Purification and characterization of UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase from Clitoria ternatea
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- Kogawa, K., Kato, N., Kazuma, K. et al. Planta (2007) 226: 1501. doi:10.1007/s00425-007-0584-1
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A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable kcat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent Km value for 1 (3.89 × 10−5 M), which is lower than that for 2 (1.38 × 10−4 M), renders the kcat/Km value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent Km value for UDP-glucose (6.18 × 10−3 M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10−3 M) with saturated 1, the kcat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center.
KeywordsAnthocyanin 3′-O-glucosyltransferaseAnthocyanin 3′,5′-O-glucosyltransferaseAnthocyanin 5′-O-glucosyltransferaseAnthocyanidin glycoside biosynthesisClitoriaPolyacylated anthocyanins
UDP-glucose: anthocyanin 3′-O-glucosyltransferase
UDP-glucose: anthocyanidin 3-O-glucosyltransferase
UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase
UDP-glucose: anthocyanin 5′-O-glucosyltransferase
Flower colors ranging from reddish to bluish are mainly due to the various structures of anthocyanins which accumulate in the flowers. They are the glycosides or acylated glycosides of anthocyanidin aglycones. Although anthocyanin structure is of primary importance, there are other factors which influence flower color by interacting with anthocyanin molecules including some flavone and flavonol glycosides, metal ions, and vacuolar pH (Brouillard and Dnagles 1994).
The blue flowers in many plants such as Clitoria ternatea, Delphinium hybridum, Eustoma grandiflora, Gentiana triflora, contain delphinidin as anthocyanidin aglycone which are generally glycosylated, and acylated by aromatic organic acids (Honda and Saito 2002). These modifications are important enabling the plant to produce blue flowers because the mauve flower line of C. ternatea accumulates delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) which lacks the modifications (Kazuma et al. 2003). The effect of acylation can be explained as the intramolecular stacking between delphinidin chromophore and aromatic-acyl residue(s), which produces a bathochromic shift in the visible light absorption wavelength of anthocyanins. Thus, polyacylation of anthocyanins with aromatic-acyl groups is one of the mechanisms to produce blue flowers. The acylation at anthocyanin’s B-ring sugar residue (3′-O-glucosyl residue) has been suggested as the most appropriate site for the bluish color in the G. triflora anthocyanin, gentiodelphin (Yoshida et al. 2000).
Anthocyanin glycosyltransferases are important for the flower colors and the stability of the anthocyanins because they decide the positions where glycosyl residues are transferred. Among them, anthocyanin B-ring glucosyltransferases are of interest in relation to the blue flower color. Recently, anthocyanin 3′-O-glucosyltransferase was isolated (Fukuchi-Mizutani et al. 2003) from G. triflora. However, no anthocyanin 5′-O-glucosyltransferase activity has been reported. In this study, we purified anthocyanin 3′,5′-O-glucosyltransferase, a bifunctional enzyme which has both anthocyanin 3′- and 5′-O-glucosyltransferase activities, for the first time from C. ternatea and characterized its properties in detail.
Materials and methods
For HPLC analysis of reaction mixtures, a Waters 600s HPLC system equipped with a 717 autosampler and a 996 photodiode array detector was used with a Develosil ODS-UG-5 column (1.5 i.d. × 250 mm; Nomura Chemical Co. Ltd., Seto, Japan). For DEAE Toyopearl and Mono-Q chromatography, a Fast Protein Liquid Chromatography (FPLC) system equipped with a P-500 pump, a UV-1 detector, a 50-ml superloop, and a Frac-100 fraction collector (Amersham Biosciences, Piscataway, NJ, USA) was used. Protein concentration was determined by the Bradford method (Bradford 1976) with BSA as a standard.
Seeds of C. ternatea cv. Double Blue (DB) were purchased from Sakata Seed Ltd., Yokohama, Japan, and plants were cultivated in a greenhouse. Flower buds were harvested just before blooming (40-45 mm length) and stored at −80°C until use.
Standard assay conditions and analysis of the reaction mixture
For enzymatic assays, delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2) was used as the substrate and production of ternatin C5 (3) was measured. The standard assay mixture consisted of 2.0 mM of 2 in 40% MeCN containing 0.05 M Trifluoroacetic acid (TFA) (4 μl), 5.0 mM UDP-glucose in distilled water (4 μl), enzyme solution (8 μl), 1.0 M potassium phosphate buffer (pH 7.5, 2 μl), and distilled water (2 μl) in a final volume of 20 μl. The reaction was initiated by adding the anthocyanin solution to the rest of the assay mixture. The reaction was terminated by the addition of 4 μl of 1.0 M HCl. For detection of the activity, the reaction times of 10 min were used. However, for determining the initial velocity, three reaction times of 30, 60, and 90 s were used. Prior to HPLC analysis, the reaction was mixed with 5% MeCN containing 0.05 M TFA (24 μl). The reaction mixtures were analyzed by HPLC with a 14–86% linear gradient of solvent B (40% MeCN in aq. 0.05 M TFA) in solvent A (5% MeCN in aq. 0.05 M TFA) for 20 min at a flow rate of 0.5 ml/min.
Purification of UA3′5′GT
The buffer systems used were: buffer A, 100 mM Tris–HCl, pH 7.5; buffer B, 20 mM Tris–HCl, pH 7.5; buffer C, 20 mM Tris–HCl, pH 7.5, containing 1.0 M NaCl; buffer D, 5.0 mM Mes, pH 7.0; buffer E, 5.0 mM Tris–HCl, pH 8.0, containing 10 mM UDP-glucose; buffer F, 2.0 mM sodium/potassium phosphate, pH 7.5; buffer G, 15 mM sodium/potassium phosphate, pH 7.5; and buffer H, 30 mM sodium/potassium phosphate, pH 7.5. Dithiothreitol (DTT) and p-Amidinophenylmethylsulfonylfluoride (pAPMSF) were added to each buffer just before use to final concentrations of 5.0 mM and 10 μM, respectively (buffer A), or 1.0 mM and 10 μM, respectively (buffers B–H).
All the purification procedures were carried out at 4°C. Powdered frozen petals (1,035 g) mixed with polyvinylpolypyrrolidone (51 g) were extracted three times with buffer A. The extract was stirred with Dowex 1 × 2 (ca. 1 kg) (Muromachi Technos Co. Ltd., Tokyo, Japan) to remove pigments. After filtered, the extract was brought to (NH4)2SO4 precipitation. The 35–70% (NH4)2SO4 fraction was retrieved and desalted with a Sephadex G-25 (Amersham Biosciences) column (void volume 38 ml). The protein solution (333 ml) in buffer B was brought to DEAE-Toyopearl 650M (Tosoh Co., Tokyo, Japan) anion exchange chromatography (three times, using ca. 100 ml at once). The elution with a 0–20% linear gradient of buffer C in buffer B for 45 min at a flow rate of 8 ml/min using an FPLC system could gave an active fraction in 6.6–10.2% buffer C. The proteins were retrieved by 90% (NH4)2SO4 saturation and desalted with the Sephadex G-25 column. Two halves of the protein solution in buffer D (ca. 17 ml each) were passed through two columns (10 mm i.d. × 120 mm, containing 10 ml of the resin) of Reactive Yellow 86 resin (Sigma, St. Louis, MO, USA) in parallel. After washing with buffer D (52.5 ml each), an active fraction was eluted with buffer E (50 ml each), and ca. 14-times concentrated with Amicon Ultra-15 10K NMWL units (Millipore, Bedford, MA, USA) followed by buffer-exchange to buffer F with PD-10 (Amersham Biosciences) columns. Cellulofine HAp (Seikagaku Co., Tokyo, Japan) column (10 i.d. × 75 mm, containing 5 ml of the resin) chromatography gave an active fraction by eluting with buffer H (30 ml) after the column was successively washed with buffers F and G (30 ml each). The active fraction was 60-times concentrated with an Amicon Ultra-15 10K NMWL unit followed by buffer-exchange to buffer B with a NAP-10 column (Amersham Biosciences). A column chromatography with pre-packed Mono Q HR 5/5 column (0.5 i.d. × 5 cm; Amersham Biosciences) gave an active fraction in 13.1–14.4% buffer C when it was eluted with a 0–25% linear gradient of buffer C in buffer B for 60 min at a flow rate of 1.0 ml/min using an FPLC system. The active fraction was finally concentrated to 2.7 ml by ultrafiltration in an Ultracent-30 unit (Tosoh Co.). UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) was purified several times to obtain enough amount of enzyme for determining enzyme characteristics.
Determination of molecular mass
The molecular mass of the protein was determined by SDS-PAGE and gel filtration. Gel filtration was performed on a calibrated Superdex 75 HR 10/30 column (Pharmacia, Kalamazoo, MI, USA) with 20 mM Tris–HCl buffer of pH 7.5 containing 150 mM NaCl at a flow rate of 0.5 ml/min. The column was calibrated with molecular mass standards of albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease (13.7 kDa).
Preparation and identification of substrates and products
1 (Kazuma et al. 2003), 2, 3, and delphinidin 3,3′-di-O-β-glucoside (10) (Kazuma et al. 2004), cyanidin 3-O-(6″-O-malonyl)-β-glucoside (5) and petunidin 3-O-(6″-O-malonyl)-β-glucoside (7) (Kogawa et al. 2007), and delphinidin 3,3′,5′-tri-O-β-glucoside (11) (Terahara et al. 1990) were prepared in our laboratory. Cyanidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (6) and petunidin 3-O-(6″-O-malonyl)-β-glucoside 5′-O-β-glucoside (8) were prepared from 5 to 7, respectively, with the glucosyltransferase activities in the crude petal extract of C. ternatea cv. DB in the presence of UDP-glucose. Delphinidin 3,5-di-O-β-glucoside (12) was prepared by alkaline hydrolysis of a crude acidic methanolic extract from flowers of Verbena hybrida cv. Tapien Blue–Violet, which accumulates delphinidin 3,5-di-O-(6-O-acetyl)-β-glucoside (13) (Toki et al. 1991). Spectroscopic and other chemical data for 6, 8, and 12 were shown in Fig. 1.
Determination of optimum pH, metal ion requirements, effects of chemical reagents, and substrate specificities
The same conditions as those of the standard assay were used unless otherwise stated. For all reactions, the concentration of UDP-glucose was 150 mM, and the purified UA3′5′GT solution (19.6 μg/ml) was used. Three buffer systems with various pHs (1.0 M acetate buffers of pHs 4.0, 5.0, and 5.5; 1.0 M potassium phosphate buffers of pHs 6.0, 6.5, 7.0, 7.5, and 8.0; 1.0 M Tris–HCl buffers of pHs 7.5, 8.0, 8.5, and 9.0) were used to determine the optimum pH, and three reaction times of 60, 90, and 120 s were used.
To assess the metal ion requirements, 10 mM aqueous solutions of MgCl2, CaCl2, FeCl2, CuCl2, ZnCl2, and ethylenediaminetetraacetic acid (EDTA) were prepared. To the standard reaction mixture, 2.0 μl of each solution were added instead of distilled water. Reaction times of 60, 90, and 120 s were used. To assess the effects of chemical reagents, 10 mM solutions of p-chloromercuribenzoic acid (PCMB), N-ethylmaleimide (NEM), iodoacetate, diethylpyrocarbonate (DEPC), and dithioerythritol (DTE) were prepared. To the standard reaction mixture, 2.0 μl of each solution were added instead of distilled water and reaction times of 60, 90, and 120 s were used.
To determine the substrate specificities, 2.0 mM solutions of 1, 2, pelargonidin 3-O-(6″-O-malonyl)-β-glucoside (4), 5, 6, 7, delphinidin 3-O-β-glucoside (9), 10, 11, and 12 were prepared, and the reaction times of 30, 60, and 90 s were used.
Detection of UA3′GT and UA3GT activities
The same conditions as those of the standard assay were used except for the substrates. For the detection of UA3′GT activity, 1 was used for the substrate, and the production of 2 was measured. For the detection of UA3GT activity, delphinidin chloride was used for the substrate, and the production of delphinidin 3-O-glucoside was measured.
Steady-state kinetic analyses of UA3′5′GT
The reaction conditions were the same as the standard assay conditions except for the reaction time and substrate concentrations. Three reaction times of 30, 60, and 90 s were used to determine the initial velocity. When parameters for 1 or 2 were determined, protein solutions of 20.7 and 19.6 μg/ml were used, respectively. The dependence of the activity on various concentrations of UDP-glucose (0.1–30 mM) was analyzed at a fixed concentration (400 μM) of 2. The UDP-glucose concentration was held constant (30 mM), and the concentration of 2 was varied (10–400 μM). Then, the UDP-glucose concentration was varied (0.1–30 mM) while holding the concentration of 1 fixed (400 μM). Finally, the concentration of 1 was varied (10–400 μM) while holding the UDP-glucose concentration constant (30 mM). Hanes–Woolf plots produced from the data sets were used to calculate apparent Km and Vmax values. To calculate turnover numbers, the molecular weight of 50.8 kDa from SDS-PAGE was used.
Purification of UA3′5′GT and molecular mass
Purification of UA3′5′GT
Total activity (pkat)
Specific activity (pkat mg/protein)
Purification for each stepa (-fold)
Total activity (pkat)
Specific activity (pkat mg/protein)
Purification for each stepa (-fold)
Total activity (pkat)
Specific activity (pkat mg/protein)
Reactive yellow 86
Substrate specificities of UA3′5′GT
Specific activity (pkat mg/protein)
Relative activity (%)
Although there is no simple correlation of the enzymatic activity and the acid dissociation constant (pKa) of acceptor compounds, it was still of interest whether the reaction was accelerated or decelerated by the acidity of the 3′ or 5′ OH groups. The only structural differences among 1, 2, 5, and 7 are in the substituent group of the B-ring at the meta position of the OH group that participates in the reaction: R1 = OH for 1, R1 = O-glucosyl for 2, R1 = methoxy for 7, and no substitution (R2 = H) for 5 (Fig. 1). All of these substituent groups accelerated the reaction 2.3–5.1-fold (in terms of relative activity) compared to 5. The OH group that participates in the reaction in these anthocyanins might be more acidic than that for 5 because m-OH and m-methoxy groups generally work as electron-withdrawing groups as seen in resorcinol and m-methoxyphenol (Gawron et al. 1952). A slightly more acidic OH group on the acceptor anthocyanin (1, 2, and 7) might experience an easier formation of a phenoxide nucleophile than 5, resulting in acceleration of the reaction.
Finally, none of the demalonylated anthocyanins were favored substrates. 9 and 10 decreased the specific activity by about 30-fold and 26-fold, respectively. In addition, the enzyme could not use 12. Although UDP-galactose was not used as a donor substrate, several recombinant glucosyltransferases have been reported to use UDP-galactose (Offen et al. 2006).
Optimum pH, ion requirements, and effects of chemical reagents
UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase has both UA3′GT and UA5′GT activities. For its UA5′GT activity, it showed an optimum pH of 7.5–8.0 [pH 4.5 (0 nkat/mg), 5.0 (8.8), and 5.5 (26.0) for acetate buffer; pH 6.0 (122.2 nkat/mg), 6.5 (158.5), 7.0 (165.4), and 7.5 (213.9) for potassium phosphate buffer; pH 7.5 (148.7 nkat/mg), 8.0 (213.9), 8.5 (128.7), and 9.0 (88.9) for Tris–HCl buffer].
None of the tested metal ions promoted its activity. The enzyme was completely inhibited by Cu2+ ion (0 nkat/mg protein; 0% of the original specific activity of 35.3 nkat/mg protein), but moderately inhibited by Zn2+ ion (22.0; 62%) by which many flavonoid glycosyltransferases are strongly inhibited. Other metal ions such as Mg2+ (31.1; 88%), Ca2+ (28.9; 82%), and Fe2+ (25.8; 73%) as well as EDTA (26.3; 75%) reduced the enzymatic activity to some extent.
Several chemical reagents were tested for their effects on the enzyme activity. PCMB completely inhibited the activity (0 nkat/mg protein; 0% of the original specific activity of 141.5 nkat/mg protein). In addition, NEM strongly reduced the activity (22.7; 16%). The same level of inhibition was observed by DEPC (23.2; 16%). Iodoacetate (119.1; 84%) and DTE (117.4; 83%) moderately inhibited the activity as compared to the other reagents tested here.
Finally, for its UA3′GT activity, it similarly affected by the buffer’s pHs, the metal ions, and chemical reagents when comparing to UA5′GT activity (data not shown).
Steady-state kinetic analyses of purified UA3′5′GT
Steady-state kinetic parameters for purified UA3′5′GT
Vmax (nkat mg/protein)
1.38 × 10−4
3.9 × 104
3.89 × 10−5
1.1 × 105
6.18 × 10−3
7.9 × 102
1.49 × 10−3
3.4 × 103
A noticeably large apparent Km value for UDP-glucose (6.18 mM) was obtained when 2 was saturated. This value is much larger than that reported for flavonoid O-glycosyltransferases, although values higher than 1 mM have been reported for several glycosyltransferases from Vitis vinifera (1.2 mM; Do et al. 1995) and Vigna mungo (1.67 mM; Ishikura and Mato 1993). The apparent Km value (1.49 mM) for UDP-glucose (when the 1 was saturated) was also greater than 1 mM, although it was 4.1 times lower than that for UDP-glucose (when 1 was saturated). Despite these differences in the apparent Km values, the kcat values were almost the same. This suggests that the structure of the anthocyanins does not affect the rate of the product formation from the enzyme-substrate (ES) complex, but lowers the rate of UDP-glucose binding to the enzyme by 4.3-fold (in terms of kcat/Km). The binding of UDP-glucose is very likely to be inhibited by 2 with its 3′-O-glucosyl group probably located in the UDP-glucose binding site because 2 is not only a substrate but also a product converted from 1.
UDP-glucose: anthocyanin 5′-O-glucosyltransferase has been of interest because it is one of the key enzymes for producing polyacylated anthocyanins with 3′,5′-di-O-acylglucosyl groups such as lobelinins, delphinidin 3,7,3′,5′-tetra-O-p-coumaroylglucoside, and ternatins which accumulate in the blue flowers of Lobelia erinus (Kondo et al. 1989), Dianella spp. (Bloor 2001), and C. ternatea (Terahara et al. 1998), respectively. Among these anthocyanins, only ternatins of C. ternatea have been studied for their biosynthetic pathways. In this study, UA3′5′GT was first purified as UA5′GT, however, it was also found to possess UA3′GT activity as well. Since it had the similar kinetic parameters for the two neighboring intermediates 1 and 2 (Table 3) in the ternatin biosynthetic pathway (Fig. 2), both intermediates are probably the substrates for this enzyme. In addition, the mauve flower line of C. ternatea loses not only UA5′GT but also UA3′GT activities (Kazuma et al. 2004), which can be explained by the lack of this bi-functional UA3′5′GT. This enzyme is, thus, crucial for the blue color expression in C. ternatea. Bi-functional glucosyltransferases are rare among glucosyltransferases, however, another bi-functional anthocyanidin glucosyltransferase, UDP-glucose: anthocyanidin 5,3-O-glucosyltransferase which catalyzes the transfer of two glucosyl groups to the 5- and 3-OH groups of cyanidin, was identified recently from roses (Ogata et al. 2005).
It would be disadvantageous if the inhibition of UDP-glucose binding were mediated by 2 in planta. Thus, there might be a mechanism that cancels the inhibition by the 3′-O-glucosyl group: UDP-glucose might bind first and 2 next, resulting in an ordered bi–bi mechanism that can help to prevent product inhibition by 2. An ordered bi–bi mechanism was reported for a flavonol-ring-B O-glucosyltransferase from Chrysosplenium americanum (Khouri and Ibrahim 1984).
Finally, UA3′GT of G. triflora transfers one glucosyl group to 3′-OH of 12 (Fukuchi-Mizutani et al. 2003) while it was not reported to be able to use the product, delphinidin 3,5,3′-tri-O-β-glucoside, as a substrate. UA3′5′GT is unique in terms of reusing the product 2 as a substrate. A possible explanation for the mechanism behind this uniqueness is that its substrate binding pocket has an enough space for the 3′-O-glucosyl group of 2 at the opposite side of UDP-glucose binding site, which allows 2 to bind to the enzyme in the substrate conformation (Fig. 4c). The three-dimensional structure of UA3′5′GT should enable clarification of mechanism.