functional differentiation of the glycosyltransferases ... · contribute to the chemical diversity...

Functional Differentiation of the Glycosyltransferases ThatContribute to the Chemical Diversity of Bioactive FlavonolGlycosides in Grapevines (Vitis vinifera) W OA

Eiichiro Ono,a Yu Homma,b Manabu Horikawa,c Satoshi Kunikane-Doi,b Haruna Imai,b Seiji Takahashi,b

Yosuke Kawai,d Masaji Ishiguro,e Yuko Fukui,f and Toru Nakayamab,1

a Core Research Group, R&D Planning Division, Suntory Holdings Ltd., Shimamoto, Mishima, Osaka 618-8503, Japanb Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japanc Suntory Institute for Bioorganic Research, Shimamoto, Mishima, Osaka 618-8503, Japand College of Life Sciences, Institute of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japane Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Akiha, Niigata 956-8603,

Japanf Institute for Health Care Science, Suntory Wellness Ltd., Shimamoto, Mishima, Osaka 618-8503, Japan

We identified two glycosyltransferases that contribute to the structural diversification of flavonol glycosides in grapevine

(Vitis vinifera): glycosyltransferase 5 (Vv GT5) and Vv GT6. Biochemical analyses showed that Vv GT5 is a UDP-glucuronic

acid:flavonol-3-O-glucuronosyltransferase (GAT), and Vv GT6 is a bifunctional UDP-glucose/UDP-galactose:flavonol-3-O-

glucosyltransferase/galactosyltransferase. The Vv GT5 and Vv GT6 genes have very high sequence similarity (91%) and are

located in tandem on chromosome 11, suggesting that one of these genes arose from the other by gene duplication. Both of

these enzymes were expressed in accordance with flavonol synthase gene expression and flavonoid distribution patterns in

this plant, corroborating their significance in flavonol glycoside biosynthesis. The determinant of the specificity of Vv GT5

for UDP-glucuronic acid was found to be Arg-140, which corresponded to none of the determinants previously identified for

other plant GATs in primary structures, providing another example of convergent evolution of plant GAT. We also analyzed

the determinants of the sugar donor specificity of Vv GT6. Gln-373 and Pro-19 were found to play important roles in the

bifunctional specificity of the enzyme. The results presented here suggest that the sugar donor specificities of these Vv GTs

could be determined by a limited number of amino acid substitutions in the primary structures of protein duplicates,

illustrating the plasticity of plant glycosyltransferases in acquiring new sugar donor specificities.

INTRODUCTION

Grapevine (Vitis vinifera) (Figure 1A) has been one of the most

important fruit crops from the beginning of human civilization

(McGovern et al., 1997). Dietary intake of grapevine-based

products results in diverse biological effects that are beneficial

to human health, in part due to polyphenols, such as flavonoids

(e.g., flavonols, proanthocyanidins, and anthocyanins) (Renaud

and de Lorgeril, 1992; Corder et al., 2001, 2006; Baur et al.,

2006).

Flavonols, such as kaempferol, quercetin, and isorhamnetin,

are an important class of bioactive flavonoids, which are most

abundant as their 3-O-glycosides (i.e., glucoside, glucuronoside,

galactoside, and rutinoside) in the berries, seeds, and leaves of

grapevine (Hmamouchi et al., 1996; Castillo-Munoz et al., 2009).

In this plant, biosynthesis of flavonol glycosides primarily serves

to protect the plant fromexcessUV light (Price et al., 1995;Matus

et al., 2009). Flavonol aglycons are biosynthesized from dihy-

droflavonols by the action of flavonol synthase (Vv FLS), a

2-oxoglutarate-dependent dioxygenase, which is temporally

regulated by anR2R3-Myb transcription factor, VvMYBF1 (Fujita

et al., 2006; Czemmel et al., 2009). Subsequently, glycosylation

of flavonols enhances their water solubility, such that high

concentrations of flavonols can accumulate in plant cells.

From a food chemistry perspective, flavonol glycosides define

the color of wine grapes and products by acting as copigments

(Yoshitama et al., 1992; Boulton, 2001; Schwarz et al., 2005).

Thus, they are important phytochemicals for viticulture and

enology. Moreover, they also have important biomedical activ-

ities. The activities that are unique to flavonol glycosides and are

not associated with the aglycon forms include the following: the

cytoprotective effects of quercetin 3-O-glucoside (isoquercitrin)

by induction of cholesterol biosynthesis (Soundararajan et al.,

2008), antiatherosclerotic activity of quercetin glucuronides that

are specifically incorporated by macrophages (Kawai et al.,

2008), and antidepressant effects of quercetin 3-O-glucuronide

(miquelianin), quercetin 3-O-glucoside, and quercetin 3-O-

galactoside (hyperoside) (Butterweck et al., 2000; Jurgenliemk

1Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions to Authors (www.plantcell.org) is: Toru Nakayama([email protected]).WOnline version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.110.074625

The Plant Cell, Vol. 22: 2856–2871, August 2010, www.plantcell.org ã 2010 American Society of Plant Biologists

et al., 2003). Importantly, the bioactivities of these compounds

are greatly modulated by their glycon structures (Jurgenliemk

et al., 2003; Soundararajan et al., 2008).

Glycosylation of flavonoids in plant cells usually takes place in

a regio-(or position-)specific manner after the completion of

aglycon biosynthesis (Heller and Forkmann, 1994), providing a

basis for the structural diversification of these important metab-

olites. Glycosylation is catalyzed byglycosyltransferases (UGTs),

which generally catalyze transfer of the glycosyl group from

nucleoside diphosphate–activated sugars (e.g., UDP-sugars) to

Figure 1. Phytogenetics of V. vinifera Flavonoid-3-O-Glycosyltransferases and the Related UGTs.

(A) Grapes (cv Cabernet Sauvignon) at a vineyard in the TOMI NO OKA Winery, Yamanashi Prefecture, Japan.

(B) An unrooted phylogenetic tree was constructed as described in Methods. The alignment used for this analysis is available as Supplemental Data Set

1 online. The percentages of replicate trees, in which the associated taxa clustered together in the bootstrap test (1000 replicates), are shown. Bar = 0.1

amino acid substitutions per site. Resveratrol glucosyltransferase (Vl RSgt) was used as an outgroup sequence (Hall and De Luca, 2007). The branches

shown in red and black indicate the foreground and background lineages, respectively, of the UGT gene clusters on the basis of a branch-specific

model with the dN/dS ratios of v1 = 0.34 and v0 = 0.22, as suggested by the maximum likelihood analysis (Yang, 2007). Vv, Vitis vinifera; Vl, Vitis

labrusca; Ph, Petunia hybrida; Fi, Forsythia intermedia; At, Arabidopsis thaliana; Ac, Aralia cordata; Vm, Vigna mungo; Ih, Iris hollandica; Zm, Zea mays;

Hv, Hordeum vulgare. Ih, Zm, and Hv are monocots.

(C) Graphical map of Vv GT genes (shown in blue) in the grapevine genome. Accession numbers of Vv GTs are shown below each gene. PN, Pinot Noir;

CS, Cabernet Sauvignon; SR, Shiraz.

Flavonol Glycosyltransferases of Grapes 2857

acceptor molecules (Campbell et al., 1997; Coutinho et al., 2003;

Bowles et al., 2005; Wang, 2009). The UGTs that are involved in

plant secondary metabolism, including flavonoid biosynthesis,

have been assigned to family 1 of UGTs (Vogt and Jones, 2000;

Bowles et al., 2005). These UGTs are characterized by a unique,

well-conserved sequence of;45–amino acid residues (called a

PSPG box) (Vogt and Jones, 2000) and a catalytic mechanism

that inverts the anomeric configuration of a transferred sugar

(Campbell et al., 1997; Wang, 2009). Phylogenetic analyses of

plant family 1 UGTs show several phylogenetic clusters, which

appear to be characterized by the regiospecificity of their gly-

cosyl transfer to flavonoids (Vogt and Jones, 2000; Sawada et al.,

2005; Noguchi et al., 2007). Moreover, the differentiation of the

sugar donor specificity of UGTs appears to have occurred in a

lineage-specific manner after establishment of general regio-

specificity for the glycosyl acceptor (Yonekura-Sakakibara et al.,

2007; Noguchi et al., 2009a; Ono et al., 2010).

The grapevine genome contains as many as 240 UGT genes

(The French-Italian Public Consortium for Grapevine Genome

Characterization, 2007), and of these, the only grapevine UGT

that has been functionally characterized is Vv GT1, which cat-

alyzes the 3-O-specific glucosylation of anthocyanidin to pro-

duce anthocyanidin 3-O-glucoside and is involved in the

coloration of grape skin (Ford et al., 1998). This study aimed at

identifying the UGTs that are responsible for the observed

structural diversity of flavonol glycosides in grapevine. To that

end, the genome of grapevine cv Pinot Noir (PN42004 genotype)

was screened in silico for genes that are related to Vv GT1 and

therefore potentially encode flavonoid-3-O-glycosyltransferases.

Seven such Vv GT1–related genes were identified, two of which

were heterologously expressed in Escherichia coli cells as cat-

alytically active proteins. These enzymes, termed Vv GT5 and Vv

GT6, have very similar primary structures and are encoded in

tandem on the same chromosome, strongly suggesting that one

evolved from the other through gene duplication. Biochemical

analyses of these enzymes revealed that Vv GT5 is a UDP-

glucuronic acid:flavonol-3-O-glucuronosyltransferase, whereas

Vv GT6 is a bifunctional UDP-glucose/UDP-galactose:flavonol-

3-O-glucosyltransferase/galactosyltransferase. Detailed analy-

ses of these VvGTs led us to propose a scenario for evolution of a

new sugar donor specificity of UGT after gene duplication,

illustrating the plasticity of UGTs that enhance the chemical

diversity of plant secondary metabolites.

RESULTS

Screening of V. vinifera Flavonoid-

3-O-Glycosyltransferase Genes

In an effort to the identify genes that encode flavonol-3-O-

glycosyltransferases in grapevine, the cv Pinot Noir (PN42004

genotype) genome sequence (The French-Italian Public Con-

sortium for Grapevine Genome Characterization, 2007) was

screened in silico using the Vv GT1 nucleotide sequence. We

identified seven UGT-related genes with appreciable sequence

identity to VvGT1: VvGT2, VvGT2-like, VvGT3, VvGT4, VvGT4-

like, Vv GT5, and Vv GT6 (Table 1; see Supplemental Table

1 online). Each of these Vv GT genes was predicted to have a

single intron. Phylogenetic analysis revealed that each of these

genes is related to the GT1 family phylogenetic cluster (cluster I)

of flavonoid 3-O-glycosyltransferases, as expected (Vogt and

Jones, 2000; Sawada et al., 2005; Noguchi et al., 2007). More-

over, these genes formed a subcluster that was distinct from the

Vv GT1–related subcluster (Figure 1B). Comparison of these

sequences with the physical map of the PN42004 genotype

allowed us to clarify their location on the genome, as summarized

in Figure 1C. VvGT2, VvGT4, as well as a set of copies of VvGT2

and VvGT4 (termed VvGT2-like and VvGT4-like with nucleotide

sequences that are 99.3 and 98.2% identical to those of the Vv

GT2 and Vv GT4 genes, respectively; see Supplemental Table

1 online) are located in the same orientation on chromosome 6.

However, the Vv GT4-like gene was incomplete in length and

should therefore be considered a pseudogene. Vv GT3, Vv GT5,

and Vv GT6 were located in the same orientation on chromo-

some 11, where particularly high sequence identity (91%) was

noted between Vv GT5 and Vv GT6 (see Supplemental Table

1 online). These three Vv GT genes alternate with two puta-

tive flavonol sulfotransferase genes (Figure 1C). The commit-

tee responsible for UDP-glucuronosyltransferase nomenclature

(http://somflinders.edu.au/FUSA/ClinPharm/UGT/) assigned

these Vv GTs as follows: Vv GT1, UGT78A5; Vv GT2, UGT78A6;

Vv GT2-like, UGT78A7; Vv GT3, UGT78A10; Vv GT4, UGT78A8;

Vv GT4-like, UGT78A9; Vv GT5, UGT78A11; and Vv GT6,

UGT78A12.

Biochemical Characterization of Vv GT5 and Vv GT6

Among the Vv GT genes identified, Vv GT5 and Vv GT6 were

heterologously expressed in E. coli as soluble and catalytically

active proteins. The specificity and catalytic properties of these

recombinant proteins were analyzed using a wide variety of

phenolics as glycosyl acceptors and UDP-glucose (UDP-Glc),

UDP-glucuronic acid (UDP-GA), UDP-galactose (UDP-Gal), and

UDP-rhamnose as sugar donors.

Vv GT5 displayed a strong glucuronosyl transfer activity from

UDP-GA to flavonols (kaempferol, quercetin, and isorhamnetin)

(Figure 2). The relative activities for flavonols (100mM), examined

using 100 mM UDP-GA as the sugar donor, were as follows:

quercetin, 100%; kaempferol, 51.5%; and isorhamnetin, 5.6%.

The following 14 phenolic compoundswere inert as glucuronosyl

acceptors: pelargonidin, cyanidin, and delphinidin (anthocyani-

dins), apigenin and luteolin (flavones), (+)-catechin (a flavan-3-ol),

genistein (an isoflavone), naringenin (a flavanone), ferulic acid,

caffeic acid, and salicylic acid (aromatic carboxylic acids),

capsaicin (a capsaicinoid), trans-resveratrol (a stilbene), and

esculetin (a coumarin). Moreover, UDP-Glc, UDP-Gal, and UDP-

rhamnose were inert as sugar donors. Thus, Vv GT5 was highly

specific for both flavonols and UDP-GA. Vv GT5–catalyzed

transfer of glucuronic acid to quercetin produced a single trans-

fer product, which was clearly identified as quercetin 3-O-

glucuronide based on its cochromatography with an authentic

sample and absorption spectral characteristics (lmax, 356 nm;

see Supplemental Table 2 online for HPLC and spectrophoto-

metric identification of regioisomers of quercetin monoglucu-

ronides). Therefore, this enzyme was defined as a UDP-GA:

2858 The Plant Cell

flavonol-3-O-glucuronosyltransferase (termed hereafter flavonol-

3GAT). The optimal pH and temperature for Vv GT5–catalyzed

glucuronosyl transfer to quercetinwere9.1 and458C, respectively.The enzymatic reaction followed Michaelis-Menten kinetics,

and the steady state kinetic parameters were determined at pH

9.1 and 308C as summarized in Table 2. The enzyme displayed

high kcat values for quercetin and kaempferol, which were 10- to

100-fold higher than the values reported for many other UGTs at

optimal pH (e.g., Noguchi et al., 2007).

Vv GT6 catalyzed glucosyl transfer from UDP-Glc to flavonols.

Remarkably, this enzyme also displayed a strong galactosyl

transfer activity (126%of glucosyl transfer activity) fromUDP-Gal

to flavonols (Figure 2). UDP-GAandUDP-rhamnosewere inert as

sugar donors. The relative activities for flavonols (100 mM) with

UDP-Glc (100mM)were as follows: quercetin, 100%; kaempferol,

84.6%; and, isorhamnetin, 61.2%. The relative activities for

flavonols (100 mM) with UDP-Gal (100 mM) were as follows:

quercetin, 100%; kaempferol, 110%; and, isorhamnetin, 26.9%.

Other phenolics (see above) were all inert as glycosyl acceptors,

indicating that the enzyme is also highly specific for flavonols. Vv

GT6–catalyzed transfer of glucose to quercetin produced a

single transfer product, which was identified as quercetin 3-O-

glucoside based on its coelution with an authentic sample and

NMR spectra. The optimal pH and temperature for Vv GT6–

catalyzed glucosyl transfer to quercetin was 8.0 and 358C,respectively.

Both the glucosyl and galactosyl transfer reactions catalyzed

by Vv GT6 followedMichaelis-Menten kinetics when the flavonol

concentration was <150 mM; substrate inhibition was observed

when high concentrations (higher than 150 mM) of flavonol were

used. Apparent steady state kinetic parameters were deter-

mined at pH 7.4 and 308C, as summarized in Table 3 (for GlcT

activity and GalT activity). Based on the kcat/Km values, the best

substrates for the GlcT and GalT activities of Vv GT6 were

kaempferol (0.115 s21·mM21) and quercetin (0.304 s21·mM21),

respectively. When Vv GT6 activity was assayed in the presence

of both UDP-Glc and UDP-Gal (1 mM each), both quercetin 3-O-

glucoside and quercetin 3-O-galactoside were produced in an

approximate molar ratio of 1:1 (see Supplemental Figure 1 on-

line). These results show that this enzyme is bifunctional, cata-

lyzing both glucosyl and galactosyl transfer to flavonols, and can

be defined as a UDP-Glc/UDP-Gal:flavonol 3-O-glucosyltrans-

ferase/galactosyltransferase (termed hereafter flavonol-3GlcT/

GalT).

Quantitative RT-PCR

To evaluate the functional significance of Vv GT5 and Vv GT6 in

the biosynthesis of flavonol glycosides in the grapevine plant,

transcription levels of Vv GT5 and Vv GT6 in an array of organs

and tissues of cv Pinot Noir, including the leaves, stems, and

developing berries, were quantified by quantitative RT-PCR

using total RNA. The results were compared with those of Vv

GT1 (Ford et al., 1998), which is responsible for anthocyanin

biosynthesis in grapevine berry skin and Vv FLS1 (also termed Vv

FLS4), which is responsible for flavonol biosynthesis (Fujita et al.,

2006; Czemmel et al., 2009) (Figure 3, left panels). The Vv GT5

transcript was abundant in berry skin and increased at the onset

of pigmentation (veraison), with the highest level of transcription

detected in the fully pigmented skin (exocarp) of mature berries

(Figure 3, second panel). An appreciable amount of transcript

was also found in leaves and petioles, and only small amounts of

transcript were found in seeds and berry flesh (mesocarp). The

pattern of Vv GT6 expression was similar to that of Vv GT5

expression (Figure 3, third panel). Moreover, the expression

profiles of both Vv GT5 and Vv GT6 were similar to that of Vv

FLS1. By contrast, Vv GT1 expression was highly specific for

berry skin, and the highest transcription level was found in fully

pigmented berries (Figure 3, first panel). Organ/tissue specific-

ities of Vv GT cDNA expression in another grapevine cultivar (cv

Cabernet Sauvignon) were also analyzed (Figure 3, right panels).

In Cabernet Sauvignon, considerable amounts of Vv GT5, Vv

GT6, and Vv FLS1 transcript were found in both leaves and

petioles (Figure 3, second through fourth panels) in addition to

berry skin. By contrast, VvGT1 expressionwas highly specific for

berry skin (Figure 3, first panel), as was the case for cv Pinot Noir.

Flavonoid Analysis

To validate further the functional significance of Vv GT5 and Vv

GT6 in the biosyntheses of both the 3-O-glucuronide and the

3-O-glucoside/galactoside of quercetin, the contents of these

Table 1. Sequence Identities of Vv GT Proteins

Protein Length (aa)a

Sequence Identity (%) to

Vv GT1 Vv GT2 Vv GT2-Like Vv GT3 Vv GT4 Vv GT4-Like Vv GT5 Vv GT6

Vv GT1 452b 100 51.3 51.3 56.3 55.9 56.5 53.8 55.0

Vv GT2 445b 100.0 98.2 60.1 73.3 72.3 57.6 58.7

Vv GT2-like 445b 100.0 59.7 73.3 71.9 57.6 58.7

Vv GT3 531b 100.0 62.6 68.2 64.0 64.1

Vv GT4 455b 100.0 98.3 61.6 62.3

Vv GT4-like 295c 100.0 64.1 63.9

Vv GT5 459b 100.0 88.2

Vv GT6 458b 100.0

aAmino acids.bFull length.cN-terminally truncated.


Figure 2. Enzymatic Analyses of Recombinant Vv GT5 and Vv GT6 Proteins for Glycosylation of Quercetin.

(A) HPLC chart for standard quercetin (top), enzyme reaction with quercetin, UDP-GA, and Vv GT5 (middle), and standard quercetin 3-O-glucuronide

(bottom).

(B) HPLC chart for the enzyme reaction with quercetin, UDP-Gal, and Vv GT6 (top), standard quercetin 3-O-galactoside (second), the enzyme reaction

with quercetin, UDP-Glc, and Vv GT6 (third), and standard quercetin 3-O-glucoside (bottom). Asterisk indicates product peak for each enzyme reaction.

2860 The Plant Cell

quercetin 3-O-glycosides in grapevine leaves and berries (cv

Pinot Noir, Cabernet Sauvignon, and Shiraz) were analyzed by

liquid chromatography–mass spectrometry (LC-MS) and HPLC

(Table 4). It must be noted that, for the 3-O-glucoside and the

3-O-galactoside of quercetin, only the sum of both stereoiso-

mers was determined because our analytical systems could not

separate these isomers in crude samples. Both isomers are

present in the leaves and berry skins (exocarp) of grapevine and

in red wines (Hmamouchi et al., 1996; Downey et al., 2007;

Castillo-Munoz et al., 2009).

Results of this study showed that the leaves and berries of

these cultivars each contained quercetin glycosides. In particu-

lar, very large amounts of quercetin 3-O-glucuronide and quer-

cetin 3-O-glucoside/galactoside were found in leaves of cv Pinot

Noir (9.7 and 3.2 mg/g fresh weight, respectively). Berry skins of

both varieties contained appreciable amounts (a total of 600 to

780 mg/g fresh weight) of these quercetin 3-O-glycosides. More-

over, the quercetin glycosides were also observed in dried fruits.

These results, along with the biochemical properties and en-

zyme expression profiles, corroborate the involvement of Vv GT5

and Vv GT6 in the biosyntheses of quercetin 3-O-glucuronide,

3-O-glucoside, and 3-O-galactoside.

Identification of a Residue That Critically Determines the

Sugar Donor Specificity of Vv GT5

Previous studies of two GATs, Bp UGAT (UGT94B1) of the red

daisy flower (Bellis perennis) (Sawada et al., 2005; Osmani et al.,

2008) and Pf F7GAT (UGT88D7) of Perilla frutescens (Noguchi

et al., 2009a), showed that Arg-25 in Bp UGAT and Arg-350 in Pf

F7GAT play an important role in recognition of UDP-GA. These

results suggest plasticity of the mechanisms that alter the sugar

donor specificity of UGTs. To examine themolecular basis for the

specificity of Vv GT5 for UDP-GA, a three-dimensional structural

model of Vv GT5 docked with UDP-GA and a flavonol (kaemp-

ferol) was constructed using the crystal structure of Vv GT1 as a

template (Figure 4A) (Offen et al., 2006). The structural model

predicted an interaction between the guanidinium group of Arg-

140 of Vv GT5 and the carboxyl group of the glucuronic acid

moiety of bound UDP-GA. A sequence comparison showed that

Arg-140 of Vv GT5 is replaced by a Trp residue in each UGT

examined, which, for example, corresponds to Trp-140 in VvGT1

and Vv GT6 (Figure 4C). In the reported crystal structure of the

ternary complex of Vv GT1 (Figure 4B), Trp-140 does not point

toward the bound UDP-2-deoxy-2-fluoroglucose. These obser-

vations imply the importance of Arg-140 for recognition of UDP-

GA in the Vv GT5–catalyzed glucuronosyl transfer. To validate

this possibility, Arg-140 was replaced with a Trp residue, and the

catalytic properties of the resultant mutant, R140W, were exam-

ined. Unlike the wild-type Vv GT5, the R140W mutant displayed

strong glucosyl and galactosyl transfer activities, but no detect-

able glucuronosyl transfer activity (i.e., the relative activity was

<0.01% of the glucosyl transfer activity). These results indicate

that the enzyme was converted from a GAT to a bifunctional

GlcT/GalT as a result of the single R140W substitution. Kinetic

analyses revealed that both the kcat of glucuronosyl transfer and

the affinity for UDP-GA were greatly reduced by this substitution

(Table 2), while the Km for the glucuronosyl acceptor (quercetin)

was only slightly affected. When quercetin was used as a

glycosyl acceptor, the kcat values for the glucosyl and galactosyl

transfer activities of the mutant were 41 and 6% of the wild-type

glucuronosyl transfer activity, respectively (Table 2). The Km

values for quercetin in the mutant (9.69 mM for glucosyl and 10.6

mM for galactosyl transfer) were somewhat larger than the Km for

wild-type glucuronosyl transfer activity (5.60 mM). The Km values

of themutant for UDP-Glc (77.2mM) and UDP-Gal (225mM)were

Table 2. Kinetic Parameters of Vv GT5 and Its R140W Mutanta

Substrate kcat (s�1) Km (mM)

kcat/Km

(s�1 mM�1)

Wild-Type Vv GT5 (GAT Activity)

Quercetinb 7.16 6 0.37 5.60 6 1.76 1.280

Quercetinc 1.63 6 0.26 54.7 6 17.4 0.030

Kaempferolb 6.05 6 0.05 10.8 6 0.4 0.560

Isorhamnetinb 0.413 6 0.003 7.92 6 0.32 0.052

UDP-GAb,d 17.7 6 2.9 0.405

Vv GT5-R140W (GAT Activity)b

Quercetinb

UDP-GAe

0.00016 6 0.00002 29.0 6 15.5 5.52 3 10�6

0.0015 3 10�6

Vv GT5-R140W (GlcT activity)b

Quercetinb 2.94 6 0.11 9.69 6 1.50 0.303

Kaempferolb 1.04 6 0.01 1.96 6 0.17 0.531

Isorhamnetinb 0.148 6 0.004 5.85 6 0.86 0.025

UDP-Glcd 77.2 6 8.4 0.038

Vv GT5-R140W (GalT activity)b

Quercetinb 0.429 6 0.020 10.6 6 1.5 0.041

Kaempferolb 0.395 6 0.005 3.02 6 0.19 0.131

Isorhamnetinb 0.0243 6 0.0007 11.9 6 0.9 0.002

UDP-Gald 225 6 21 0.002

aOnly flavonols were accepted as sugar acceptor substrates.bKinetic parameters were determined at pH 9.1 with the appropriate

UDP-sugar (1.0 mM), as described in Methods.cKinetic parameters were determined at pH 7.0, as described in

Methods.d100 mM quercetin was used as the sugar acceptor.eKm value was too large to be determined precisely.

Table 3. Kinetic Parameters of Vv GT6a

Substrate kcat (s�1) Km (mM) kcat/Km (s�1 mM�1)

GlcT Activity

Quercetin 0.760 6 0.023 9.24 6 1.20 0.082

Kaempferol 0.473 6 0.013 4.11 6 0.47 0.115

Isorhamnetin 0.273 6 0.017 7.43 6 1.62 0.037

UDP-Glcb 28.5 6 3.9 0.027

GalT Activity

Quercetin 1.09 6 0.001 3.59 6 0.01 0.304

Kaempferol 0.725 6 0.044 10.4 6 1.9 0.070

Isorhamnetin 0.707 6 0.019 5.04 6 0.49 0.140

UDP-Galb 37.2 6 2.2 0.029

aKinetic parameters were determined at pH 7.4 with the appropriate

UDP-sugar (1.0 mM), as described in Methods. Only flavonols were

accepted as sugar acceptor substrates.b100 mM quercetin was used as the sugar acceptor.


greater than the Km of the wild type for UDP-GA (17.7 mM).

Overall, the specificity constants (i.e., the kcat/Km values) for

quercetin in glucosyl and galactosyl transfer activities of the

mutant were 24 and 3%, respectively, of the specificity constant

for wild-type glucuronosyl transfer activity. The specificity con-

stants (with quercetin) for UDP-Glc and UDP-Gal of the mutant

were 9.4 and 0.5% of the value for UDP-GA of the wild type. It is

noteworthy that, although the wild type showed the highest

activity toward quercetin, the R140Wmutant showed the highest

activity toward kaempferol, with a specificity constant of glucosyl

transfer activity (0.531 s21·mM21) that was comparable to that of

wild-type glucuronosyl transfer activity for the same substrate

(0.560 s21·mM21).

Analysis of the Specificity Determinant for Sugar Donors in

Vv GT6

Because of the successful conversion of Vv GT5 from a GAT to

a GlcT/GalT by an R140W substitution (see above) and the

close evolutionary relationship predicted between Vv GT5 and

Vv GT6, we examined whether the sugar donor specificity of

Vv GT6 is converted to that of GAT upon W140R substitution.

Thus, we prepared aW140Rmutant of Vv GT6 and examined its

catalytic activity. When quercetin was used as a glycosyl

acceptor, the mutant showed no GlcT, GalT, or GAT activity,

indicating that the mutant could not use UDP-Glc, UDP-Gal, or

UDP-GA.

Figure 3. Gene Expression Analysis of V. vinifera Flavonoid-3-O-Glycosyltransferases.

Expression analysis was performed on each organ using quantitative RT-PCR (n = 3). These graphs show the expression of Vv GT1, Vv GT5, Vv GT6,

and Vv FLS1 relative to that of the reference gene, V. vinifera UBIQUITIN2. Means6 SE are shown. PN, Pinot Noir; CS, Cabernet Sauvignon; L, leaf; Pt,

petiole; Sd, seed; M, mesocarp (fruit flesh); E-y, exocarp (fruit skin)-young, -m (medium), and -f (fully mature).

2862 The Plant Cell

Vv GT6 is a native bifunctional enzyme that displays GlcT and

GalT activity at comparable levels. Because most cluster I UGTs

that have been functionally characterized are monofunctional

(in most cases GlcTs), it is of interest to identify the structural

characteristics that make Vv GT6 bifunctional. We explored

characteristics that are unique to flavonoid GlcTs and those

unique to flavonoid GalTs by means of sequence comparison as

well as molecular modeling of a variety of flavonoid GlcTs and

GalTs. Previous site-directed mutagenesis studies using a fla-

vonoid GlcT and a flavonoid GalT showed that an amino acid

residue located at the C-terminal position of the PSPG box

sequence (Gln and His, respectively; see Figure 4C) critically

determines the sugar donor specificity of these enzymes: Gln at

that position is important for the maximal catalytic efficiency of

GlcT activity, whereas His is required for GalT activity (Kubo

et al., 2004); Vv GT6 has a Gln reside (Gln-373) at this position.

Moreover, position 19 of Vv GT6 is uniquely occupied by a Pro

residue, whereas the corresponding position in known flavonoid

GlcTs and GalTs is occupied by Thr or Ser (Figure 4C). Molecular

modeling of aMichaelis complex of VvGT6 predicted that Pro-19

is in close proximity to the bound sugar moiety and potentially

determines the sugar donor specificity of the enzyme.

To examine the effects of these amino acid substitutions on the

GlcT/GalT bifunctionality of Vv GT6, we prepared single mutants

of Vv GT6 (i.e., Q373H and P19T). The Q373H substitution

essentially abolished the GlcT activity of this enzyme (relative

activity was 1.4% of wild-type activity). Upon P19T substitution,

the ratio of the GlcT and GalT activities of the enzyme (GlcT:

GalT = 100:79) was reversed from that of the wild type (GlcT:

GalT = 80:100).

To analyze further the effects of the Q373H and P19T substi-

tutions on the preference of Vv GT6 for sugar donors, detailed

kinetic studies of these mutants were performed (Table 5). The

Q373H substitution of VvGT6 resulted in a 5.4-fold enhancement

of kcat for GalT activity, with a large (29-fold) decrease in the kcatfor GlcT activity. This amino acid substitution also increased

the Km values for UDP-Glc (5.4-fold), UDP-Gal (11.9-fold),

and quercetin (7-fold with UDP-Gal and 2.4-fold with UDP-Glc).

As a result, the specificity constant for the galactosyl transfer

activity of the mutant (0.23 s21·mM21) was comparable to that

of the wild-type enzyme (0.30 s21·mM21), while the glucosyl

transfer activity of the mutant was severely impaired

(0.0012 s21·mM21 versus 0.083 s21·mM21). Thus, bifunctional

Vv GT6 was converted into a monofunctional GaT by a single

Q373H substitution.

The P19T substitution of Vv GT6 resulted in 3.2- and 5.7-fold

decreases in the kcat values for GlcT and GalT activity, respec-

tively, with slight increases (2.2- and 3.2-fold) in the Km values for

UDP-Glc and UDP-Gal, respectively. Unlike wild-type enzyme,

the GlcT activity of the mutant was higher than its GalT activity,

judging from the kcat values (i.e., the kcat,GlcT/kcat,GalT value of

P19T was 1.26; the ratio for the wild-type enzyme was 0.70)

and kcat/Km values [i.e., (kcat/Km)UDP-Glc/(kcat/Km)UDP-Gal of P19T

was 2.38; the value of the wild-type enzyme was 0.93;

(kcat/Km)GlcT/(kcat/Km)GalT for quercetin for P19T was 0.78 and

the value for the wild-type enzyme was 0.27] (Table 6). For

comparison, the Vv GT5-R140W-T19P mutant, which mimicked

Vv GT6, was prepared, and kcat and kcat/Km values of GlcT and

GalT activities of the mutant were compared with those of Vv

GT5-R140W (Table 5). The results showed that kcat,GlcT/kcat,GalT

and (kcat/Km)UDP-Glc/(kcat/Km)UDP-Gal values of Vv GT5-R140W-

T19P (2.12 and 2.95, respectively; Table 6) were lower than the

values of Vv GT5-R140W (6.85 and 19.0, respectively), indicating

relative enhancement of GalT activity for Vv GT5-R140W follow-

ing the T19P substitution.

DISCUSSION

Functional Significance of Vv GT5 and Vv GT6 in

Biosynthesis and Structural Diversification of Flavonol

Glycosides in Grapevines

In grapevines, the structural diversity of secondary metabolites

is, in part, illustrated by the occurrence of a variety of flavonol

3-O-glycosides, which include 3-O-glucuronide, 3-O-glucoside,

3-O-galactoside, and 3-O-rutinoside of quercetin, implying that a

variety of UGTs with different sugar donor specificities enhance

the structural diversity of flavonols. This study successfully

identified two such UGTs (Vv GT5 and Vv GT6), where Vv GT5

is a flavonol-3GAT and VvGT6 is a UGT that exhibits comparable

3GlcT and 3GalT activity.

A previous study showed relatively high flavonol GlcT activities

in the leaves and berries of cv Shiraz aswell as in white grapevine

lacking anthocyanin (cvMuscat of Alexandria), with activity ratios

of flavonol GlcT:anthocyanidin GlcT ranging from 1:1.1 (for cv

Muscat of Alexandria) and 1:24 (for cv Shiraz) (Ford et al., 1998).

A recombinant Vv GT1 displayed flavonol GlcT activity, which

was significantly lower than its activity toward anthocyanins

(activity ratio of flavonol GlcT:anthocyanin GlcT = 1:48) and

therefore does not account for the flavonol GlcT activity ob-

served in planta (Ford et al., 1998). Because Vv GT6 is highly

specific for flavonols and is expressed in the leaves and exocarp

of berries, whereas VvGT1 is preferentially expressed in exocarp

(Figure 3), Vv GT6 is responsible for flavonol 3-O-GlcT activity in

planta, while Vv GT1 is an UDP-glucose:anthocyanidin 3-O-GlcT

Table 4. Analysis of Quercetin 3-O-Glycosides in the Leaves and the

Berry Skins (Exocarps) of Grapevines

Cultivar

Organ/Tissue

Quercetin

3-O-glucoside +

3-O-galactoside

(mg/g fresh weight)

Quercetin

3-O-glucuronide

(mg/g fresh weight)

Pinot Noir

Leaves 3.242 9.724

Exocarp 0.418 0.367

Cabernet Sauvignon

Leaves 0.045 0.255

Exocarp 0.178 0.422

Shiraz

Exocarp 0.057 0.219

Dried fruits 0.029 0.030

Contents of quercetin 3-O-glycosides in grapevine leaves and berry

skins were determined by LC-MS and HPLC. For experimental details,

see Methods.


Figure 4. Homology Docking and Multiple Alignment of Vv GTs.

(A) A structural model of Vv GT5 docked with UDP-GA and a flavonol (kaempferol).

(B) The crystal structure of Vv GT1 complexed with UDP-2-deoxy-2-fluoroglucose (2F-UDPGlc) and a flavonol (kaempferol).

(C)Multiple alignment of UGTs from V. vinifera and other plants (Arabidopsis thaliana, Petunia hybrida,B. perennis, P. frutescens, Scutellaria baicalensis,

and A. cordata). The multiple alignment was performed using a ClustalW program packaged in MACVECTOR 7.2.2 software (Accelrys) (Thompson

et al., 1994). The amino acids that are involved in sugar donor specificity are highlighted in yellow.

2864 The Plant Cell

(Ford et al., 1998). Thus, the results of this study show that

anthocyanidins and flavonols are glycosylated at the same

position (3-hydroxy group) by different, but structurally related,

UGT enzymes.

The VvGT5 and VvGT6 cDNAswere consistently expressed in

both the leaves and pigmented berry skins of different cultivars,

where quercetin 3-O-glucuronide, quercetin 3-O-glucoside, and

quercetin 3-O-galactoside accumulate. The spatial and temporal

patterns of Vv GT5 and Vv GT6 cDNA expression were very

similar and resembled those of Vv FLS1. These results, along

with the fact that Vv GT5 and Vv GT6 genes are located in

proximity to one another on the same chromosome, strongly

suggest that expression of these two UGT genes is coordinately

controlled by common transcription factors in grapevines, which

probably include the flavonol-specific MYB transcription factor

Vv MYBF1 (Czemmel et al., 2009).

Based on the absence of any appreciable signal sequences for

translocation to organelles or secretion in primary structures, it is

likely that both Vv GT5 and Vv GT6 are cytosolic enzymes. In this

context, the optimal pH for Vv GT5 activity (pH 9.1) was appre-

ciably higher than probable cytosolic pHs (7.0 to 7.5), with a high

specificity constant (e.g., 1.28 mM21·s21 for quercetin). It is

noteworthy, in this regard, that a UGT of Concord grape berries

(Vitis labrusca), Vl RSgt, also optimally catalyzes the glucosyla-

tion of both flavonoids and trans-resveratrol at pH 9.0, but at pH

6.0, it glucosylates exclusively hydroxybenzoic and hydroxycin-

namic acids (Hall and De Luca, 2007). However, Vv GT5 showed

no glycosyl transfer activity toward these aromatic carboxylic

acids at pH 6.0. The kcat/Km value of Vv GT5 at pH 7.0 (0.029

mM21·s21 for quercetin) was;2% of the value at pH 9.1, which

was comparable to the values reported for many other UGTs at

their optimum pH. For example, the kcat/Km of a UGT of Glycine

Table 5. Kinetic Parameters of Vv GT6-Q373H, Vv GT6-P19T, and Vv GT5-R140W-T19P as Analyzed with Quercetin as a Sugar Acceptor

Substrate kcat (s�1) Km (mM) kcat/Km (s�1 mM�1)

Vv GT6-Q373H (GlcT activity)

Quercetina 0.026 6 0.002 22.6 6 14.9 0.0012

UDP-Glcb 157 6 34 0.00017

Vv GT6-Q373H (GalT activity)

Quercetina 5.89 6 0.74 25.1 6 11.9 0.23

UDP-Galb 439 6 55 0.0134

Vv GT6-P19T (GlcT activity)

Quercetina 0.24 6 0.02 34.9 6 7.9 0.0069

UDP-Glcb 62.3 6 9.5 0.0038

Vv GT6-P19T (GalT activity)

Quercetina 0.19 6 0.02 21.5 6 7.3 0.0088

UDP-Galb 118 6 7.3 0.0016

Vv GT5-R140W-T19P (GlcT activity)

Quercetina 1.40 6 0.03 120 6 59 0.012

UDP-Glcb 251 6 58 0.0056

Vv GT5-R140W-T19P (GalT activity)

Quercetina 0.66 6 0.06 47.3 6 12 0.014

UDP-Galb 337 6 21 0.0019

aKinetic parameters were determined at pH 7.4 with the appropriate UDP-sugar (1.0 mM), as described in Methods.b100 mM quercetin was used as the sugar acceptor.

Table 6. Ratios of Kinetic Parameters of GlcT and GalT Activities

Enzyme kcat,GlcT/kcat,GalT Km,GlcT/Km,GalT (kcat/Km)GlcT/(kcat/Km)GalT

Vv GT6-Q373Ha 0.0044 0.90 (for quercetin)

0.36 (for UDP-sugar)

0.005 (for quercetin)


Vv GT6-P19Ta 1.26 1.62 (for quercetin)




Wild-type Vv GT6b 0.70 2.57 (for quercetin)




Vv GT5-R140Wc 6.85 0.91 (for quercetin)



19 (for UDP-sugar)

Vv GT5-R140W-T19Pa 2.12 2.53 (for quercetin)




aValues for the Vv GT6-Q373H, Vv GT6-P19T, and Vv GT5-R140W-T19P mutants were calculated using the data presented in Table 5.bValues for the wild-type enzyme were calculated using the data presented in Table 3.cValues for the Vv GT5-R140W mutant were calculated using the data presented in Table 2.


max (Gm IF7GlcT, UGT88E3) for genistein is 0.006 mM21·s21

(Noguchi et al., 2007). Hence, the specificity constant of Vv GT5

should be sufficiently high in the cytosol to fulfill its biosynthetic

role.

Specificity Determinant of Vv GT5 for UDP-GA

The results of biochemical and molecular modeling studies of

Vv GT5 and its mutant showed that Arg-140 critically determines

the ability of Vv GT5 to use UGP-GA as the sugar donor. This

conclusion corroborates the recent hypothesis that an Arg

residue proximal to the carboxylate of UDP-GA is the biochem-

ical basis of GATs of both plant and animal origin (Noguchi

et al., 2009a). Arg-140 does not correspond to Arg-25 of Bp

UGAT (UGT94B1) (Osmani et al., 2008) or Arg-350 of Pf F7GAT

(UGT88D7) (Noguchi et al., 2009a) in primary structure, providing

another example of convergent evolution of plant GATs. This

illustrates the positional plasticity of the crucial Arg residue in

acquiring specificity for UDP-GA in UGT catalysis (see Supple-

mental Figure 2 online).

It is important to note that, from the perspective of kinetics, the

change in Vv GT5 sugar donor specificity observed after the

R140W substitution arose from changes in both kcat and Km

(Table 2). The observed diminution of the kcat value of Vv GT5

GAT activity upon R140W substitution corresponded to desta-

bilization of the transition state of glucuronosyl transfer by

27.1 kJ·mol21 (see Supplemental Results 1 online). Thus, the

predicted Arg-140–mediated salt bridge is also of mechanistic

significance in specific stabilization of the transition state of

GAT catalysis, by which both rate acceleration and specificity of

GAT catalysis are attained. This role of Arg-140 is consistent with

a generally accepted theory of enzymatic catalysis, which is called

the “transition state fitting theory” (Pauling, 1948; Copeland, 2000;

see Supplemental Results 1 online for details). Moreover, in the

model of the Vv GT5-R140W docked with ligands (see Supple-

mental Figure 3 online), due to the absence of Arg-140, the

carboxylate of UDP-GA points to His-20, which corresponds to

the catalytically important His-20 of Vv GT1 (Offen et al., 2006).

This interaction takes His-20 away from the 3-OH group of

flavonol substrates and likely impairs the role of His-20. This may

also account for the absence of GAT activity in the Vv GT5-

R140W mutant.

Molecular modeling also suggested that Arg-140 plays an-

other role in the determination of the sugar donor specificity

of Vv GT5, which may account for the emergence of strong

GlcT/GalT activities as a result of a single R140W substitution.

In the modeled structures of Vv GT5 and of the Michaelis Vv

GT5-UDP-Glc complex, it is possible to create a salt bridge

between Arg-140 and Asp-119. This Asp residue, which corre-

sponds to Asp-119 of Vv GT1, might play an important role in

UGT catalysis (Offen et al., 2006), providing the possibility that

the free VvGT5 enzymemight have latent UGT activity due to an

interaction between Asp-119 and Arg-140. Upon UDP-GA

binding to the enzyme, the electrostatic interaction of Arg-140

likely shifts from an interaction with Asp-119 to one with the

carboxylate group of bound UDP-GA (Figure 4A), thereby

permitting Asp-119 to participate in UGT catalysis. However,

binding of UDP-Glc (or UDP-Gal) keeps Asp-119 bound to the

salt bridge with Arg-140; hence, the enzyme remains latent in

terms of GlcT/GalT activity. Therefore, it has been proposed

that Arg-140 is important not only because of its role in UDP-GA

recognition, but also because it provides a mechanism that

keeps GlcT/GalT inactive.

Specificity Determinants of Vv GT6 for Sugar Donors

Judging from the occurrence of Gln at the C-terminal position of

the PSPG box sequence (Mato et al., 1998; Miller et al., 1999;

Kubo et al., 2004), the bifunctional Vv GT6 could be considered a

variant of GlcT with an enhanced GalT activity. Replacement of

Gln-373 with His consistently resulted in loss of Vv GT6 activity

toward UDP-Glc, without substantial loss of GalT activity, giving

rise to a monofunctional GalT (Table 5). By molecular modeling,

the effect of the Q373H substitution could be explained in terms

of a slight shift in the binding of the sugar donor molecule in

the enzyme upon this substitution (see Supplemental Figure 4

online). It is important to note, however, that Vv GT1 also

displayed low GalT activity, which was ;8% of its GlcT activity

(in terms of kcat/Km value); and unlike the cases of Vv GT6 and

other enzymes (Kubo et al., 2004), a Q375H substitution in Vv

GT1 does not improve its GalT activity (Offen et al., 2006). Thus,

the effect of the Gln/His substitution at that position on sugar

donor specificity differs among UGTs. This observation could be

explained by the fact that specificity for neutral sugar donors

Figure 5. Proposed Gene Duplication Evolutionary Process for a GAT

Gene and a Bifunctional GlcT/GalT Gene from a Common Putative GlcT

Gene in V. vinifera.

Horizontal arrow and double-headed arrow represent the evolutionary

history of the Vv GT genes, with the red and blue arrowheads showing

mutational events that occurred only in ancestral Vv GT5 and Vv GT6

genes, respectively. The results of this study suggest that a common

ancestral gene of Vv GT5 and Vv GT6 encoded a putative GlcT (gray

box), in which positions 19, 140, and 373 were occupied by Thr, Trp, and

Gln, respectively. After gene duplication, which is shown by the thick

vertical arrow, mutational events occurred in the respective ancestral

genes. Among these mutations, a mutation resulting in W140R substi-

tution in the ancestral Vv GT5 gene destined it to evolve into a GAT (pink

box), while the mutation resulting in a T19P substitution in the ancestral

Vv GT6 gene led to its evolution into a bifunctional GlcT/GalT (light-blue

box). The results of mutational studies show that Vv GT5 could be con-

verted into a GlcT/GalT by a R140W mutation, suggesting the revers-

ibility of the evolutionary process in terms of sugar donor specificity, as

shown by the double-headed arrow. Our results show that Vv GT6 could

no longer be converted into GAT by a W140R mutation.

2866 The Plant Cell

(such as UDP-Glc and UDP-Gal) is not established by a single

amino acid residue, but is attained by a combination of multiple

amino acid residues, which vary among the different UGTs.

We also showed that Pro-19 of Vv GT6 (Figure 4C) plays a

unique role in enhancing the relative GalT activity of this enzyme.

TheGlcT activity of the P19Tmutant of VvGT6 exceeded its GalT

activity (in terms of both kcat and kcat/Km; Table 5), in contrast with

the wild-type enzyme, for which GalT activity exceeded GlcT

activity (Table 3). In this context, Vv GT5-R140W, which has a

primary structure that is very similar to that of Vv GT6, also

showed both GlcT and GalT activity (Table 2); however, the GlcT

activity of Vv GT5-R140W was higher than its GalT activity (e.g.,

kcat,GlcT/kcat,GalT = 6.85; Table 6), probably due to the absence

of a Pro residue at position 19 (Figure 4C). Consistently, T19P

substitution of Vv GT5-R140W resulted in a relative enhance-

ment of its GalT activity (Table 6). Because Pro-19 is adjacent to

His-20, which is the proposed catalytic residue of Vv GT1, and is

also predicted to be in close proximity to the bound sugar moiety

in themodeled structures (for example, see Supplemental Figure

4 online), substitution of Pro-19with Thr (or another residue) likely

affects the preference of this enzyme for sugar donors.

Evolution of Sugar Donor Specificity

Vv GT5 and Vv GT6 constitute a set of very similar genes, which

are located in tandem on chromosome 11 (Figure 1). Thus, these

genes are likely paralogs, where one gene results from the gene

duplication of the other. This is consistent with a theory that

explains the evolution of enzyme function (Ober, 2005; Des

Marais and Rausher, 2008; Matsuno et al., 2009), which states

that a set of enzymes with similar (but different) catalytic func-

tions can be generated by gene duplication. As implicated from

the genomic map and the phylogenetic tree (Figure 1), gene

duplication of cluster I UGTs appears to have occurred in the

grapevine genome, resulting in a small subcluster of functionally

differentiated UGTs. The observed structural variations in the

primary structures of grapevine Vv GTs (Table 1; see Supple-

mental Table 1 online) suggest that these genes probably have

experienced neofunctionalization (or nonfunctionalization) after

gene duplication. Maximum likelihood analysis (Yang, 2007) of

the cluster I UGTs (Figure 1) suggested that, in the V. vinifera

genome, differentiation of Vv GT genes has more recently

occurred in a more positive manner than that of other cluster I

enzymes due to either positive Darwinian selection or relaxation

of functional constraints (see Supplemental Results 2 online for

details of the analysis).

Maximum parsimony analysis (see Supplemental Figure 5

online) indicates that Vv GT5 and Vv GT6 might have been

derived from a common ancestral GlcT gene and differentiated

via subsequent mutations (Figure 5). The ancestral Trp140 (TGG)

residue was replaced by Arg (AGG) in Vv GT5, whereas the

ancestral Thr19 (ACC) residue was replaced by Pro (CCC) in Vv

GT6 via single nonsynonymous substitutions (see Supplemental

Figure 5 online). It is important to note, however, that acquisition

of an Arg residue proximal to the carboxylate of UDP-GA is

necessary, but not sufficient, for GAT function because the

W140R mutant of Vv GT6 was devoid of GAT activity, as in the

case of our recent mutational studies of Pf F7GlcT (UGT88A7)

and Gm IF7GlcT (UGT88E3) (Noguchi et al., 2009a). It also was

noteworthy that the GalT activity of the Vv GT5-R140W mutant

was much lower than its GlcT activity and the fact that the T19P

mutation of the VvGT5-R140Wmutant enhanced itsGalT activity

mimicked the evolutionary process of Vv GT6 for acquiring

bifunctional specificity (Figure 5). The bifunctionality of Vv GT6

can be regarded as a promiscuous state with respect to sugar

donor specificity, as indicated by previous directed evolution

studies (Aharoni et al., 2005; Bloom and Arnold, 2009), which

show that enzymes tend to go through a promiscuous state prior

to neofunctionalization.

Finally, although flavonol glycosides in grapevine and grape-

vine products should be of biological, nutritional, and food

chemical significance, how their glycon structures modulate

the functional properties and bioactivities of these secondary

metabolites remains to be thoroughly examined. Characteriza-

tion of Vv GT5 and Vv GT6 now make it possible to produce

efficiently flavonol 3-O-glucuronides and 3-O-galactosides, in

addition to flavonol 3-O-glucosides, in large amounts, facilitating

examination of unanswered questions in future studies.

METHODS

Plant Materials

Plant samples of Vitis vinifera cv Cabernet Sauvignon and cv Pinot Noir

were obtained at a vineyard in the TOMI NO OKA Winery, Yamanashi,

Japan, and those of V. vinifera cv Shiraz were kindly provided by N. Goto-

Yamamoto, National Research Institute of Brewing, Hiroshima, Japan.

Chemicals

Quercetin, kaempferol, isorhamnetin, quercetin 3-O-glucoside, quercetin

3-O-galactoside, quercetin 3-O-glucuronide, kaempferol 3-O-glucoside,

and isorhamnetin 3-O-glucoside were available from TransMIT Flavo-

noidforschung. UDP-Glc, UDP-Gal, and UDP-GA were products of

Sigma-Aldrich. UDP-rhamnose was a generous gift from Keiko Yonekura-

Sakakibara, RIKEN, Yokohama, Japan. Pelargonidin, cyanidin, delphini-

din, apigenin, luteolin, (+)-catechin, genistein, naringenin, ferulic acid,

caffeic acid, salicylic acid, capsaicin, trans-resveratrol, and esculetin

were obtained as described previously (Sawada et al., 2005; Noguchi

et al., 2007, 2008, 2009b).

Screening of V. vinifera Flavonoid-3-O-Glycosyltransferase Genes

The V. vinifera cv Pinot Noir (PN42004 genotype) genome database

(http://genomics.research.iasma.it/iasma/) provided by Instituto Agrario

Samn Michele all’Adige was screened using standard BLAST algorithms

(Altschul et al., 1990) and the Vv GT1 sequence (Ford et al., 1998).

Total RNA was prepared from leaves (0.1 g) of V. vinifera cv Cabernet

Sauvignon using commercially available kits (FruitMate RNA purification

kit and Fast Pure RN kit from TaKaRa Bio). cDNA synthesis was

performed using the Superscript first-strand synthesis system for RT-

PCR (Invitrogen) starting with total RNA (1 mg) according to the manu-

facturer’s guidelines. Using the cDNA library as a template, VvGT5 cDNA

was amplified by PCR with ExTaq polymerase (TaKaRa Bio) using

the primers 59-CACCCATATGACTACCACCGCCAGCTCCAT-39 and

59-AGATCTCTACTTATTGGTGTCCAAAGGTA-39. The thermal cycling

conditions were 948C for 3 min followed by 35 cycles of PCR (one cycle

consisted of 948C for 1 min, 508C for 1 min, and 728C for 2 min). The PCR

product, 1.4 kb in length, was gel purified and cloned into pENTER-

Directional-TOPO vector (Invitrogen). The DNA insert was sequenced by


the primer walking method using synthetic primers with an Applied

Biosystemsmodel 3100 sequencer to confirm the nucleotide sequence of

Vv GT5. The Vv GT6 cDNA was obtained essentially as described above

except that the PCR primers used were 59-CACCCATATGACTGC-

CACCGCGAGCTCCATG-39 and 59-AGATCTCTACTTATTGGTACTCA-

AATGTAACT-39.

Heterologous Expression and Purification of the

Expressed Products

The pENTR-Directional-TOPO vector containing Vv GT5 cDNA was

digested with BglII, followed by partial digestion with NdeI to obtain the

full-length Vv GT5 cDNA. The Vv GT5 cDNA was ligated into a pET15b

vector (Novagen) that had previously been digested withNdeI andBglII to

obtain the plasmid pET15b-Vv GT5, which encoded an N-terminal in-

frame fusion of Vv GT5 with a His6 tag. Escherichia coli BL21 (DE3) cells

were transformed with the resulting plasmid. The Vv GT6 cDNA was

excised from the recombinant pENTER-Directional-TOPO vector, as

described above. The Vv GT6 cDNA was ligated into a pColdI vector

(TaKaRa) that had previously been digested with NdeI and BglII to obtain

the plasmid pColdI-Vv GT6, which encoded anN-terminal in-frame fusion

of Vv GT6 with a His6 tag. E. coli BL21 cells were transformed with the

resulting plasmid.

Heterologous expression of Vv GT5 cDNA was performed as follows.

After the transformants were precultured at 378C for 16 h in Luria-Bertani

broth containing 50 mg/mL ampicillin, the culture was used to inoculate

the same medium (1000 mL). After cultivating the cells at 378C until

the optical turbidity at 600 nm (OD600) of the culture reached 0.5, isopropyl

1-b-D-thiogalactoside was added to the medium at a final concentration

of 0.5 mM, followed by cultivation at 188C for 20 h. The cells were then

harvested by centrifugation (10 min, 5000g). Heterologous expression of

Vv GT6 cDNAs was performed as follows. After cultivating the trans-

formant cells at 378C until the OD600 of the culture reached 0.5, the culture

was immediately chilled on ice for 10 min, followed by the addition of

isopropyl 1-b-D-thiogalactoside to the medium at a final concentration of

0.5 mM and further cultivation at 158C for 24 h. The cells were then

harvested by centrifugation (10 min, 5000g).

All subsequent operations were conducted at 0 to 48C. The harvested

cells were suspended in buffer A (20 mM HEPES-NaOH, pH 7.5,

containing 14 mM 2-mercaptoethanol) supplemented with 20 mM imid-

azole. The cell suspension was sonicated eight times for 15 min, and the

resulting cell debris was removed by centrifugation (15,000g, 15 min)

followed by filtration with a 0.22-mmfilter (Millipore). The supernatant was

applied to a 1-mL His SpinTrap (GE Healthcare Bio-Sciences) that had

been equilibrated with buffer A containing 20 mM imidazole, followed by

centrifugation (70g, 30 s). The column was washed with 5 mL of equil-

ibration buffer, followed by washing with 5 mL of buffer A containing 100

mM imidazole and then with 5 mL of buffer A containing 500 mM

imidazole. The enzyme-containing fractions were concentrated using a

Microcon YM-30 ultrafiltration device (Millipore), followed by substitution

with buffer A. SDS-PAGE was performed according to the method of

Laemmli (1970), and the proteins in the gels were visualized using

Coomassie Brilliant Blue R 250.

Phylogenetic Analysis

The nucleotide sequences of VvGTs and the relatedUGTs were aligned by

takingconsideration of codonposition usingClustalWbundled inMEGA4.1

(Thompson et al., 1994; Tamura et al., 2007). All positions containing gaps

and missing data were eliminated from the further analysis. The unrooted

phylogenetic trees of them were reconstructed by neighbor-joining

methods from translated amino acid sequences. The alignment used to

generate the tree shown in Figure 1B is available as Supplemental Data Set

1 online. The neighbor-joining treewas reconstructed byMEGA4.1with the

matrix of the evolutionary distances calculated by Poisson correction for

the multiple substitutions. The reliability of reconstructed tree was evalu-

ated by bootstrap test with 1000 replicates.

Test for Positive Selection

To assess the degree of natural selection on Vv GTs, the rate ratio of

nonsynonymous to synonymous substitution (v) was estimated by the

codon substitutionmodel implemented in CODEMLprogramof PAML 4.3

package (Yang, 2007). The likelihood analysis of branch-specific model

(two-ratio model; model = 2, NS sites = 0) was performed to estimate the

ratio v for the ancestral lineages of VvGT2;6 (foreground branches) and

the ratio v for all other branches (background branches). The likelihood

analysis of the one-ratiomodel (M0model; model = 0, NS sites = 0), which

assumes the same ratio for all branches, was also performed as a null

model comparing with the branch-specific model.

Site-Directed Mutagenesis

According to the previously published method of Noguchi et al. (2007), in

vitro mutagenesis of the Vv GT5 and Vv GT6 genes was performed using

recombinant PCR with the pENTR-Directional-TOPO vector (Invitrogen)

containing the wild-type cDNAs as the templates and the specific

mutagenic oligonucleotide primers shown below to obtain the following

site-directed mutants: Vv GT5-R140W, Vv GT6-P19T, Vv GT5-R140W-

T19P, and Vv GT6-Q373H. The amplified fragments were digested with

NdeI and BglII, and the resulting DNA fragments were ligated with pET-

15b (Novagen), as described above. Individual mutation was verified by

DNA sequencing of both strands: VvGT5-R140W-Fw, 59-GGGTGGC-

AATTTGGACTGCTG-39; VvGT5-R140W-Rv, 59-CAGCAGTCCAAATT-

GCCACCC-39; VvGT5 T19P R140W-Fw, 59-TTCCCCCCCCATACAGC-39;

VvGT5 T19P R140W-Rv, 59-TAGCTGTATGGGGGGGG-39; VvGT6-P19T-Fw,

59-TTCCCCACCCATGCAGCTA-39; VvGT6-P19T-Rv, 59-TAGCTGCATGG-

GTGGGGAA-39; VvGT6-S142A-Fw, 59-ATTTGGACTGCTGCGCTCTGC-

TAC-39; VvGT6-S142A-Rv, 59-TGAGCAGAGCGCAGCAGTCCAAAT-39;

VvGT6-Q373H-Fw, 59-TTCTTTGGAGATCATTGTATCGACA-39; VvGT6-

Q373H-Rv, 59-TGTCGATACAATGATCTCCAAAGAA-39.

Enzyme Assays

The standard reaction mixture (100 mL) consisted of 100 mM sugar

acceptor, 1 mMUDP-sugar, 50 mM glycine-NaOH (pH 9.1, for Vv GT5) or

50 mM HEPES-NaOH (pH 7.4, for Vv GT6), 14 mM 2-mercaptoethanol,

and enzyme (typically 50 ng). Prior to addition of the enzyme, the mixture

was preincubated at 308C for 15 min, and the reaction was started by

addition of the enzyme. After incubation at 308C for 15 min, the reaction

was stopped by the addition of 100 mL of 40% (by volume) acetonitrile

containing 0.1% (by volume) trifluoroacetic acid. After centrifugation, the

supernatant (100 mL) was subjected to reversed-phase HPLC using a

Gilson 305 system equipped with an online Gilsonmodel 231 autosample

injector.

HPLC conditions for the analysis of flavonols and their glycosides were

as follows: column, YMC J’sphere ODS M80 (4.6 3 150 mm; YMC Co.);

flow rate, 0.7 mL/min; solvent A, 0.2% (by volume) formic acid in water;

solvent B, 0.2% formic acid in a 9:1 (by volume)mixture of acetonitrile and

water. After injection (100 mL) into a column that had been equilibrated

with 10% solvent B (by volume), the column was initially developed

isocratically with 10% solvent B for 3 min, followed by a linear gradient

from 10 to 40% solvent B for 10 min and then by a linear gradient from 40

to 90% solvent B for 10 min. The column was then washed isocratically

with 90%solvent B for 1min, followed by a linear gradient from 90 to 10%

solvent B for 1 min. There was a 10-min delay before the next injection to

2868 The Plant Cell

ensure re-equilibration of the column. The chromatogramswere obtained

with detection at 360 nm.

HPLC conditions for the analysis of anthocyanidins and their glyco-

sides were as follows: column, Asahipak ODP-50 4E (4.6 3 250 mm;

Shodex); flow rate, 0.7 mL/min; solvent A, 0.5% (by volume) trifluoro-

acetic acid in water; solvent B, 0.5% trifluoroacetic acid in a 1:1 (by

volume) mixture of acetonitrile and water. After injection (100 mL) into a

column that had been equilibrated with 30% solvent B (by volume), the

column was initially developed isocratically with 30% solvent B for 3 min,

followed by a linear gradient from 30 to 65% solvent B for 10 min and

then by a linear gradient from 65 to 100% solvent B for 1 min. The column

was then washed isocratically with 100% solvent B for 5 min, followed by

a linear gradient from 100 to 30% solvent B for 1 min. The column was

equilibrated with 30% solvent B for 10 min before the next injection. The

chromatograms were obtained with detection at 520 nm.

Peak identification of each component was confirmed post-run by

photodiode array spectroscopy analysis from 220 to 600 nm (for antho-

cyanin) using a Shimadzu SPD M20A system. Regioisomers of flavonol

glucuronides were also identified by their lmax values (see Supplemental

Table 2 online). The amounts of flavonoids were determined from peak

integration using authentic samples for calibration.

The HPLC conditions used for the analyses of the phenolics flavones,

isoflavones, flavanones, coumarins, capsaicin, caffeic acid, ferulic acid,

salicylic acid, and trans-resveratrol have been described previously

(Noguchi et al., 2008, 2009b).

Enzyme Kinetics

Assays of the initial velocity of UGT-catalyzed reactions were performed

under steady state conditions using a standard assay system (see above)

and various substrate concentrations. The apparent Km and kcat values

and their standard errors for the sugar donors and acceptors in the

presence of a saturating concentration of the counter substrate were

determined by fitting the initial velocity data to the Michaelis-Menten

equation using a nonlinear regression method (Leatherbarrow, 1990).

Quantitative RT-PCR

Total RNA was prepared from an array of organs and tissues, including

the leaves, stems, and developing berries, of cv Cabernet Sauvignon and

cv Pinot Noir using the RNeasy plant mini kit (Qiagen). Total RNA was

treated with DNase I (RNase-free; TaKaRa). First-strand cDNA synthesis

was performed starting with 0.5 mg of total RNA using a kit (Transcriptor

first-strand cDNA synthesis kit; Roche). The VvGT5, VvGT6, VvGT1, and

Vv FLS1 cDNAs in the mixture were quantified by quantitative real-time

PCR with the specific primers: Vv GT5, 59-GCTCCATCTCCTCTGCT-

CAAA-39 and 59-GAAAGCACAAGGTCCTCT-39; Vv GT6, 59-GGTTCCC-

TGGTTGGCAATTT-39 and 59-GCACCCGCCCCACAACCTT-39; Vv GT1,

59-CCCACCGCCGGTTATACC-39 and 59-CGACCGAGGTGGGTTTTCT-3;

and, Vv FLS1, 59-AAACCACCTACTTACAGAGG-39 and 59-ACCTAACCC-

CAGTGACAGAC-39. Real-timePCRwasperformedusing a7500Real-Time

PCR system (Applied Biosystems) and a QuantiTect SYBR Green RT-PCR

Kit (Qiagen). Relative transcription levels were analyzed by a DDCt method

(Applied Biosystems) after normalization to transcription of an internal

standard (cDNA encoding V. vinifera ubiquitin 2), which was quantified

using the primers 59-TCCAGGACAAGGAAGGGATTC-39 and 59-GCCA-

TCCTCAAGCTGCTTTC-39. The results arepresentedas theaverage6SEof

three independent determinations.

Flavonoid Analysis

Weighed quantities of frozen organs and tissues of grapevine (1 to 2 g)

were dried in a lyophilizer. The flavonoids were extracted from the dried

samples with 8 to 16 mL of 0.1% trifluoroacetic acid (by volume) in a 1:1

mixture of acetonitrile and water as follows: 0.1 g fresh weight/mL with

sonication (38 kHz) for 20 min. In the case of dried fruits, a sample (10 g)

was pulverized in liquid nitrogen using a mortar. The flavonoids were

extracted with 20 mL of 0.1% trifluoroacetic acid (by volume) in a 1:1

mixture of acetonitrile and water. The extract was diluted with water (two

equivalents), embedded on a water-equilibrated C-18 Sep-Pak column

(Vac20cc; Waters), and eluted with 20 mL of 9:1 mixture of acetonitrile

and water after washing with 50 mL water. The extracts were centrifuged

at 1000g for 10 min, and the supernatant was filtered through a 0.45-mm

filter and analyzed for flavonoids by LC-MS using an LCMS-IT-TOF

apparatus (Shimadzu) equipped with a YMC-Pack polymer C18 column

(2.0 3 150 mm; s6mm, YMC). HPLC was performed at a flow rate of

0.2 mL/min using 0.1% (by volume) formic acid in water as solvent A and

0.1% formic acid in a 9:1 (by volume) mixture of acetonitrile and water as

solvent B. After injection of sample into a column that had been equil-

ibrated with 20% solvent B (by volume), the columnwas developedwith a

linear gradient from 20 to 60% solvent B for 15 min and then was washed

isocratically with 60% solvent B for 10 min. The chromatograms were

obtained with detection over a wavelength range of 220 to 500 nm using

an SPD-M10A photodiode array detector (Shimadzu). The online mass

spectrometry analyses (scan range, 100 to 1000 m/z) were performed

using the interfaces in negative mode with 4.5 kV and in positive mode

with 3.5 kV applied to the spray tip. Identification was based on a

combination of HPLC retention time and UV and mass spectrometry

spectral data. The m/z value for quercetin 3-O-glucuronide was 477.077

[M–H]–. The 3-O-glucoside and galactoside of quercetin in crude samples

could not be separated from one another under these analytical condi-

tions; therefore, these compounds were identified as quercetin 3-O-

glucoside/galactoside (m/z 463.09 [M–H]–). Flavonols were quantified by

absorption at 350 nm.

Homology Modeling

The crystal structure of Vv GT1 was used to construct homology models

of Vv GT5 and Vv GT6 using the Homology module installed in the Insight

II molecular modeling system (Molecular Simulations). The UDP-Glc

bound in Vv GT1 was inserted into the constructed Vv GT5 and Vv GT6

models. The glucose portion of UDP-Glc was replaced with glucuronic

acid or galactose moieties as needed. Kaempferol was placed in the

sugar acceptor binding site. Two water molecules in the crystal structure

were inserted into the model structures to form hydrogen bonds with the

substrates, UDP-Glc or kaempferol, and with amino acid residues of Vv

GT1. Before optimization, initial model structures of the corresponding

mutants (e.g., the R140W mutant of Vv GT5 or the Q373H and P19T

mutants of Vv GT6) were constructed by replacing the relative amino acid

residues of Vv GT5 and Vv GT6, respectively. Structure optimization of

each model was performed using the molecular mechanics and molec-

ular dynamics simulation program Discover3 (Molecular Simulations).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome

Initiative or Genbank/EMBL/DDBJ databases under the following acces-

sion numbers: Vv GT1 (UGT78A5), AAB81683 (cv Shiraz); Vv GT2

(UGT78A6), CAO67330 (cv Pinot Noir); Vv GT2-like (UGT78A7),

CAO67328 (cv Pinot Noir); Vv GT3 (UGT78A10), CAO43305 (cv Pinot

Noir); Vv GT4 (UGT78A8), CAO67332 (cv Pinot Noir); Vv GT4-like

(UGT78A9), CAO67329 (cv Pinot Noir); Vv GT5 (UGT78A11), CAN74919

(cv Pinot Noir) and AB499074 (cv Cabernet Sauvignon); Vv GT6

(UGT78A12), AB499075 (cv Cabernet Sauvignon); Vv GT5 promoter (1.5

kb), AB541989; Vv GT6 promoter (1.5 kb), AB541990; At3GlcT

(UGT78D2), AAM91139; Ph3GlcT, BAA89008; Ph3GalT, AAD55985; Pf

F7GAT (UGT88D7), BAG31948; Pf 7GlcT (UGT88A7), AB362992; Gm

IF7GlcT (UGT88E3), BAF64416; Ac GalT, BAD06514; Bp UGAT


(UGT94B1), BAD77944; Vl RSgt, ABH03018; Vm 3GalT, AB009370; Ih

3GlcT, BAD83701; Zm 3GlcT (Bronze 1), AAV64215; and V. vinifera

ubiquitin 2, CAO48597. Vv GT1 structural information is available in the

Protein Data Bank (pdb code 2C1Z)

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Bifunctional Activities of Vv GT6.

Supplemental Figure 2. Superposition of Structural Models of

Michaelis Complexes of GATs from Different Phylogenetic Clusters

(Vv GT5, Pf F7GAT [UGT88D7 of Perilla frutescens], and Bp UGAT

[UGT94B1 of Bellis perennis]).

Supplemental Figure 3. A Structural Model of the R140W Mutant of

Vv GT5 Docked with UDP-GA and Kaempferol.

Supplemental Figure 4. A Structural Model of the Q373H Mutant of

Vv GT6 Docked with UDP-Gal.

Supplemental Figure 5. Maximum Parsimony Analysis of the Vv GT

Cluster.

Supplemental Table 1. Sequence Identity among Vv GT cDNAs.

Supplemental Table 2. HPLC Analysis of Quercetin Monoglucu-

ronides and 3-O-Glucoside.

Supplemental Data Set 1. Text File of the Alignment Used to

Generate the Phylogenetic Tree Shown in Figure 1B.

Supplemental Results 1. Thermodynamic Consideration of the

Effects of R140W Substitution on Kinetic Parameters of Vv GT5.

Supplemental Results 2. Maximum Likelihood Analysis of Vv GT

Genes.

ACKNOWLEDGMENTS

We thank N. Goto-Yamamoto (National Research Institute of Brewing,

Hiroshima, Japan) for providing a plant sample of V. vinifera cv Shiraz

and N. Watanabe and S. Fujimoto (Suntory Liquors Ltd., TOMI NO OKA

Winery, Yamanashi, Japan) for the gifts of cv Cabernet Sauvignon and

cv Pinot Noir. We thank Keiko Yonekura-Sakakibara (RIKEN, Yokohama,

Japan) for providing UDP-rhamnose. We also thank A. Ohgaki (Suntory

Wellness), Y. Matsuo, N. Tsuruoka, and H. Toyonaga (Suntory Holdings)

for their technical support.

Received February 11, 2010; revised June 20, 2010; accepted July 8,

2010; published August 6, 2010.

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DOI 10.1105/tpc.110.074625; originally published online August 6, 2010; 2010;22;2856-2871Plant Cell

Yosuke Kawai, Masaji Ishiguro, Yuko Fukui and Toru NakayamaEiichiro Ono, Yu Homma, Manabu Horikawa, Satoshi Kunikane-Doi, Haruna Imai, Seiji Takahashi,

)Vitis viniferaof Bioactive Flavonol Glycosides in Grapevines (Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity

This information is current as of August 14, 2019

Supplemental Data /content/suppl/2010/07/15/tpc.110.074625.DC1.html

References /content/22/8/2856.full.html#ref-list-1

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