleotide-sugar binding site (
      • Li Y.
      • Baldauf S.
      • Lim E-K.
      • Bowles D.J.
      ,
      • Vogt T.
      • Jones P.
      ,
      • Vogt T.
      • Jones P.
      ,
      • Keegstra K.
      • Raikhel N.
      ). In an extensive search for similarity of the entire amino acid sequence in data bases and the previous literature, the highest identity was found with the UDP-rhamnose:flavonoid rhamnosyltransferase of Citrus maxima (40%) (
      • Bar-Peled M.
      • Lewinsohn E.
      • Fluhr R.
      • Gressel J.
      ) and the UDP-rhamnose:anthocyanidin-3-glucoside rhamnosyltransferase of Petunia × hybrida (28%) (
      • Kroon J.
      • Souer E.
      • de Graaf A.
      • Xue Y.
      • Mol J.
      • Koes R.
      ). A lower sequence similarity was found with anthocyanin 5-glucosyltransferases from several plant species (identity, 15–20%) (
      • Yamazaki M.
      • Gong Z.
      • Fukuchi-Mizutani M.
      • Fukui Y.
      • Tanaka Y.
      • Kusumi T.
      • Saito K.
      ). However, despite the similarity in glycosyl donor specificity, BpUGAT showed only a very low sequence similarity to vertebrate UGATs and all known plant UGATs (NpUGT (
      • Iwai H.
      • Masaoka N.
      • Ishii T.
      • Satoh S.
      ), PsUGT1 (
      • Woo H-H.
      • Orbach M.J.
      • Hirsch A.M.
      • Hawes M.C.
      ), and UBGAT (GenBank™/DDBJ/EBI accession number BAC98300)).
      The BpUGAT cDNA was expressed under the control of the GAL1 promoter in the S. cerevisiae YPH499 cells as a soluble, catalytically active protein at a level of ∼1 mg/liter of culture broth. The recombinant protein could be purified to homogeneity (Fig. 1) from crude extracts of transformant yeast cells using procedures that were essentially the same as those used for the purification of the native BpUGAT from daisy flowers. The recombinant protein showed a strong BpUGAT activity; catalytic and molecular properties of the recombinant enzyme were essentially identical to those of the native BpUGAT (Table II), thus confirming that the cloned cDNA codes for BpUGAT.

      Quantitative Real Time PCR

      To evaluate the functional significance of BpUGAT in the coloration of red daisy flowers, the organ specificity and developmental changes in BpUGAT expression in the daisy plant were analyzed by quantitative real time PCR (Fig. 2A). Flower development was divided into five morphologically different stages. The BpUGAT transcript was identified in petals of red flowers, and the highest level of the transcription was at stage 4 of flower development. The transcript was not identified in the petals of white flowers. No or only a negligible amount of the transcript could be found in the sepal, stem, and leaf. Such temporal and spatial expressions of BpUGAT are consistent with the trend observed for BpUGAT activity and the pigmentation pattern in the B. perennis plant (Fig. 2B). Thus, the expression of BpUGAT is temporally regulated during flower development, as is the case for the genes responsible for anthocyanin biosynthesis, and is consistent with the temporal and spatial distributions of enzyme activity and pigment. These results are consistent with the role of the enzyme in the production of glucuronosylated anthocyanins that are the origin of the red coloration of daisy flowers (
      • Saito N.
      • Toki K.
      • Honda T.
      • Kawase K.
      ,
      • Toki K.
      • Saito N.
      • Honda T.
      ).
      Figure thumbnail gr3
      Fig. 2Quantitative real time PCR of BpUGAT. A, the relative transcription levels of BpUGAT (white bars) in individual organs of B. perennis plant, i.e. petals at five developmental stages of flowers (for definition of flower stages, see “Experimental Procedures”), sepal, stem, leaf, and tubular corolla (yellow in color) were determined by real time PCR. White petal, white petals, taken from fully open flowers of an acyanic cultivar of daisy, were also analyzed for the transcription of BpUGAT. Average values of four independent determinations of transcription levels are presented with error bars indicating ±S.E. B, specific activities of BpUGAT in crude extracts of organs of B. perennis (gray bars). BpUGAT activity was assayed as described under “Experimental Procedures.” Plus and minus signs indicate the presence and absence of red pigmentation in individual organs of the daisy, respectively.

      DISCUSSION

      In this study, primary structure and phylogenetics of a novel UGAT that is involved in plant secondary metabolism (flower pigment biosynthesis) were clarified along with its molecular properties and specificity. Sequence comparison studies revealed that BpUGAT belongs to the glycosyltransferase superfamily (
      • Campbell J.A.
      • Davies G.J.
      • Bulone V.
      • Henrissat B.
      ) and is most closely related to the GT1 family, which consists of vertebrate UGATs that are involved in glucuronide conjugation and PSPGs (
      • Vogt T.
      • Jones P.
      ). It showed only low sequence similarity to vertebrate UGATs despite its similarity in glycosyl donor specificity and appears to belong to the subfamily (clade) of PSPGs (Fig. 3) (
      • Vogt T.
      • Jones P.
      ). It is very important to note here that PSPGs play crucial roles in the metabolism of endobiotics and xenobiotics in plants (
      • Vogt T.
      • Jones P.
      ,
      • Hefner T.
      • Arend J.
      • Warzecha H.
      • Siems K.
      • Stockigt J.
      ), and their functions are essentially parallel with those of vertebrate UGATs (
      • Lim E-K.
      • Bowles D.J.
      ). Namely plant systems retain secondary metabolites intracellularly and may also be exposed to a wide range of xenobiotic compounds. The accumulation of such compounds in plant cells may interfere with the physiological functions of the host plants, particularly if the compounds are toxic or precipitate due to a low solubility. PSPG-catalyzed glycosylation enhances the solubility of these compounds and allows their storage within vacuoles, thus maintaining the metabolic homeostasis of host plants (
      • Lim E-K.
      • Bowles D.J.
      ,
      • Vogt T.
      • Jones P.
      ). Thus, the enzymatic glycosylation by these family GT1 members confers greater water solubility on the substrate, facilitating product accumulation in vacuoles (in PSPGs and BpUGAT) or the product excretion from cells (in the case of vertebrate UGATs). The present phylogenetic analysis of the UBGAT sequence (GenBank™/DDBJ/EBI accession number BAC98300) revealed that this plant UGAT should also be a member of PSPG (Fig. 3), consistent with its role (e.g. solubilization of the aglycon) in baicalin synthesis. Thus, it could be concluded that BpUGAT and UBGAT are variants of PSPG that have been diversified to show an altered glycosyl donor specificity to fulfill the common role of the GT1 family in respective plant species.
      Figure thumbnail gr4
      Fig. 3Molecular phylogenetic tree of the GT1 family members of the glycosyltransferase superfamily. The tree was constructed by the neighbor-joining method. The lengths of the lines indicate the relative distances between nodes. Numbers indicate bootstrap values greater than 800. Clusters I, II, and III of PSPGs (
      • Vogt T.
      • Jones P.
      ) are shown with gray circles. PSPGs used for the alignment are as follows and are shown with GenBank™ accession numbers in parentheses unless otherwise indicated: Nierembergia_RhmT, rhamnosyltransferase of Nierembergia sp. (AB078511); Petunia_RhmT, UDP rhamnose:anthocyanidin 3-O-glucoside rhamnosyltransferase of Petunia × hybrida (EBI accession numbers X71059 and X71060); BpUGAT, UDP-glucuronic acid:anthocyanidin 3-O-glucoside 2″-O-β-glucuronosyltransferase (this study, AB190262); Citrus_RhmT, flavonoid rhamnosyltransferase of C. maxima (AY048882); Petunia_5GT, anthocyanin 5-O-glucosyltransferase of P. hybrida (AB027455); Verbena_5GT, UDP-glucose:anthocyanin 5-O-glucosyltransferase of Verbena × hybrida (BAA36423); Torenia_5GT, anthocyanin 5-O-glucosyltransferase of Torenia hybrida (BAC54093); Perilla_5GT, UDP-glucose:anthocyanin 5-O-glucosyltransferase of Perilla frutescens (AB013596); PsUGT1, UDP-glucuronosyltransferase of P. sativum (AF034743); S_baicalensis_7GT, UDP-glucose:flavonoid 7-O-glucosyltransferase of S. baicalensis (BAA83484); Betanidin_5GT, betanidin 5-O-glucosyltransferase of Dorotheanthus bellidiformis (Y18871); Gentiana_3′GT, anthocyanin 3′-O-glucosyltransferase of Gentiana triflora (BAC54092); tomato_twi1, a wound-induced gene product (twi1) of Lycopersicon esculentum (X85138); N_tabacum_IS5a, salicylate-induced glucosyltransferase (IS5a) of Nicotiana tabacum (U32644); N_tabacum_IS10a, salicylate-induced glucosyltransferase (IS10a) of N. tabacum (U32643); Arbutin_synthase, arbutin synthase of Rauvolfia serpentina (AJ310148); UBGAT, UDP-glucuronic acid:baicalein 7-O-glucuronosyltransferase of S. baicalensis (BAC98300); Barley_3GT, UDP-glucose:flavonol 3-O-glucosyltransferase (bronze 1 homolog) of Hordeum vulgare subsp. vulgare (X15694); Maize_3GT, UDP-glucose:flavonol 3-O-glucosyltransferase (bz1 gene product) of Zea mays (X13501 and M37640); Grape_3GT, UDP-glucose:flavonoid 3-O-glucosyltransferase of Vitis vinifera (AF000371); Ipomoea_3GT, UDP-glucose:flavonoid 3-O-glucosyltransferase of Ipomoea purpurea (AF028237); Eggplant_3GT, UDP-glucose:flavonoid 3-O-glucosyltransferase of Solanum melongena (AF028237); Petunia_3GT, anthocyanidin 3-O-glucosyltransferase of P. hybrida (AB027454); Gentiana_3GT, UDP-glucose:flavonoid-3-O-glucosyltransferase Gentiana triflora (D85186); Perilla_3GT, flavonoid 3-O-glucosyltransferase of P. frutescens (AB002818). The following enzymes are UGAT isozymes of humans that are involved in glucuronide conjugation: UGT1A1 (P22309), UGT1A3 (P35503), UGT1A6 (P19224), UGT2A1 (CAB41974), UGT2B7 (P16662), UGT2B10 (P36537), and UGT2B15 (A48633).
      Consistent with a closer relationship of BpUGAT with PSPGs, BpUGAT is more similar in its enzymatic properties to other PSPGs (including UBGAT) than to vertebrate UGATs. For example, BpUGAT is a monomeric, soluble enzyme that does not require any divalent metal ion for its catalytic activity. BpUGAT is most likely to be a cytosolic enzyme, judging from the apparent absence of sugar chains in the enzyme, its optimum pH for activity (pH 8.0), and pH stability (pH 7.5) (see “Results”), and the result of a PSORT analysis of the BpUGAT sequence (not shown). For comparison, UGT1A1, a vertebrate enzyme that is involved in glucuronide conjugation, is an endoplasmic reticulum-localized, membrane-bound, multimeric enzyme that shows an absolute requirement of Mg2+ (or Mn2+) ion for activity (
      • Radominska-Pandya A.
      • Czernik P.J.
      • Little J.M.
      • Battaglia E.
      • Mackenzie P.I.
      ). BpUGAT shows a high specific activity (1300 nmol–1 mg–1 s–1 at 30 °C), more than 1,000 times higher than those of vertebrate UGATs. Moreover BpUGAT is specific for anthocyanidin 3-O-glucosides, whereas UGT1A1 shows a broad specificity toward lipophilic compounds (see Supplemental Fig. 2S for example). It should be noted, however, that some PSPGs that are involved in xenobiotic metabolism also show low acceptor specificities (
      • Vogt T.
      • Jones P.
      ,
      • Hefner T.
      • Arend J.
      • Warzecha H.
      • Siems K.
      • Stockigt J.
      ), and the strict acceptor specificity of BpUGAT should be closely related to its specialized role in the biosynthesis of flower pigment. It should also be noted that the enzymatic properties of UDP-glucuronate:soyasapogenol UGAT of Glycine max (
      • Kurosawa Y.
      • Takahara H.
      • Shiraiwa M.
      ) significantly differ from those of BpUGAT and UBGAT but are rather similar to those of vertebrate UGATs. For example, UDP-glucuronate:soyasapogenol UGAT appears to be a membrane-bound microsomal enzyme and requires Mg2+ ion for catalytic activity (
      • Kurosawa Y.
      • Takahara H.
      • Shiraiwa M.
      ). This might be related to a difference in their phylogenetic positions. An elucidation of the primary structure of UDP-glucuronate: soyasapogenol UGAT is awaited with much interest.
      Phylogenetic analyses of PSPG sequences have shown that several separated clusters in the PSPG subfamily exist with members sharing high sequence identities (>40%) and common trends of acceptor specificity with each other (e.g. clusters I, II, and III; see Fig. 3) (
      • Vogt T.
      • Jones P.
      ). BpUGAT and UBGAT do not belong to any of these known clusters. Moreover BpUGAT is distantly related to UBGAT and appears to be more closely related to flavonoid rhamnosyltransferases (
      • Bar-Peled M.
      • Lewinsohn E.
      • Fluhr R.
      • Gressel J.
      ,
      • Kroon J.
      • Souer E.
      • de Graaf A.
      • Xue Y.
      • Mol J.
      • Koes R.
      ) (Fig. 3). Most notably, the specificities of BpUGAT and these rhamnosyltransferases are distinct from members of the any known clusters (I, II, and III) and UBGAT since they do not catalyze the transfer of a glycosyl unit to an aglycon moiety but catalyze the transfer to a sugar moiety of flavonoid 3-O-glucosides. Thus, BpUGAT and these rhamnosyltransferases appear to constitute another class of enzymes that represent a fourth cluster of the family GT1 PSPGs. Further identification of other BpUGAT-related PSPGs and their specificities will be needed to verify this prediction.
      The biosynthetic pathways leading to anthocyanidin 3-O-glucosides have been well characterized and are conserved in plant species. On the other hand, the modification of anthocyanidin 3-O-glucosides, including acylation (e.g. malonylation) and further glycosylation (e.g. glucuronosylation), varies with plant species. The specificity of BpUGAT provides important information relative to the order of anthocyanin modification in daisy flower. Both Cy3MG and cyanidin 3-O-glucoside were good substrates for BpUGAT (see Table II), suggesting that these anthocyanins may serve as physiological glucuronosyl acceptors in vivo. Cyanidin 3-O-2″-O-β-glucuronosyl-6″-O-malonylglucoside accounts for more than 85 mol % of the total anthocyanins in the daisy flowers used in this study, whereas cyanidin 3-O-2″-O-β-glucuronosylglucoside was virtually absent.
      S. Sawada, unpublished results.
      These results suggest that the anthocyanin malonyltransferase of daisy flowers should be able to malonylate cyanidin 3-O-2″-O-β-glucuronosylglucoside as well as cyanidin 3-O-glucoside, and the late stage of biosynthesis of this flower pigment may constitute a metabolic grid (
      • Yamazaki M.
      • Gong Z.
      • Fukuchi-Mizutani M.
      • Fukui Y.
      • Tanaka Y.
      • Kusumi T.
      • Saito K.
      ) where either 6″-O-malonylation or 2″-O-glucuronosylation may take place first. A similar metabolic grid has been proposed for anthocyanin modifications (6″-aromatic acylation and 5‴-glucosylation) in red Perilla leaves (
      • Yamazaki M.
      • Gong Z.
      • Fukuchi-Mizutani M.
      • Fukui Y.
      • Tanaka Y.
      • Kusumi T.
      • Saito K.
      ).

      Acknowledgments

      We thank Akio Noguchi for valuable help and discussion.

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