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• W2108650147 abstract "We report here the cloning and optimized expression at 16 °C and the characterization of a Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, an enzyme responsible for a late step in grapevine anthocyanin biosynthesis. The properties of this and other UDP-glucose:flavonoid 3-O-glucosyltransferases, homologues of the product encoded by the maize Bronze-1locus, are a matter of conjecture. The availability of a purified recombinant enzyme allowed for the unambiguous determination of the characteristics of a flavonoid 3-O-glucosyltransferase. Kinetic analyses showed that kcat for glucosylation of cyanidin, an anthocyanidin substrate, is 48 times higher than for glucosylation of the flavonol quercetin, whereasKm values are similar for both substrates. Activity toward other classes of substrates is absent. Cu2+ ions strongly inhibit the action of this and other glucosyltransferases; however, we suggest that this phenomenon in large part is due to Cu2+-mediated substrate degradation rather than inhibition of the enzyme. Additional lines of complementary biochemical data also indicated that in the case of V. vinifera, the principal, if not only, role of UDP-glucose:flavonoid 3-O-glucosyltransferases is to glucosylate anthocyanidins in red fruit during ripening. Other glucosyltransferases with a much higher relative activity toward quercetin are suggested to glucosylate flavonols in a distinct spatial and temporal pattern. It should be considered whether gene products homologous to Bronze-1 in some cases more accurately should be referred to as UDP-glucose:anthocyanidin 3-O-glucosyltransferases. We report here the cloning and optimized expression at 16 °C and the characterization of a Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, an enzyme responsible for a late step in grapevine anthocyanin biosynthesis. The properties of this and other UDP-glucose:flavonoid 3-O-glucosyltransferases, homologues of the product encoded by the maize Bronze-1locus, are a matter of conjecture. The availability of a purified recombinant enzyme allowed for the unambiguous determination of the characteristics of a flavonoid 3-O-glucosyltransferase. Kinetic analyses showed that kcat for glucosylation of cyanidin, an anthocyanidin substrate, is 48 times higher than for glucosylation of the flavonol quercetin, whereasKm values are similar for both substrates. Activity toward other classes of substrates is absent. Cu2+ ions strongly inhibit the action of this and other glucosyltransferases; however, we suggest that this phenomenon in large part is due to Cu2+-mediated substrate degradation rather than inhibition of the enzyme. Additional lines of complementary biochemical data also indicated that in the case of V. vinifera, the principal, if not only, role of UDP-glucose:flavonoid 3-O-glucosyltransferases is to glucosylate anthocyanidins in red fruit during ripening. Other glucosyltransferases with a much higher relative activity toward quercetin are suggested to glucosylate flavonols in a distinct spatial and temporal pattern. It should be considered whether gene products homologous to Bronze-1 in some cases more accurately should be referred to as UDP-glucose:anthocyanidin 3-O-glucosyltransferases. Phytochemists have identified some 70,000 plant chemicals, many thousands of which are glycosylated (1Harborne J.B. Williams C.A. Harborne J.B. The Flavonoids: Advances in Research Since 1980. Chapman and Hall Ltd., London1988: 303-328Crossref Google Scholar). From a chemical point of view, two main properties differ between the glycosylated secondary products and their respective aglycones, as glycosylation invariably results in enhanced water solubility and lower chemical reactivity. Glycosylated compounds are therefore often thought of as transportable storage compounds or indeed waste/detoxification products assumed to lack physiological activity (2Bandurski R.S. Cohen J.D. Slovin J. Reinecke D.M. Davies P.J. Plant Hormones: Physiology, Biochemistry, and Molecular Biology. 2nd Ed. Kluwer Academic Publishers Group, Dordrecht, Netherlands1995: 39-65Crossref Google Scholar). Despite the widespread occurrence of glycosylated secondary metabolites, including flavonols (3Williams C.A. Harborne J.B. Harborne J.B. The Flavonoids: Advances in Research Since 1986. Chapman & Hall Ltd., London1993: 337-385Crossref Google Scholar), anthocyanins (4Brouillard R. Dangles O. Harborne J.B. The Flavonoids: Advances in Research Since 1986. Chapman & Hall Ltd., London1993: 565-588Crossref Google Scholar), monoterpenes (5Williams P.J. Strauss C.R. Wilson B. Am. J. Enol. Vitic. 1981; 32: 230-235Google Scholar), plant hormones (2Bandurski R.S. Cohen J.D. Slovin J. Reinecke D.M. Davies P.J. Plant Hormones: Physiology, Biochemistry, and Molecular Biology. 2nd Ed. Kluwer Academic Publishers Group, Dordrecht, Netherlands1995: 39-65Crossref Google Scholar), and metabolites of systemic fungicides (6Coleman J.O.D. Blake-Kalff M.M.A. Davies T.G.E. Trends Plant Sci. 1997; 2: 144-151Abstract Full Text PDF Scopus (506) Google Scholar), isolation and characterization of purified enzymes responsible for their metabolism have only been reported in a couple of select instances (7Bar-Peled M. Lewinsohn E. Fluhr R. Gressel J. J. Biol. Chem. 1991; 266: 20953-20959Abstract Full Text PDF PubMed Google Scholar, 8Kowalczyk S. Bandurski R.S. Biochem. J. 1991; 279: 509-514Crossref PubMed Scopus (40) Google Scholar). The most widely studied groups of plant glycosyltransferases are those associated with the biosynthesis of flavonoid glucosides, including flavonol glucosides, flavanone glucosides, and anthocyanins (3Williams C.A. Harborne J.B. Harborne J.B. The Flavonoids: Advances in Research Since 1986. Chapman & Hall Ltd., London1993: 337-385Crossref Google Scholar, 4Brouillard R. Dangles O. Harborne J.B. The Flavonoids: Advances in Research Since 1986. Chapman & Hall Ltd., London1993: 565-588Crossref Google Scholar, 9Lewinsohn E. Britsch L. Mazur Y. Gressel J. Plant Physiol. (Bethesda). 1989; 91: 1323-1328Crossref PubMed Google Scholar, 10McIntosh C.A. Mansell R.L. Phytochemistry. 1990; 29: 1533-1538Crossref Scopus (40) Google Scholar). Earliest reports included the detection of an anthocyanidin and flavonol glucosylating activity in endosperm extracts of maize (Zea mays), (11Larson R.L. Phytochemistry. 1971; 10: 3073-3076Crossref Scopus (28) Google Scholar, 12Larson R.L. Lonergan C.M. Planta. 1972; 103: 361-364Crossref PubMed Scopus (15) Google Scholar, 13Larson R.L. Lonergan C.M. Cereal Res. Commun. 1973; 1: 13-22Google Scholar). This work formed the basis for identification by transposon tagging of the gene product of the maize Bronze-1 locus (14Fedoroff N.V. Furtek D.B. Nelson O.E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3825-3829Crossref PubMed Google Scholar). cDNAs encoding flavonoid glucosyltransferases have been isolated from a number of plant species utilizing homology to the maizeBronze-1 cDNA. In the grapevine, Sparvoli and co-workers (15Sparvoli F. Martin C. Scienza A. Gavazzi G. Tonelli C. Plant Mol. Biol. 1994; 24: 743-755Crossref PubMed Scopus (430) Google Scholar) cloned a partial cDNA from the variety Lambrusco based upon homology to a putative flavonoid glucosyltransferase ofAntirhinnum majus, itself identified by homology to the maize cDNA (16Martin C.R. Prescott A. Mackay S. Bartlett J. Vrijlandt E. Plant J. 1991; 1: 37-49Crossref PubMed Scopus (342) Google Scholar). Recently, Boss et al. (17Boss P.K. Davies C. Robinson S.P. Plant Physiol. (Bethesda). 1996; 111: 1059-1066Crossref PubMed Scopus (602) Google Scholar, 18Boss P.K. Davies C. Robinson S.P. Aust. J. Grape Wine Res. 1996; 2: 163-170Crossref Scopus (139) Google Scholar, 19Boss P.K. Davies C. Robinson S.P. Plant Mol. Biol. 1996; 32: 565-569Crossref PubMed Scopus (327) Google Scholar) used the “Sparvoli” cDNA to detect the expression of anthocyanidin glucosyltransferase mRNA during the development of berries ofVitis vinifera variety Shiraz and to show that red and white grapes, and color mutants (sports) of several varieties, differed in whether or not they express the UFGT 1The abbreviations used are: UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase; rUFGT, recombinant UDP-glucose:flavonoid 3-O-glucosyltransferase; IMAC, immobilized metal affinity chromatography; UDP-glucose, uridine-5′-diphosphoglucose; HPLC, high pressure liquid chromatography; PIPES, 1,4-piperazinediethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. gene. Although several reports on the characteristics of expression levels and transgenesis of the UFGT gene have appeared, a clear picture of the properties of the corresponding gene product is not available. Thus, despite the suspected pivotal role played by UFGT in pigment accumulation, this enzyme has never been subjected to a thorough characterization in a purified and guaranteed homogeneous state. Indeed, a confusing picture of the nature and extent of flavonoid glucosyltransferases has arisen in the very extensive literature on these activities. For example, Jonsson et al. (20Jonsson L.M.V. Arsman M.E.G. Bastiaannet J. Donker-Koopman W.E. Gerats A.G.M. Schram A.W. Z. Naturforsch. 1984; 39: 559-564Crossref Scopus (16) Google Scholar), Teusch et al. (21Teusch M. Forkmann G. Seyffert W. Z. Naturforsch. 1986; 41: 699-706Crossref Scopus (14) Google Scholar), and Hrazdina (22Hrazdina G. Biochim. Biophys. Acta. 1988; 955: 301-309Crossref Scopus (28) Google Scholar) conclude a common identity of UDP-glucose:anthocyanidin 3-O-glucosyltransferase and UDP-glucose:flavonol 3-O-glucosyltransferase with the enzyme significantly more active toward the flavonols. In contrast, preliminary analysis of aGentiana triflora recombinant UFGT in crude bacterial lysates indicated a preference for anthocyanins over flavonols (23Tanaka Y. Yonekura K. Fukichi-Mizutani M. Fukui Y. Fujiwara H. Ashikari T. Kusumi T. Plant Cell Physiol. 1996; 37: 711-716Crossref PubMed Scopus (125) Google Scholar). Additionally Do et al. (24Do C.B. Cormier F. Nicholas Y. Plant Sci. 1995; 112: 43-51Crossref Scopus (24) Google Scholar), recently concluded that a partially purified UFGT from grapes exhibits activity with anthocyanins but not at all with flavonols. The confusing picture concerning the multiplicity and properties of anthocyanidin/flavonol 3-O-glucosyltransferases undoubtedly stems from the repeated inability of investigators to purify any such enzyme to homogeneity. Additionally, the highly labile nature of anthocyanidins but not of flavonol substrates at the basic pH optimum of most glucosyltransferases has inevitably contributed further to the confusion regarding the relative activities and observed specificities of these enzymes. We report here the cloning of a cDNA encoding a full-length UFGT from grapes of V. vinifera, the world's largest fruit crop with annual production of 60 million tons (25Kanellis A.K. Roubelakis-Angelakis K.A. Seymour G. Taylor S.J. Tucker G. Biochemistry of Fruit Ripening. Chapman & Hall Ltd., London1993: 189-234Crossref Google Scholar). Following development of a protocol which employs very slow growth of Escherichia coli at 16 °C, expression of the cDNA and subsequent purification of a histidine-tagged rUFGT in an active form has permitted, for the first time, the unambiguous determination of the properties of a member of this ubiquitous enzyme family and allowed us to highlight some of the pitfalls that could contribute to erroneous conclusions regarding anthocyanidin glucosyltransferases. It has been demonstrated that UFGT exhibits much higher catalytic efficiency against anthocyanidins than against flavonols such as quercetin and kaempferol. Additional data also strongly indicate that in the case of grapes, the principal role of UFGT is to glucosylate anthocyanidins in red fruit, whereas products of other genes serve to glucosylate flavonols in a distinct spatial and temporal pattern. All biochemicals were of analytical grade or higher. Flavonoid substrates and authentic glucosides were obtained from Fluka, Castle Hill, New South Wales, Australia; Apin Chemicals, Oxford, UK; Carl Roth GmbH, Karlsrühe Germany; Extrasynthèse, Genay, France; and Indofine, Somerville, NJ. Employing mRNA isolated from whole V. vinifera cv. Shiraz grape berries 10 weeks after flowering (26Davies C. Robinson S.P. Plant Physiol. (Bethesda). 1996; 111: 275-283Crossref PubMed Scopus (298) Google Scholar), a Superscript Choice cDNA synthesis kit (Life Technologies, Inc.), λZAPII arms pre-digested with EcoRI (Stratagene) and EcoRI adaptors, a cDNA library was prepared according to the manufacturer's instructions by C. Davies (CSIRO Plant Industry, Adelaide, Australia). An aliquot of the amplified library, consisting of approximately 150,000 plaques, was screened using a 532-base pair partial cDNA clone for UFGT from grape (15Sparvoli F. Martin C. Scienza A. Gavazzi G. Tonelli C. Plant Mol. Biol. 1994; 24: 743-755Crossref PubMed Scopus (430) Google Scholar) as a probe. Hybridization was carried out overnight at 65 °C in Church-Gilbert buffer (27Church G.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7266) Google Scholar); washings, also at 65 °C, were in 2× SSC. This would allow the detection of nucleic acid species with up to approximately 30% mismatched sequences (28Meinkoth J. Wahl G. Anal. Biochem. 1984; 138: 267-284Crossref PubMed Scopus (931) Google Scholar). Two hybridizing clones were isolated, and their nucleotide sequences determined using standard protocols for chain termination sequencing (29Sanger F. Nicklen S. Coulsen A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52770) Google Scholar) of both encoding and non-encoding strands. PCR amplification reactions were carried out using 1× Vent DNA polymerase buffer, 0.2 mm dNTPs, 1 μmeach forward (UFGTF1 [5′-GCGGATCCGCATATGTCTCAAACCACCACCAACCCCCATGTGGCCGTC-3′]) and reverse (UFGTR1 [5′-GTGTCAAAACCAAAGGATGTCTAGGGATCCAAGCTTGAATTCCG-3′]) DNA primer, 2 mm MgSO4, and 0.8 units of Vent DNA polymerase (New England Biolabs) in a 20-μl final volume with approximately 1 ng of plasmid pGUT3 DNA template. Thermal cycling parameters were 94 °C, 5 min, 30 × (94 °C for 1 min, 58 °C for 1 min, and 72 °C for 2 min) and a final 5 min at 72 °C. After digestion of the 1403-base pair amplification product with restriction enzymes NdeI and BamHI, the DNA was ligated into pET14b (Novagen, Madison, WI) and used to transform competent cells of E. coli strain JM109. Plasmid was prepared from recombinant clones and used to transform the E. coli BL21 pLysS strain (30Studier F.W. J. Mol. Biol. 1991; 219: 37-44Crossref PubMed Scopus (683) Google Scholar) to yield the clone pET14-VVUFGT used in the subsequent production of recombinant UFGT. Soluble, active UFGT was expressed from pET14-VVUFGT following growth and induction at 16 °C. Briefly, L broth (100 ml) held at 16 °C and containing ampicillin and chloramphenicol was inoculated with 100 μl of overnight culture (grown at 37 °C) and grown until anA600 between 0.4 and 0.6 was reached (typically 24–40 h). Induction of recombinant protein expression was initiated by the addition of isopropyl-1-thio-β-d-galactopyranoside to 0.4 mm, and cultures were grown for a further 24 h. Cells were harvested by centrifugation at 1400 × g for 15 min, and the drained pellets stored at −20 °C. Bulk growth (1-liter scale) was carried out in 5-liter Ehrlenmeyer flasks using 5 ml of an overnight starter culture. Frozen cells (about 0.8–1.0 g per 50 ml of culture) were thawed and resuspended in lysis buffer (20 mm Tris-Cl, pH 8.0, 100 mm NaCl, 10 mm 2-mercaptoethanol, 10% v/v glycerol; 3 ml per g wet cell weight) in 10-ml tubes followed by three freeze/thaw cycles using liquid nitrogen. Phenylmethylsulfonyl fluoride was added to 1.5 mm, DNase I to 690 units ml−1, and the suspension mixed thoroughly by passage through an 18-gauge syringe needle 10 times before incubation on a mixing wheel for 20 min at room temperature. The suspension was transferred to 2-ml tubes and centrifuged for 20 min at full speed in a bench top microcentrifuge. The resulting supernatant and pellet contained, respectively, soluble and insoluble protein fractions. Recombinant UFGT was purified from the soluble fraction by IMAC using the Talon system (CLONTECH, Palo Alto, CA), utilizing the 6-histidine tag placed at the N terminus of the recombinant protein. Protein in the soluble fraction was bound to the affinity resin and eluted using a buffer containing 50 mmimidazole as described in the manufacturer's protocol. All buffers were modified to contain 10 mm 2-mercaptoethanol and 10% v/v glycerol. Using 0.5 ml of Talon resin, recombinant protein from approximately 50 ml of culture could be purified. Incorporation of 15 mm imidazole in the wash buffers resulted in a homogeneous preparation of the enzyme, albeit at the expense of yield. Glucosylation was routinely assayed in a modification of the buffer system reported by Do et al.(24Do C.B. Cormier F. Nicholas Y. Plant Sci. 1995; 112: 43-51Crossref Scopus (24) Google Scholar). In a final assay volume of 200 μl, the reaction conditions were 100 mm buffer (Tris-Cl, pH 8.0, or HEPES-OH, pH 8.0), 10 mm poly(ethylene glycol) 3400, 14 mm2-mercaptoethanol, 2 mm dithiothreitol, 9 mmUDP-glucose, 100 μm flavonol or anthocyanidin acceptor substrate and enzyme. The amount of enzyme used was varied according to the acceptor substrate under study. Except in the case of anthocyanidin substrates, assays were routinely started with the addition of the enzyme and were incubated at 30 °C for up to 6 min (anthocyanidin substrates) and 10 min (flavonol substrates). Samples were taken at two time points to ensure linearity of all data points and hence to measure initial rates of activity. Flavonols and anthocyanidins were prepared fresh as required from stock solutions stored at −20 °C. Unless stated otherwise, the solvent used for substrate dilution was 2-methoxyethanol (ethylene glycolmonomethyl ether (2-methoxyethanol)). Reactions were stopped by acidification. For flavonol acceptor substrates, 50 μl of glacial acetic acid was used, and for anthocyanidins, 150 μl of 5% HCl was added to the reaction mixture. Routinely, reaction products were analyzed by reversed-phase HPLC using a Beckman System Gold apparatus (128 diode array detector module, 126 pumps, 507E autosampler) and Gold Nouveau software. A 250 × 4.5 mm Vydac 218TP C18 column was used and maintained at 30 °C in an Eppendorf CH-30 column heater. Buffer A was 0.1% trifluoroacetic acid, buffer B was 0.085% trifluoroacetic acid in 80% acetonitrile. Glucosides were resolved using a linear gradient from A to B of 0 to 50% B at 2.22% B per min with a flow rate of 0.8 ml/min. Absorbance maxima were determined for each glucoside by examination of the spectrum of the eluting glucoside (retention times were as follows: delphinidin-3-O-glucoside 9.43 min; cyanidin-3-O-glucoside 9.99 min; pelargonidin-3-O-glucoside 10.70 min; peonidin-3-O-glucoside 10.93 min; and malvidin-3-O-glucoside 11.06 min). Integration values were calculated at the respective λmax for each glucoside (cyanidin-3-O-glucoside λmax 515 nm; delphinidin-3-O-glucoside λmax 525 nm; pelargonidin-3-O-glucoside λmax 511 nm; peonidin-3-O-glucoside λmax 516 nm; and malvidin-3-O-glucoside λmax 525 nm). The use of phosphoric acid-based buffers for HPLC separation of anthocyanins, while widely reported (21Teusch M. Forkmann G. Seyffert W. Z. Naturforsch. 1986; 41: 699-706Crossref Scopus (14) Google Scholar, 31Schwinn K.E. Davies K.M. Deroles S.C. Markham K.R. Miller R.M. Bradley J.M. Manson D.G. Given N.K. Plant Sci. 1997; 125: 53-61Crossref Scopus (39) Google Scholar), was found to be unnecessary under the conditions employed here. The radioactive donor substrate UDP-[U14C]glucose was used for the determination of the rates of synthesis of glucosides for which extinction coefficient data were not available. UDP-[U14C]glucose (10.6 GBq mmol−1, DuPont) was added to a final concentration of 2.5 μm and supplemented with “cold” UDP-glucose to 100 μm. For analysis by liquid scintillation counting or thin layer chromatography, flavonol glucosides were extracted into 500 μl of ethyl acetate to remove unincorporated donor substrate. Liquid scintillation counting of ethyl acetate extracts (200 μl, duplicates) was performed using Ultima Gold XR mixture (Canberra Packard) in a LS6000TD counter (Beckman). TLC analysis was performed upon ethyl acetate extracts following concentration at 70 °C in a Centrivap Concentrator (Labconco) under reduced pressure. Residues were dissolved into 20 μl of ethyl acetate. TLCs were run on Silica Gel 60 F254 plates (Merck) in a solvent comprising 35 parts ethyl acetate, 2 parts formic acid, 2 parts water (32Parry A.D. Edwards R. Phytochemistry. 1994; 37: 655-661Crossref Scopus (17) Google Scholar). Developed plates were dried at 80 °C for 1 h and exposed to storage phosphorimaging plates (Molecular Dynamics) prior to scanning on a Storm 860 PhosphorImager (Molecular Dynamics). Glucoside formation from anthocyanidin, phenol, and monoterpene acceptor substrates was assessed after separation using SepPak disposable reverse phase cartridges (Millipore) to remove the unincorporated UDP-[U14C]glucose prior to liquid scintillation counting or TLC assay. Briefly, acidified assay mixtures were loaded onto pre-charged SepPak cartridges, washed with 5 ml of MilliQ water, and eluted in 2–3 ml of ethanol (33Williams P.J. Cynkar W. Francis I.L. Gray J.D. Iland P.G. Coombe B.G. J. Agric. Food Chem. 1995; 43: 121-128Crossref Scopus (129) Google Scholar). After lyophilization under reduced pressure, residues were dissolved into 20 μl of ethyl acetate. For assays with anthocyanidin substrates, ethanol used for elution was acidified to 0.1% v/v HCl. Km values of the recombinant glucosyltransferase were determined for the donor substrate UDP-glucose and the acceptors quercetin, cyanidin, malvidin, and delphinidin. Additionally, Vmax was determined for quercetin and cyanidin. For the measurement of theKm for UDP-glucose, 200 μm quercetin was used as the acceptor substrate, and the concentration of UDP-glucose was varied from 0.25 to 9 mm. Acceptor substrate Km values were determined with 9 mm UDP-glucose as the donor substrate, and the concentration of the acceptor varied from 1 to 200 μm as applicable. Assays were as described above. Additionally, for the determination of the Km for cyanidin, reducing agents were omitted from the reaction mixture, and cyanidin, rather than enzyme, was added to start the reaction. Experimental data from HPLC analyses were integrated using Gold Nouveau software and for cyanidin and quercetin glucosylation, converted to specific activities by reference to standard curves obtained using the authentic glucosides. Data were transformed and plotted as Lineweaver-Burk graphs to allow calculation of Km and Vmax values. The activity of the recombinant glucosyltransferase with alternative donor and acceptor substrates was tested. Nucleotide sugars ADP-, CDP-, GDP-, and TDP-glucose, UDP-glucuronic acid, UDP-galactose, UDP-mannose, and UDP-xylose were tested for their ability to support glycosylation of quercetin. Alternative acceptor substrates tested, with the donor substrate UDP-glucose, were kaempferol, fisetin, morin, myricetin, isorhamnetin (flavonols), quercetin-3-O-glucoside (flavonol glucoside), pelargonidin, delphinidin, peonidin, and malvidin (anthocyanidins), cyanidin-3-O-glucoside (anthocyanin), apigenin (flavone), hesperitin, naringenin (flavanones), naringenin-7-O-glucoside (flavanone glucoside), biochanin A (isoflavone), resveratrol (stilbene), (+)-catechin (flavan-3-ol), taxifolin (dihydroflavonol), geraniol (monoterpene), triadimenol (1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol; a systemic fungicide), salicylic acid, gentisic acid (benzoic acids), and ferulic acid (cinnamic acid). The variation of activity with pH was studied for the acceptor substrates quercetin and cyanidin. Glucosylation was measured between pH 5.4 and 9.4 using the following buffers: MES, pH 5.4–6.2; PIPES, pH 6.2–6.6; HEPES, pH 6.0–8.0; Tris, pH 6.5–8.5; Tricine, pH 7.5–9.5; CAPS, pH 9.4. Standard assays were carried out as described, with the buffer concentration fixed at 100 mmthroughout. The effect of divalent metal ions upon the glucosylation of quercetin and cyanidin was studied using radiolabeled UDP-[U14C]glucose and liquid scintillation counting. Standard 200-μl assays performed in the absence of added reducing agents were supplemented with CuCl2, MgCl2, CaCl2, MnCl2, and ZnCl2, to final concentrations of 0.01, 0.1, 1, and 10 mm as specified. The inhibition of glucosyltransferase activity was tested for quercetin and cyanidin glucosylation. In both cases the cognate and alternative glucoside were added individually to glucosylation assays at concentrations between 1 and 100 μm. Product formation was determined by HPLC analysis. Rabbit antibodies were produced by the School of Biochemistry, La Trobe University, Melbourne. rUFGT (approximately 500 μg) was mixed with an equal volume of Freund's complete adjuvant and injected intramuscularly. The rabbit was given two more boosts at 4-week intervals before blood samples were obtained. Serum was separated from red cells by centrifugation at 3000 × gfor 15 min at 4 °C and stored in 0.02% sodium azide at 4 °C. Total soluble protein extracts of leaves and berries of grapevine varieties as specified were prepared by a modification of the method of Hawker (34Hawker J.S. Phytochemistry. 1969; 8: 9-17Crossref Scopus (88) Google Scholar, 35Hawker J.S. Phytochemistry. 1969; 8: 337-344Crossref Scopus (57) Google Scholar); full details of this protocol will appear elsewhere. 2C. M. Ford and P. B. Høj, manuscript in preparation. Following preparation, extracts were desalted on a Bio-Gel P6 column and concentrated in a stirred cell unit (Amicon) using a YM30 membrane. Proteins were resolved by SDS in 12% T, 2.63% C (w/v) Tris-Gly gels (36Fling S.P. Gregerson D.S. Anal. Biochem. 1986; 155: 83-88Crossref PubMed Scopus (787) Google Scholar) and blotted onto nitrocellulose membranes (MSI Laboratories, Westbro, MA) using a semi-dry transfer unit (LKB, Broma, Sweden) as described by Harlow and Lane (37Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 471-510Google Scholar). A dilution of antibody of 1:10,000 was used for immunoblotting experiments. Immunoblots were probed with anti-rUFGT antibodies and horseradish peroxidase-coupled goat anti-rabbit antibodies (Promega) before detection using ECLTM reagents (Amersham Pharmacia Biotech) and exposure to Hyperfilm-MPTM film (Amersham Pharmacia Biotech). By using the partial 532-base pair grape cDNA previously isolated by Sparvoli et al. (15Sparvoli F. Martin C. Scienza A. Gavazzi G. Tonelli C. Plant Mol. Biol. 1994; 24: 743-755Crossref PubMed Scopus (430) Google Scholar), two clones homologous to putative plant UFGT sequences were isolated from a screen of approximately 150,000 plaque forming units of a V. vinifera cv. Shiraz post-veraison (coloring) berry cDNA library. Clone GUT8 constitutes a full-length UFGT cDNA sequence but surprisingly contains a 77-base pair intron located at nucleotide 492, while the second clone, GUT3, was intronless and almost full length. Determination of the nucleotide sequence of both clones (GenBankTM accession numbers AF000371 and AF000372 for GUT3 and GUT8, respectively) allowed us to predict the amino acid sequence for the corresponding V. vinifera UFGT (VVUFGT; Fig. 1). Comparison of the predictedV. vinifera UFGT amino acid sequence with that of other glucosyltransferases (Fig. 1) reveals overall positional identities ranging from 42% (V. vinifera UFGT versus Bronze-1 encoded UFGT) to 25% (V. vinifera UFGTversus solanidine glucosyltransferase). Positional identity is not surprisingly greater in the C-terminal third of the protein where the suspected UDP-glucose binding domain, common to all glucosyltransferases, is located (46% V. vinifera UFGTversus Bronze-1 encoded UFGT, 35% V. viniferaUFGT versus solanidine glucosyltransferase) (38Hughes J. Hughes M.A. DNA Sequence. 1994; 5: 41-49Crossref PubMed Scopus (170) Google Scholar). Quite clearly, sequence comparisons of the N-terminal parts of these various glucosyltransferases do not allow solid conclusions to be drawn regarding the enzymic properties of the encoded enzyme, particularly because there exists very little correlation between protein/gene sequences and biochemical characterization of the encoded products. The recombinant UFGT (rUFGT) was therefore expressed as a soluble, active protein in E. coli using the pET14b 6-His fusion vector system. Proteins expressed from sequences cloned into pET14b contain an additional 20 NH2-terminal amino acid residues arising from the vector, including the 6-His “tag” used for metal-chelate affinity purification and a recognition site for the protease thrombin. Preliminary growth and induction trials at 30 and 25 °C yielded protein that was expressed mainly as insoluble inclusion bodies (data not shown). Further reduction of the growth and induction temperature to 16 °C resulted in the expression of significant amounts of a protein, the size expected for recombinant UFGT, in the soluble fraction. This is illustrated in Fig. 2, which shows an SDS-polyacrylamide gel analysis of total proteins extracted from both uninduced cells and those harvested after isopropyl-1-thio-β-d-galactopyranoside induction. The detailed analysis of this expression protocol and the general applica" @default.
• W2108650147 created "2016-06-24" @default.
• W2108650147 creator A5049330844 @default.
• W2108650147 creator A5050827633 @default.
• W2108650147 creator A5064122530 @default.
• W2108650147 date "1998-04-01" @default.
• W2108650147 modified "2023-10-12" @default.
• W2108650147 title "Cloning and Characterization of Vitis viniferaUDP-Glucose:Flavonoid 3-O-Glucosyltransferase, a Homologue of the Enzyme Encoded by the Maize Bronze-1Locus That May Primarily Serve to Glucosylate Anthocyanidins in Vivo" @default.
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