Crystal Structures of a Multifunctional Triterpene/FlavonoidGlycosyltransferase from Medicago truncatula

Hui Shao,1 Xianzhi He,1 Lahoucine Achnine, Jack W. Blount, Richard A. Dixon, and Xiaoqiang Wang2

Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401

Glycosylation is a ubiquitous reaction controlling the bioactivity and storage of plant natural products. Glycosylation of

small molecules is catalyzed by a superfamily of glycosyltransferases (GTs) in most plant species studied to date. We

present crystal structures of the UDP flavonoid/triterpene GT UGT71G1 from Medicago truncatula bound to UDP or UDP-

glucose. The structures reveal the key residues involved in the recognition of donor substrate and, by comparison with other

GT structures, suggest His-22 as the catalytic base and Asp-121 as a key residue that may assist deprotonation of the

acceptor by forming an electron transfer chain with the catalytic base. Mutagenesis confirmed the roles of these key

residues in donor substrate binding and enzyme activity. Our results provide an initial structural basis for understanding the

complex substrate specificity and regiospecificity underlying the glycosylation of plant natural products and other small

molecules. This information will direct future attempts to engineer bioactive compounds in crop plants to improve plant,

animal, and human health and to facilitate the rational design of GTs to improve the storage and stability of novel engineered

bioactive compounds.

INTRODUCTION

Glycosylation is quantitatively the most significant reaction on

earth. It is catalyzed by a superfamily of enzymes, the glycosyl-

transferases (GTs), which have been classified into >70 families

(Campbell et al., 1997) (http://afmb.cnrs-mrs.fr/CAZY/index.

html). Family 1 GT enzymes, the UDP glycosyltransferases

(UGTs), transfer UDP-activated sugar moieties to specific ac-

ceptor molecules. Twenty-seven UGTs have been identified in

the human genome (de Wildt et al., 1999; Li et al., 2001); these

play central roles in the metabolism and detoxification of foreign

chemicals such as carcinogens and hydrophobic drugs. By

contrast, plants contain a very large number of UGTs that are

involved in the glycosylation of natural products (Vogt and Jones,

2000; Jones and Vogt, 2001) and contain a highly conserved

consensus signature sequence called the PSPG motif (Hughes

and Hughes, 1994; Paquette et al., 2003). Plants collectively

synthesize >200,000 natural products, and a significant pro-

portion of them are glycosylated. Consistent with this chemical

complexity, 117 putative UGT genes have been identified in the

genome of Arabidopsis thaliana (Ross et al., 2001). Likewise,

>100 UGTs are predicted in the model legume Medicago

truncatula based on an analysis of currently available EST and

genomic sequences (Achnine et al., 2005).

Glycosylation is generally seen as a mechanism for the de-

toxification of xenobiotics or for facilitating the storage of bio-

activemolecules in plant vacuoles (Sandermann, 1992; Paquette

et al., 2003). However, in some cases, glycosylated plant natural

products exhibit higher bioactivity than their corresponding

aglycones, as in the case of the triterpene saponins (Osbourn,

2003). UGT71G1 was recently identified in M. truncatula as an

enzyme functionally involved in the biosynthesis of saponins,

although the enzyme exhibits more favorable kinetics with the

flavonoid quercetin (Achnine et al., 2005). Triterpene glycosides

have allelopathic, antimicrobial, insecticidal, and antiherbivory

activities for plant protection (Osbourn et al., 1996; Small, 1996;

Oleszek et al., 1999; Papadopoulou et al., 1999). They also have

pharmacological, anticholesterolemic, anticancer, adjuvant, and

hemolytic activities (Behboudi et al., 1999; Oh et al., 2000;

Haridas et al., 2001), act on the cardiovascular, nervous, and

digestive systems, and are used in sneezing powders, emetics,

and cough syrups to facilitate expectoration (Dennehy, 2001).

The saponins inM. truncatula are glycosides of the five triterpene

aglycones soyasapogenol B, soyasapogenol E, medicagenic

acid, hederagenin, and bayogenin (Huhman and Sumner, 2002).

These aglycones share similar structures, and UGT71G1 recog-

nizes both medicagenic acid and hederagenin as acceptors

(Figure 1) for the sugar moiety delivered from UDP-glucose. It is

not yet clear which positions on the triterpene scaffold are

glycosylated by UGT71G1;O-glycosylation at positions 3 and 28

is most common for this class of compound, butC-glycosylation

of plant natural products has also been observed, and these

products areof particular pharmaceutical interest (Clemenset al.,

2004). Understanding the structural basis for the glycosylation of

triterpene natural products will facilitate future metabolic engi-

neering of these compounds to improve the health of plants

(resistance to disease and herbivory), animals (via improved

forage quality), and humans (through drug discovery).

1 These authors contributed equally to this work.2 To whom correspondence should be addressed. E-mail xwang@noble.org; fax 580-224-6692.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Xiaoqiang Wang(xwang@noble.org).Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.105.035055.

The Plant Cell, Vol. 17, 3141–3154, November 2005, www.plantcell.orgª 2005 American Society of Plant Biologists

To date, 17 members of the GT superfamily (but none from

plants) have been analyzed structurally (Goldsmith et al., 1989;

Vrielink et al., 1994; Charnock and Davies, 1999; Gastinel

et al., 1999, 2001; Ha et al., 2000; Pedersen et al., 2000, 2003;

Unligil et al., 2000; Persson et al., 2001; Gibbons et al., 2002;

Gibson et al., 2002; Buschiazzo et al., 2004; Fritz et al., 2004;

Lobsanov et al., 2004; Mulichak et al., 2004), and these struc-

tures fall into two different topologies/folds, GT-A and GT-B

(Breton et al., 2002; Hu and Walker, 2002; Coutinho et al., 2003).

Plant UGTs have been predicted to be similar to the bacterial GT

family 1 TDP-epi-vancosaminyltransferasesGtfA, GtfB, andGtfD

from Amycolatopsis orientalis (Mulichak et al., 2001, 2003, 2004;

Hans et al., 2004; Bowles et al., 2005), which belong to the GT-B

fold comprising twoRossmann-like domains. However, bacterial

Gtfs share only ;10% sequence identity with plant UGTs,

making it very difficult to model plant glycosylation processes

without knowledge of plant UGT crystal structures.

Here, we report the structures of UGT71G1 fromM. truncatula

bound to UDP or UDP-glucose. These plant UGT crystal struc-

tures reveal the detailed interactions between the enzyme and its

donor substrate as well as the roles of the UGT signature motif in

substrate recognition and the catalytic activity of the enzyme.

Further mutational and structural analyses, and comparisons

with other GT structures, will provide a basis for understanding

the complex and often overlapping substrate specificity and

regiospecificity within the plant UGT superfamily.

RESULTS

Overall Structure

The three-dimensional structure of UGT71G1 fromM. truncatula

bound with a UDP molecule was determined using the multi-

wavelength anomalous dispersion (MAD) method with a crystal

of Se-Met–substituted enzyme and refined to 2.0-A resolution

with a native data set. Data collection, phasing, and refinement

statistics are presented in Table 1.

The structure of UGT71G1 consists of two N- and C-terminal

domains with similar Rossmann-type folds and, as predicted,

belongs to the GT-B fold (Figures 2 and 3). The N-terminal

domain contains a central seven-stranded parallel b sheet

flanked by eight a helices on both sides and a small two-

stranded b sheet. The C-terminal domain contains a six-

stranded b sheet flanked by eight a helices. The two domains

pack very tightly and form a deep cleft with a UDP molecule

bound.

The asymmetric unit contains two independent UGT71G1

molecules related by a noncrystallographic twofold axis. These

two molecules are very similar to each other, with root mean

square deviations (RMSD) of 1.04 and 1.52 A for 456 Ca atoms

and all atoms (residues 8 to 463), respectively. However, small

differences were observed between the two molecules, espe-

cially in the region near the UDP binding site in the cleft between

the N- and C-terminal domains, which included the C-terminal

part of helix Na1 (around residue Ile-20); loop Nb2-Na2 (around

residuePro-50),which is highlymobilewith highB factors (;80 A2)

and poorly defined electron density in both molecules A and B;

and the loop linker between the N- and C-terminal domains

(around residue Asn-255), the electron density of which was

poorly defined inmolecule B. The helix Na1 (P18 toH36) and loop

Nb2-Na2 and the following helix Na2 (K48 to Q66) in molecule B

shift away slightly from the C-terminal domain, with the largest

movement, 2.6 A, for the Ca atom of Leu-23, which is adjacent to

the key catalytic residue His-22 (see below). This suggests that

the flexibility in the cleft region between the two subunits allows

for movement to occur during substrate binding and catalysis.

Comparison with Other Members of the GT Superfamily

There are three known structures of family 1 GTs, all for bacterial

vancomycin UDP glucosyltransferase Gtfs. Superimposing the

structures of plant UGT71G1 and bacterial GtfD revealed strong

structural similarity, with an RMSD of 2.23 A for 242 Ca atoms,

although the sequence identity is very low (;10%) (Figure 3). The

largest difference between these two enzymes was observed

in the region between the helix Na5 and the strand Nb6 (;155

to 210) in their N-terminal domains. A two-stranded b sheet

is present in this region of the UGT71G1 structure, resembling

an arm flipped to the opposite side near the helix Ca5 of the

C-terminal domain, compared with the corresponding region in

the GtfD structure. This region forms one side of the substrate

(acceptor molecule) binding pocket, and this large difference in

Figure 1. Structures of Three Substrates of M. truncatula UGT71G1.

The known or predicted glycosylation sites are labeled with numbers: quercetin (3, 5, 7, 39, and 49), medicagenic acid (3 or 28), and hederagenin (3

or 28).

3142 The Plant Cell

conformation/folding changes the shape and size of the binding

pocket. The comparison also shows that the similarity between

the C-terminal domains of UGT71G1 and GtfD (RMSD ¼ 2.13 A

for 137 Ca atoms) is higher than that between their N-terminal

domains (RMSD ¼ 2.34 A for 120 Ca atoms), presumably

because the C-terminal domains recognize similar donor mole-

cules but the N-terminal domains recognize quite different

acceptor molecules. The UGT71G1 structure is more compact

in its N-terminal domain, presumably because its substrates,

such as medicagenic acid and quercetin, are considerably

smaller than the acceptor substrate desvancosaminyl vancomy-

cin (DVV) of GtfD.

Comparison was also performed between the UGT71G1

structure and those of other GTs with the GT-B fold. Escherichia

coliMurG (Coutinho et al., 2003) is closely related to UGT71G1 in

the overall GT classification. Superimpositions between portions

of the structures of UGT71G1 and MurG gave RMSD values of

2.27 A (199 Ca atoms of the overall structures), 2.23 A (95 Ca

atoms in the N-terminal domains), and 1.95 A (110 Ca atoms in

the C-terminal domains).

Trehalose-6-phosphate synthetase OtsA, T4 bacteriophage

b-glucosyltransferase (BGT), and glycogen synthase are rela-

tively distant from UGT71G1 in classification, although they use

the same UDP-sugar or, in the case of glycogen synthase, the

related ADP-sugar. Superimposing the overall structures of

UGT71G1 and OtsA, which have very little sequence identity,

gave an RMSD of 2.21 A (158 Ca atoms). Superimposing their

N- and C-terminal domains gave RMSD values of 2.17 A (96 Ca

atoms) and 2.27 A (70 Ca atoms), respectively; the N-terminal

rather than the C-terminal domain is more similar to the corre-

sponding domain of UGT71G1, in contrast with the situation with

UGT71G1 and GtfD or MurG. Similarly, superimposing the

N- and C-terminal domains of UGT71G1 and BGT gave RMSD

values of 2.28 A (79 Ca atoms) and 2.29 A (70 Ca atoms),

respectively.

Interactions between Enzyme and Donor Ligands

Although UDP-galactose (a nondonor) and quercetin were in-

cluded for cocrystallization with UGT71G1, the only substrate

electron density observed was for the UDP moiety of UDP-

galactose (Figure 4A) as a result of possible hydrolysis. This is

similar to the situation with the bacterial GtfD structure, in which

only TDP was observed, although GtfD was cocrystallized with

TDP-glucose (Mulichak et al., 2004). Soaking crystals of

UGT71G1 with UDP-glucose resulted in a weak electron density

map for the glucose portion of the molecule in the enzyme

structure at 2.6 A (Figure 4B). The B factor of the glucose moiety

is higher than that of the UDP moiety, indicating either flexibility

or low occupancy of the sugar attributable to the possible

hydrolysis of UDP-glucose.

UDP or UDP-glucose is almost completely buried in a long,

narrow channel mainly within the C-terminal domain of the

enzyme. In the 2.0-A structure of UGT71G1 complexed with

UDP, the enzyme interacts with UDP mainly through residues in

the UGT signature PSPG motif (residues 339 to 382) (Figure 4C,

Table 2). The uracil ring of UDP interacts through hydrogen

bonds between the N3 and O4 atoms of the ring and the main

chain oxygen and nitrogen atoms of Ala-340 and also forms

parallel p-stacking interactions with the indole ring of Trp-339.

Table 1. Data Collection, Phasing, and Refinement Statistics

Data Collection and Phasing Statistics

Native (þ UDP) Se peak (þ UDP) Se inflection (þ UDP) Se remote (þ UDP) Complex (þ UPG)

Resolution (A) 2.0 2.4 2.4 2.4 2.6

Wavelength (A) 1.5418 0.9797 0.98 0.9393 1.5418

Unique reflections 62,825 36,121 35,553 34,461 29,545

Completeness (%) 98.2 (97.7) 94.4 (81.8) 97.0 (85.0) 92.4 (78.4) 99.8 (99.7)

Rsyma (%) 4.1 (27.7) 8.2 (26.0) 5.7 (25.2) 6.0 (28.6) 5.9 (44.2)

I/s (I) 13.7 (2.7) 16.5 (2.7) 16 (2.6) 16.1 (3.1) 12.1 (2.3)

Figure of merit 0.44

Refinement Statistics

Enzyme-UDP Enzyme-UDP glucose (UPG)

R factor (%) 18.7 20.5

Rfree (%) 22.9 28.0

Number of protein atoms 7204 7204

Number of solvent atoms 728 17

Number of ligand atoms 50 (two UDP molecules) 72 (two UDP-glucose molecules)

Average B factors (A2) 31.8 56.3

RMSD from ideal values

Bond length (A) 0.0057 0.0070

Bond angle (8) 1.27 1.29

Numbers in parentheses are for the highest-resolution shell.a Rsym ¼ Shkl jI � <I>j/SI, where I is the observed intensity and <I> is the average intensity from observations of symmetry-related reflections. A subset

of the data (8%) was excluded from the refinement and used to calculate the free R value (Rfree). R factor ¼ SkFoj � jFck/SjFoj.

UDP Glycosyltransferase Structure 3143

The ribose ring of UDP makes contacts with the enzyme through

hydrogen bonds between its O2* and O3* atoms and the OE1

and OE2 atoms of Glu-365 and between its O2* atom and the

NE2 atom of Gln-342. The a-phosphate forms hydrogen bonds

through its O1A atom and the ND2 atom of Asn-361, its O2A

atom and the OG and N atoms of Ser-362, and its O3A atom and

the NE2 atom of His-357. The b-phosphate forms hydrogen

bonds through its O3B oxygen and the OH atom of Tyr-379 and

the NE2 atom of His-357 and between its O2B oxygen and the

OG and N atoms of Ser-285, the only interacting residue outside

of the UGT signature motif region.

Interactions between the enzyme and the glucose moiety of

the donor are observed in the 2.6-A structure of UGT71G1 in its

complexwith UDP-glucose (Figure 4C, Table 2). TheO29 andO39

atoms of the glucose ring form hydrogen bonds with the NE2

atom of Gln-382 and the OE1 atom of Glu-381. The O49 atom of

the glucose ring contacts both the OE2 atom of Glu-381 and the

N atom of Trp-360. On the other hand, several interactions

observed between UDP and the enzyme are not present in the

model of UDP-glucose bound to the enzyme. A small rotation

and shift in the position of the b-phosphate of UDP-glucose

weakens the interaction of the O2B and O3B atoms of

b-phosphate and the O3A atom of a-phosphate with protein

residues.

In UGT71G1 binding UDP-glucose, GtfD binding TDP, treha-

lose-6-phosphate synthetase OtsA binding UDP, MurG binding

UDP-GlcNAc, and T4 bacteriophage BGT and its mutant D100A

binding UDP or UDP-glucose, the sugar donor molecules are

observed in a similar region of the enzymes (Gibson et al., 2002;

Hu et al., 2003; Lariviere et al., 2003; Mulichak et al., 2004). The

PSPG motif of UGT71G1 and the corresponding sugar binding

regions in these different GTs share a very similar conformation,

a/b/a/b, even though the sequence identities are very low and

the detailed interactions with the sugar donors also vary. Super-

impositions show that the UGT signature motif of UGT71G1 and

the corresponding regions of GtfD and MurG share higher

structural similarity, indicative of similar binding modes with

sugar donors, and differ from those for OtsA and BGT, in which

the first b strands of their a/b/a/bmotifs are long and the second

b strands cannot interact with sugar donors (Figure 5).

The Acceptor Binding Site

A very narrow, deep pocket is observed adjacent to the UDP

binding site, mainly consisting of residues from the N-terminal

domain (Figure 6), which would appear to be the binding pocket

for acceptor molecules. The location of this pocket is similar to

that of the substrate binding site observed in the structure ofGtfD

bound with DVV and TDP (Mulichak et al., 2004). In the GtfD

structure, Asp-13 is close to both DVV and TDP and has been

proposed as the general base for the catalysis of glycosyl

transfer. His-22 occupies the corresponding position in

UGT71G1 and is 4.7 A from the C19 atom of UDP-glucose, as

shown in the structure of UGT71G1 (molecule A) complexed with

UDP-glucose. Close to it (;3 A), there is an acidic residue (Asp-

121) that may form an electron transfer chain to His-22 to help

deprotonate acceptor molecules. The sequence alignment of

multiple plant UGTs (Figure 7) shows that corresponding His and

Asp residues are conserved in most plant UGTs.

There are several potential O-glycosylation sites (five hydroxyl

groups) on the quercetin molecule, and multiple products are

formed after incubation of quercetin and UDP-glucose with

UGT71G1 (Figure 8A). All five products from the in vitro in-

cubation of recombinant UGT71G1 with UDP-glucose and

quercetin exhibited a similar UV spectrum to that of quercetin

(Figure 8B), had identicalmolecular ionmasses (462.5 to 462.9D)

consistent with quercetin monoglucoside structures, and dis-

appeared after incubation with b-glucosidase (data not shown).

This indicates that, in spite of the production of one favored

product (peak 5 in Figure 8A, identified as quercetin-39-O-

glucoside), UGT71G1 is capable of glucose transfer to each of

the five hydroxyl groups on the quercetin molecule. To probe the

structural basis of the apparently promiscuous acceptor binding

to UGT71G1, molecular docking was performed using the

Figure 2. Ribbon Diagram of the Structure of UGT71G1 with Bound UDP.

The N- and C-terminal domains are shown in orange and green, with the

secondary structures and the N and C termini labeled. The a helices and

b strands in the N- and C-terminal domains are numbered separately.

The UDP molecule is shown as a ball-and-stick model colored by atom

type (nitrogen, blue; carbon, yellow; oxygen, red; phosphorus, green).

Figures 2, 3B, 4, 5, and 6 were prepared with MOLSCRIPT (Kraulis, 1991)

and RASTER3D (Merritt and Bacon, 1997).

3144 The Plant Cell

Figure 3. Comparison of M. truncatula UGT71G1 and A. orientalis GtfD.

(A) Structure-based sequence alignment ofM. truncatula UGT71G1 and A. orientalisGtfD. The secondary structure elements observed in the UGT71G1

structure are shown above the alignment. The UGT signature motifs are enclosed in a green box. Conserved residues are highlighted. This figure was

produced with ENDscript (Gouet and Courcelle, 2002).

(B) Stereo diagram showing the superimposition of the structures of UGT71G1 (molecule A; brown) and GtfD (blue; Protein Data Bank [PDB] code

1RRV).

software GOLD. Consistent with the multiple product specificity

with quercetin and the fact that quercetinwas not observed in the

structurewhen included in the crystallization liquid or soaked into

crystals, all five hydroxyl groups of quercetin could be docked

close to His-22, which lies in the middle of the potential acceptor

channel. Three aromatic residues, Phe-148, Phe-122, and Tyr-

202, and an acidic residue, Asp-190, are located at one end of

the channel; the other end is relatively larger, containing two

aromatic residues, Phe-49 and Phe-54, and two Mets, Met-52

and Met-286. An aromatic residue, Phe-123, and an acidic

residue, Glu-89, are in the middle of the channel, opposite UDP-

glucose. The pocket is much longer and larger than quercetin,

and quercetin may be fit in easily, with any of its hydroxyl groups

located ;3 A from the NE2 atom of His-22.

The docking in the active site of the hydroxyl groups at

positions 3 or 39 of quercetin gives fitness scores slightly higher

than for the other three hydroxyls, and also more potential

interactions between quercetin and the protein and donor. In the

model with the 39-OH docked in the active site (Figure 6A),

hydrogen bonds may form between the 3-OH of quercetin and

the O1B atom of UDP-glucose and between the 5-OH of

quercetin and the OG atom of Ser-285.

The triterpene substrates hederagenin and medicagenic acid

are more hydrophobic and significantly larger than quercetin.

Compared with the docking results with quercetin, docking with

medicagenic acid and hederagenin gave lower fitness scores

and weaker interaction with the enzyme, consistent with pre-

viously reported kinetic data (Achnine et al., 2005). Themolecular

docking study for hederagenin and medicagenic acid also

suggested that O-glycosylation at position 3 would be preferred

over other positions. In the model docked with hederagenin

(Figure 6B) or medicagenic acid, the OH group at position 3

points to the NE2 atom of His-22 (;3.2 A) and is located just

above the C19 atom of UDP-glucose (;3.8 A). Similar to the

proposed general mechanism of other inverting GTs (Unligil and

Rini, 2000), His-22 would act as an effective general base to

deprotonate the OH group of the acceptor molecule, then the

newly formed nucleophilic oxyanionwould attack the C19 carbon

of the UDP-glucose directly to displace the UDP moiety. The

carboxyl groups at position 28 of the triterpene acceptors may

hydrogen bond with the main chain oxygen atom of Gly-51. The

residues Pro-18, Phe-49, Pro-53, Phe-54, Leu-85, Pro-88, Ile-92,

and Phe-123 provide a hydrophobic environment for the accep-

tor substrates. A solution of the model for docking the carboxyl

oxygen atom at position 28 of hederagenin to the active site had

a slightly lower fitness score than that for the 3-hydroxyl. In the

former case, the hydroxyl group at position 3 may interact with

the main chain oxygen atom of Gly-51.

Mutagenesis of UGT71G1

To further explore the roles of the potential key residues

identified above, structure-directed point mutants of UGT71G1

were constructed, targeting residues involved in direct contact

with the sugar donor, the proposed general base His-22, and its

partner, Asp-121. Substitution of His-22 with Ala (H22A) led to

the complete loss of enzyme activity, which is consistent with the

proposed role of His-22 as the general base. Substitution of Asp-

121 with Ala or Asn resulted in no detectable enzyme activity.

This is similar to the situation with trypsin, for which the mutant

D102N (in the catalytic triad) showed a large loss in activity

(Sprang et al., 1987). Also similar to the case of trypsin, Asp-121

in UGT71G1 is nearly buried and forms a hydrogen bond with

a catalytic His residue. Our results demonstrate that Asp-121

and its proper interaction with His-22 are essential for enzyme

activity. In themodel docked with acceptors, Asp-121 is not able

to contact the acceptor molecules directly. This finding suggests

that Asp-121 may play an important role in assisting His-22 by

initialization or activation of the reaction through electron trans-

fer. The E381A mutant also showed no detectable activity,

demonstrating the key role of Glu-381 in recognition of the sugar

donor substrate because the side chain of Glu-381 forms

hydrogen bonds with the O39 and O49 atoms of the glucose

ring of the donor (Figure 4C). By contrast, mutant A340F retained

Figure 4. Donor Molecules and Their Interactions with UGT71G1.

(A) A jFobsj � jFcalcj electron density omit map of bound UDP contoured

at 1.5 s is superimposed on a ball-and-stick model of the UDP molecule.

(B) A jFobsj � jFcalcj electron density omit map of bound UDP-glucose

contoured at 1.5 s is superimposed on a ball-and-stick model of the

UDP-glucose molecule.

(C) Stereo diagram showing interactions between bound UDP-glucose

and UGT71G1 side chains. The structure of UDP-glucose is shown as

a ball-and-stick model. Hydrogen bonding interactions are indicated by

dashed lines. Interactions observed only between UDP(-galactose) and

UGT71G1 in the 2.0-A structure are indicated by green dashed lines.

3146 The Plant Cell

activity similar to that of the wild-type enzyme reported pre-

viously (Achnine et al., 2005). Ala-340 forms hydrogen bond

interactions with the uracil ring of the donor through its main

chain oxygen and nitrogen atoms; therefore, such bonds will still

be supplied by Phe.

DISCUSSION

Comparisons between GT-B and GT-A Fold GTs

Comparison of the three-dimensional structures of UGT71G1

and several other GT-B fold enzymes revealed high similarity

despite the very low sequence identity. Gtfs and MurG, with

a similar donor binding motif, share higher similarity in their

C-terminal domains than in their N-terminal domains, suggesting

a similar sugar donor binding mode and probably a common

evolutionary origin. OtsA and BGT, with a slightly different donor

binding motif, share higher similarity in their N-terminal domains

with UGT71G1. Glycogen synthase (PDB code 1RZV) shares

higher similarity with UGT71G1 in the C-terminal domain, but its

donor binding region is closer to that of OtsA or BGT (data not

shown). This suggests that these enzymes have different sugar

donor binding modes and evolutionary origins.

GT-A fold enzymes contain only a single extended domain (or

two tightly associated and abutting domains) that recognizes

both donor and acceptor (Davies et al., 2001; Coutinho et al.,

Table 2. Interactions between Enzyme Molecules (A or B) and Donors (UDP or UPG)

Protein Residue: Atom Donor Atom

Distance (A)

Molecule A, UDP Molecule B, UDP Molecule A, UPG Molecule B, UPG

A340: N O4 3.0 3.1 2.8 3.3a

O N3, O2 2.7, 3.3 2.6, 3.3 2.6, 3.1 2.8, 2.9

Q342: NE2 O2* 3.0 3.0 3.4 3.3

E365: OE1 O2* 2.7 2.6 2.7 2.7

OE2 O2*, O3* 3.2, 2.8 3.2, 2.8 3.1, 2.9 3.0, 2.6

N361: ND2 O1A 3.1 3.2 3.1 3.2

S362: N O2A 2.8 2.9 3.1 2.9

OG O2A 2.5 2.8 2.5 2.5

H357: NE2 O3A 2.9 2.9 3.4 3.2

O3B 3.0 3.2 3.4 3.4

Y379: OH O3B 2.4 2.6 3.3 (w. O2B) 3.2(w. O2B)

S285: N O2B 2.7 2.9 3.4 3.3

OG O2B 2.7 2.7 3.5 3.3

W360: N O49 2.9 2.6

E381: OE1 O39 2.4 2.4

OE2 O49 2.6 3.0

Q382: NE2 O29 2.7 2.9

a Numbers for distances >3.3 A are underlined.

Figure 5. Comparison of Donor Binding Regions of UGT71G1 and Other GT-B Fold Enzymes.

Stereo diagram showing superimposition of the UGT signature motif regions of UGT71G1 (yellow), GtfD (red), MurG (green; PDB code 1F0K), OtsA

(cyan; PDB code 1GZ5), and BGT (black; PDB code 1J39).

UDP Glycosyltransferase Structure 3147

2003). The structures of GT-A fold enzymes and the N- and

C-terminal domains of GT-B fold enzymes are similar to each

other. Superimposition between a GT-A enzyme, human b1,

3-glucuronyltransferase I (family 43; PDB code 1FGG) (Pedersen

et al., 2000), and each domain of UGT71G1 gaveRMSDvalues of

2.3 A for 80 Ca atoms in the N-terminal domain and 1.96 A for 40

Ca atoms in the C-terminal domain. This finding suggests that

GT-A and GT-B enzymes are evolutionarily related. During

evolution, two domain enzymes were probably produced, with

one domain recognizing similar donors and another adapting to

divergent acceptors in the natural environment.

Substrate Binding and Catalysis by UGT71G1

Kubo et al. (2004) reported that a single mutation, H374Q, in

Aralia cordata UDP-galactose:anthocyanin galactosyltransfer-

ase (corresponding to Gln-382 in UGT71G1) switched the pre-

ferred donor from UDP-galactose to UDP-glucose and

suggested that the last residue, Gln or His, in this signature motif

would define the sugar donor specificity. However, the Scutel-

laria baicalensisUDP-glucose:flavonoid glucosyltransferasemu-

tant Q382H does not acquire galactosyltransferase activity,

suggesting that other residues might also play roles in sugar

recognition and specificity in plant UGTs. In the structure of

UGT71G1 bound with UDP-glucose, the O29 and O39 atoms of

the glucose ring of the donor form hydrogen bonds with the NE2

atom of Gln-382 and the OE1 atom of Glu-381, respectively. The

O49 atom of the glucose ring contacts both the OE2 atom of Glu-

381 and the N atom of Trp-360. If a UDP-galactose is placed in

the position of the UDP-glucose in the structure of UGT71G1, the

O49 atomof galactose points away from theOE2atomofGlu-381

and can no longer form a hydrogen bond. This suggests that Glu-

381 may be a key residue for sugar recognition and specificity.

A structural model for catalysis by plant UGTs has been

proposed based on a modeling and mutagenesis study of

UGT73A5, a betanidin glucosyltransferase from Dorotheanthus

bellidiformis (Hans et al., 2004). However, the low identity

(;14%) between UGT73A5 and the bacterial GtfB used as the

basis for the homology modeling made it difficult to model

regions with insertion or deletion of amino acid residues. Nev-

ertheless, His-22 was identified as a key residue for enzyme

activity, although it was oriented differently from that shown in

the crystal structure of UGT71G1, such that the acceptor binding

pocket was predicted to be in a different location from that

suggested by our docking results. The position of the binding

pocket for the sugar donor was predicted correctly, but the sugar

Figure 6. The Putative Acceptor Binding Pocket.

Stereo diagram showing quercetin (A) and hederagenin (B) docked into the proposed binding pockets. Quercetin, hederagenin, and UDP-glucose are

shown as ball-and-stick models. Some protein residues in the acceptor binding pocket are labeled and shown in cyan as bond models. Distances (A)

between the OH group of acceptors and the atom NE2 of His-22 or the atom C19 of UDP-glucose are labeled and indicated with dashed lines.

3148 The Plant Cell

Figure 7. Comparison of the Donor and Acceptor Regions of Functionally Characterized Plant UGTs.

Sequence alignment of 39 plant UGTs, showing the acceptor binding region (first three fragments: 1 to 55, 81 to 95, and 117 to 151) and the donor

binding region (last two fragments: 282 to 286 and 339 to 382). Identical residues are highlighted, and similar residues are enclosed in boxes. Residues

His-22 and Asp-121 are marked with asterisks. The alignment was performed using ClustalX (Thompson et al., 1997). The plant UGTs includeMedicago

truncatula UGT71G1 and UGT73K1; Allium cepa UGT73G1 and UGT73J1; Aralia cordata GaT; Bellis perennis UGAT; Brassica napus SGT1;

Catharanthus roseus UGT2; Citrus maxima 1,2RhaT; Crocus sativus UGTCs2; Dorotheanthus bellidiformis UGT73A5; Gentiana triflora 39GT;

Glycyrrhiza echinata UGT73F1; Nicotiana tabacum GT1a, GT2, and GT3; Phaseolus lunatus ZOG1; Phaseolus vulgaris ZOX1; Sorghum bicolor

UGT85B1; Stevia rebaudiana UGT74G1, UGT76G1, and UGT85C2; and Zea mays cisZOC1 and cisZOG2. All others are from Arabidopsis

thaliana.

was predicted to lie in the opposite orientation to that revealed

from our crystal structure, and His-370 (His-357 in UGT71G1)

interacted with the phosphates rather than the uracil ring of the

donor molecule. Based on the modeling study, Glu-394 was

predicted to be part of a catalytic triad. However, as revealed in

the crystal structure of UGT71G1, Glu-381 (corresponding to

Glu-394 in UGT73A5) contacts the glucose moiety of the donor

and plays an important role in sugar recognition and specificity.

Rather thanGlu-381, the highly conserved Asp residue (Asp-121)

located in the acceptor binding pocket of UGT71G1 may be

essential for enzyme catalysis.

In the model revealed by our crystal structures, Asp-121

interacts with His-22 at a distance of ;3 A. His-22 is close to

the glucose ring of the sugar donor and the docked acceptor.

The imidazole group of His-22 acts as a general base to depro-

tonate an OH group on the acceptor molecule and transfers the

proton to Asp-121. The deprotonated acceptor, as a nucleophilic

oxyanion, can then attack at the C19 carbon center of the UDP-

glucose from the opposite side of the sugar ring, with displace-

ment of the UDP moiety. The crystal structure of UGT71G1

presented here thus provides a basis for understanding the

catalytic mechanism for glycosyl transfer and also provides

a starting point for homology modeling of diverse plant UGTs.

Substrate Specificity in the Plant UGT Superfamily

Most of the residues in direct contact with donormolecules, such

as Trp-339, Gln-342, His-357, and Glu-365, are conserved in

all 39 plant UGTs functionally characterized to date (Figure 7). In

the position corresponding to Glu-381 of UGT71G1, Asp is

frequently observed, which may enter into a similar interaction

with the donor. At the position corresponding to Gln-382 of

UGT71G1, Gln is conserved in glucosyltransferases, His is

present and conserved in galactosyltransferases, and an Asn

residue is observed in Arabidopsis UGT78D1, which uses UDP-

rhamnose as the sugar donor. Residues at these two positions

interact with the sugar moiety of the donor and therefore would

be predicted to be critical for sugar specificity.

The apparent lack of stringent structural requirements for

acceptor binding and catalysis is consistent with the evolution of

varied and broad substrate specificities of many plant GTs (Vogt

and Jones, 2000; Bowles et al., 2005). Of five UGTs that we have

characterized recently from M. truncatula (Achnine et al., 2005),

all have multiple in vitro substrates, suggesting multiple potential

in vivo substrates. UGT71G1 can catalyze the glucosylation of

two triterpenes (medicagenic acid and hederagenin), the flavonol

quercetin, and the isoflavones genistein and biochanin A. For

assessment of in vivo function, therefore, it is necessary to

understand which potential substrates are present in the cell

types in which the enzyme is expressed. In the case of methyl

jasmonate–induced Medicago cell suspension cultures, such

analysis points to UGT71G1 glycosylating medicagenic acid

rather than the catalytically preferred substrates biochanin A or

quercetin (Achnine et al., 2005).

When considered in terms of the multiple products formed

from each potential substrate, such apparent promiscuity raises

two important questions: why do higher plants possess so many

different UGTs, and how are their activities regulated in vivo to

Figure 8. Glucosylation of Quercetin by Recombinant UGT71G1.

(A) HPLC trace showing the substrate quercetin (Q) and the five products (numbered 1 to 5). Solid line, complete reaction mixture; dashed line,

standards for quercetin (Q), quercetin-3-O-glucoside (peak 3), and quercetin-49-O-glucoside (peak 4). mAU, milli-absorption unit.

(B) UV absorption spectra of quercetin (Q) and products 1 to 5.

3150 The Plant Cell

allow for the production of a more limited number of products

than suggested by their in vitro substrate/product specificities?

The importance of UGTs for the detoxification of xenobiotics

(allelochemicals before the human introduction of synthetic

herbicides and pesticides) may have driven the evolution of

new forms of the enzyme differing in residues around the

acceptor binding pocket.

It has been suggested that UGTsmay be associated in enzyme

complexes with the early enzymes of the pathway in which they

are involved, and such metabolic channeling would essentially

limit a particular GT to a specific pathway irrespective of the

enzyme’s in vitro substrate specificity (Jorgensen et al., 2005).

The presence and/or nature of physical interactions between any

UGT and a related biosynthetic enzyme remain unknown. The

structures described in this work provide a basis for the explo-

ration of such interactions.

METHODS

Expression and Purification of UGT71G1

The UGT71G1 cDNA fromMedicago truncatula (Achnine et al., 2005) was

cloned into the pET28a expression vector (Novagen) with a hexahistidine

tag and a thrombin cleavage site. Escherichia coli BL21(DE3) cells

transformed with the plasmid were grown at 378C in Luria-Bertani

medium containing 50 mg/mL kanamycin until A600 ¼ 0.6 to 0.8. Cultures

were induced with 0.5 mM isopropyl 1-thio-b-galactopyranoside and

grown overnight at 168C. Cells were pelleted and resuspended in lysis

buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, and

10 mM b-mercaptoethanol). After lysis with a French press and centri-

fugation at 12,000 rpm at 48C for 20min, Ni2þ-nitrilotriacetic acid agarose

was added to the supernatant containing the target proteins. After

incubation for 40 to 60 min, the mixture was transferred into a disposable

column and washed extensively with lysis buffer (;50 column volumes).

The His-tagged proteins were eluted with elution buffer (50 mM Tris-HCl,

pH 8.0, 500 mM NaCl, 250 mM imidazole, and 10 mM b-mercaptoetha-

nol). Incubationwith biotinylated thrombin during overnight dialysis at 48C

against 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 10 mM b-mercap-

toethanol cleaved the N-terminal His tag. Dialyzed proteins were in-

cubated with streptavidin agarose and Ni2þ-nitrilotriacetic acid agarose

to remove thrombin and the cleaved His tag, respectively. The proteins

were further purified on Resource Q and Superdex-200 gel filtration

columns (AmershamPharmacia Biotech) and concentrated to 5mg/mL in

10 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 5 mM b-mercaptoethanol.

Se-Met–substituted UGT71G1 was prepared by expressing the re-

combinant protein in E. coli B834(DE3) cells (Novagen) grown in M9

minimal medium supplemented with Se-Met (Doublie, 1997). The pro-

cedure for expression and purification was similar to the procedure

described above for the native protein.

Crystallization and Data Collection

Crystals of UGT71G1 were grown from hanging drops by the vapor

diffusion method. UGT71G1 protein at a concentration of 5 mg/mL was

mixed with 5 mM UDP-galactose and 5 mM quercetin at a 2:1 (v/v) ratio,

then mixed with an equal volume of reservoir solution containing 40%

(w/v) polyethylene glycol 3350, 0.2 M ammonium acetate, and 0.1 M

sodium citrate, pH 5.6. The mixture was equilibrated over the reservoir

solution at 208C. Crystals typically grew over 2 to 5 d with dimensions of

0.1 3 0.1 3 0.2 mm.

Before data collection, the crystals were flash-cooled to �1808C. Data

from a native protein crystal were measured to 2.0 A with a Raxis IVþþ

image plate detector and a RU3H x-ray source. The crystal belonged to

space group P21 (a ¼ 53.7 A, b ¼ 90.6 A, c ¼ 101.8 A, b ¼ 102.68). There

were two molecules per crystallographic asymmetric unit, with 47%

solvent content and a Matthews coefficient VM of 2.6 A3/D. A 2.4-A MAD

data set from a Se-Met derivative crystal, using three wavelengths, was

collected at the Gulf Coast Protein Crystallography Consortium beamline

using a MAR CCD detector.

Crystals of UGT71G1 complexed with UDP-glucose were obtained by

soaking the crystals described above with mother liquid containing

saturated UDP-glucose. Five changes of this solution were made during

the 3-d soaking period, the last being a few hours before freezing the

crystal for data collection. A 2.6-A diffraction data set was collected with

a Raxis IVþþ image plate detector and a RU3H x-ray source.

All data sets were processed using denzo and scalepack or HKL2000

(Otwinowski and Minor, 1997).

Structure Determination and Refinement

The structure of UGT71G1 was determined using the MAD method. The

MAD data (40 to 2.4 A) were analyzed with SOLVE (Terwilliger and

Berendzen, 1999), and 20 of 24 Se sites were located, yielding an overall

figure of merit of 0.44. The program RESOLVE (Terwilliger, 2000) was

used for electron density modification, phase extension to 2.0 A by

combining with the native data, and automated model building. Interac-

tive model building and crystallographic refinement were performed

using the programs O (Jones et al., 1991) and CNS (Brunger et al., 1998),

respectively. A bulk solvent correction was applied. B factors were

refined individually for the structure of UGT71G1 complexed with UDP at

2.0 A, and grouped B factor refinement was performed for the structure of

UGT71G1 complexed with UDP-glucose at 2.6 A. Water molecules were

added with Arp/wArp (Lamzin et al., 2001) and checked manually for

inclusion. In themodels, the first two and seven amino acid residues in the

N terminus were not observed inmolecules A andB, respectively, and the

last two residues in the C terminus were disordered in both molecules.

The program PROCHECK (Laskowski et al., 1993) was used to check

themodels. All backbone f-c torsion angles are within allowed regions of

the Ramachandran plot.

Molecular Docking

The automated docking program GOLD was used to dock acceptors into

the UGT71G1 active site (Jones et al., 1997). Default genetic algorithm

parameters for controlling the operation of the docking process were

used. All docking calculations were restricted to the predicted binding

pocket by defining the active site with residue His-22. For quercetin,

medicagenic acid, and hederagenin, there are several potential glyco-

sylation sites, and a distance constraint was used to define each potential

site for calculation. GOLDscore was used to identify the lowest energy

docking results. The hydrogen bonds and van der Waals contacts

between ligands and enzyme were analyzed to identify the optimal

binding mode.

Mutagenesis and Enzyme Assay

Site-directed mutants of UGT71G1 were constructed using the Quik-

Change strategy (Stratagene). The mutant proteins were expressed and

purified using the same procedures described above for native protein

except for the thrombin cleavage. Enzyme assay was performed essen-

tially according to a reported method (Lim et al., 2003). Each GT activity

assaymix (200mL) contains 2mg of recombinant protein, 50mMTris-HCl,

pH 7.0, 14 mM b-mercaptoethanol, 5 mM UDP-glucose, and 1 mM

quercetin. For mutants, increasing concentrations of substrates (2 mM

quercetin and 10 mMUDP-glucose, or 5 mM quercetin and 25 mMUDP-

glucose) and proteins (4 mg) were also used. The reaction was performed

UDP Glycosyltransferase Structure 3151

at 308C for 1 h and stoppedby the addition of 20mL of trichloroacetic acid,

quick-frozen, and stored at �208C before HPLC analysis with a Waters

Spherisorb 5u ODS2 C18 reverse-phase column (250 3 4.6 mm) on an

AgilentHP1100HPLCdevice equippedwith anautosampler, a quaternary

pump, and a diode array detector. Elution was as described previously

(Achnine et al., 2005).

Accession Numbers

The coordinates and structure factors for the structures of UGT71G1 in

complexes with UDP and UDP-glucose have been deposited to the PDB

with codes 2ACV and 2ACW, respectively. The plant UGTs included in the

alignment (Figure 7) and their accession numbers are as follows:

Medicago truncatula UGT71G1 (accession number AAW56092) and

UGT73K1 (AAW56091); Allium cepa UGT73G1 (AAP88406) and

UGT73J1 (AAP88407); Aralia cordata GaT (BAD06514); Bellis perennis

UGAT (BAD77944); Brassica napus SGT1 (AAF98390); Catharanthus

roseus UGT2 (BAD29722); Citrus maxima 1,2RhaT (AAL06646); Crocus

sativus UGTCs2 (AAP94878); Dorotheanthus bellidiformis UGT73A5

(CAB56231); Gentiana triflora 39GT (BAC54092); Glycyrrhiza echinata

UGT73F1 (BAC78438); Nicotiana tabacum GT1a (BAB60721), GT2

(BAB88935), and GT3 (BAB88934); Phaseolus lunatus ZOG1

(AAD04166); Phaseolus vulgaris ZOX1 (AAD51778); Sorghum bicolor

UGT85B1 (AAF17077); Stevia rebaudiana UGT74G1 (AAR06920),

UGT76G1 (AAR06912), and UGT85C2 (AAR06916); Zea mays cisZOC1

(AAK53551) and cisZOG2 (AAL92460); and Arabidopsis thaliana

UGT71C1 (AAC35226), UGT72B1 (AAB61023), UGT72E2 (BAA97275),

UGT73C5 (AAD20156), UGT73C6 (AAD20155), UGT74B1 (AAC00570),

UGT74F2 (AAB64024), UGT84A2 (BAB02351), UGT84A1 (CAB10326),

UGT84B1 (AAB87119), UGT76C1 (BAB10792), UGT76C2 (AAN28835),

UGT78D1 (AAF19756), UGT85A1 (AAF18537), and UGT91B1

(BAA98174).

ACKNOWLEDGMENTS

We thank S. Liu for initial protein purification and crystallization

screening, J. Lin for technical assistance, L.W. Sumner, Z. Lei, and D.

Huhman for mass spectrometric analysis, N. Duke (Structural Biology

Center) and I. Koshelev (Industrial Macromolecular Crystallography

Association–Collaborative Access Team) at the Advanced Photon

Source (Argonne National Laboratory) for assistance with data collection

of initial crystals, H. Bellamy and D. Neau of the Gulf Coast Protein

Crystallography Consortium beamline of the Center for Advanced

Microstructures and Devices (Louisiana State University) for help with

Se-Met MAD data collection, and T.M.T. Hall and P. Xu for critical

reading of the manuscript. This work was supported by National

Science Foundation Grant 0416883 and the Samuel Roberts Noble

Foundation.

Received June 8, 2005; revised August 21, 2005; accepted September

19, 2005; published October 7, 2005.

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3154 The Plant Cell

DOI 10.1105/tpc.105.035055; originally published online October 7, 2005; 2005;17;3141-3154Plant Cell

Hui Shao, Xianzhi He, Lahoucine Achnine, Jack W. Blount, Richard A. Dixon and Xiaoqiang Wangtruncatula

MedicagoCrystal Structures of a Multifunctional Triterpene/Flavonoid Glycosyltransferase from

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