Characterization and synthetic application of a novel β1,3-galactosyltransferase from Escherichia coli O55:H7

Article abstract of DOI:10.1016/j.bmc.2009.06.005

A β1,3-galactosyltransferase (WbgO) was identified in Escherichia coli O55:H7. Its function was confirmed by radioactive activity assay and structure analysis of the disaccharide synthesized with the recombinant enzyme. WbgO requires a divalent metal ion,

Full text of DOI:10.1016/j.bmc.2009.06.005

Bioorganic & Medicinal Chemistry 17 (2009) 4910–4915
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Characterization and synthetic application of a novel
b1,3-galactosyltransferase from Escherichia coli O55:H7
Xian-wei Liu a,b, Chengfeng Xia b,c, Lei Li a,b, Wan-yi Guan a,b, Nicholas Pettit b, Hou-cheng Zhang a,
b
Min Chen a, , Peng George Wang
*
a National Glycoengineering Research Center and The State Key Laboratory of Microbial Technology, Shandong University, LuNeng Keji Dasha B 604, Shanda Nanlu 29-1, Jinan,
Shandong 250100, China
b Departments of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43202, USA
c State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A b1,3-galactosyltransferase (WbgO) was identified in Escherichia coli O55:H7. Its function was confirmed
by radioactive activity assay and structure analysis of the disaccharide synthesized with the recombinant
enzyme. WbgO requires a divalent metal ion, either Mn2+ or Mg2+, for its activity and is active between
pH 6.0–8.0 with a pH optimum of 7.0. N-acetylglucosamine (GlcNAc) and oligosaccharides with GlcNAc at
the non-reducing end were shown to be its preferred substrates and it can be used for the synthesis of
type 1 glycan chains from these substrates. Together with a recombinant bacterial GlcNAc-transferase,
benzyl b-lacto-N-tetraoside was synthesized with the purified WbgO to demonstrate the synthetic utility
of WbgO.
Received 15 April 2009
Revised 2 June 2009
Accepted 3 June 2009
Available online 9 June 2009
Keywords:
O-Antigen, Galactosyltransferase
Type 1 chain
Ó 2009 Elsevier Ltd. All rights reserved.
Lacto-N-tetraose
1. Introduction
The glycosyltransferase-catalyzed synthesis of Galb1,3GlcNAc
requires a b1,3-galactosyltransferase (b1,3-GalT). Various genes
Lacto-N-biose I disaccharide (2-acetamindo-2-deoxy-3-O-b-
D
-
encoding b1,3-GalT using GlcNAc-R as an acceptor have been identi-
fied in bothhuman and other high eukaryotesand the corresponding
b1,3-GalThavebeencharacterized.12,13 AbacterialGlcNAcb1,3-GalT
has also been identified in Streptococcus agalactiae but has yet to be
biochemically characterized.14 A recombinant human b1,3-GalT has
beenusedto synthesize multi-mgquantitiesof lacto-N-biose I disac-
charide and its analogs.15 But this enzyme was expressed in insect
cells which were costly and inefficient. Herein we reported a novel
bacterial enzyme expressed in Escherichia coli which provides a bet-
ter source for this type of glycosyltransferase. It is also worthwhile to
mention that an alternative enzymatic route has been reported on
kilogram-scale production of lacto-N-biose I disaccharide with a lac-
to-N-biose phosphorylase,16 however practical methods for the pro-
duction of other Galb1,3GlcNAc containing oligosaccharides such as
LNT are still lacking.
E. coli O55:H7 strains are classified as enteropathogenic E. coli
(EPEC). EPEC is the most common cause of prolonged diarrhea in
infants in developing countries.17,18 In addition, E. coli O55:H7 is
known to be the closest relative of enterohaemorrhagic E. coli
(EHEC) O157:H7,19 which can cause non-bloody diarrhea, bloody
diarrhea and hemolytic uraemic syndrome diarrhea and is further-
more one of the dominant intestinal pathogens in developed coun-
tries.20 The O-polysaccharide structure of E. coli O55:H7 has been
determined,21 which is a repeating structure of a pentasaccharide
containing a Galb1,3GlcNAc motif (Fig. 1). Among the five different
galactopyranosyl-b- -glucopyranose, Galb1,3GlcNAc), also known
D
as type 1 chain, is the precursor of a number of important carbohy-
drate epitopes in human body, such as Lewis A, Lewis B and sialyl
Lewis A (SLea) antigens.1 These epitopes are involved in a variety of
biological processes including pathogen adhesion,2 fertilization3
and tumor metastasis.4 For example, as a fundamental ligand for
E-Selectin, SLea mediates the trafficking and recruitment of
blood–borne leukocytes during normal and pathological inflamma-
tory responses.5 SLea is also considered as a tumor-associated anti-
gen6,7 and becomes the most frequently applied serum tumor
marker for diagnosis of cancers in the digestive organs.8
The Galb1,3GlcNAc motif is also present in other important
oligosaccharides, such as lacto-N-tetraose (Galb1,3GlcNAcb1,
3Galb1,4Glc, LNT). LNT and their fucosylated and/or sialylated
derivativesare among someof themajor componentsin humanmilk
oligosaccharides.9 Some of them share similar structures with the
glycan ligands recognized by human pathogens and are proposed
to protect infants against these pathogens via competitive inhibition
of the binding between the pathogens and the epithelial ligands.10,11
Thus, usage of these oligosaccharides as anti-infection therapeutics
can be logically suggested.
* Corresponding author. Tel./fax: +86 531 88366078.
E-mail address: chenmin@sdu.edu.cn (M. Chen).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.005

X. Liu et al. / Bioorg. Med. Chem. 17 (2009) 4910–4915
4911
Table 1
OH
O
OH
OH
HO
HO
The radioactivity reading from galactosyltransferase activity assay for GST-fused
WbgO, WbgP and WbgMa
O
HO
OH
O
O
OH
O
O
Acceptors
Enzymes
AcHN
HO
O
O
O
None
WbgO
WbgP
WbgM
n
AcHN
O
OH
None
GlcNAc
GalNAc
57
—
—
252
1053
499
150
161
167
161
137
158
HO
6)GlcNAcβ(1,3)Galα(1,3)GalNAcβ(1
a
The activity assays were performed in a total volume of 50
l
L with 50 mM Tris–
I β(1,3)
Gal
HCl (pH 7.5), 5 mM MnCl2, 0.3 mM UDP-Gal, 0.3
2 mM acceptor and 10 L purified protein (about 5 lg protein) at room temperature
overnight. The acceptor and/or enzymes were omitted in the control reaction. The
total radioactivity was 9529 cpm.
l
M UDP-[6-3H]Gal (10,000 cpm),
l
I α(1,2)
Col
Figure 1. Structure of E. coli O55:H7 O-antigen repeating unit. The glycosidic bond
catalyzed by b1,3-galactosyltransferase (WbgO) is indicated by an arrow. Col stands
for colitose.
fied by GST affinity chromatography (Fig. 2). Then GalT activity of
these enzymes was explored using GlcNAc and N-acetylgalactos-
amine (GalNAc) as acceptors. Among them, only GST-WbgO
showed significant GalT activity towards GlcNAc and GalNAc
(Table 1). To further determine the function of WbgO, a small scale
reaction was performed with p-nitrophenyl b-GlcNAc 1 as the
sugar acceptor (Scheme 1). The structure of the resultant p-nitro-
phenyl lacto-N-bioside (lacto-N-biose-b-O-pNP) 2 was analyzed
by ESI-MS and NMR spectroscopy. The formation of the b1,3 link-
age was confirmed by both 1D and 2D NMR. According to the
HMQC and 13C NMR, the d value of C-3 down shifted to 81.9, which
indicated that this hydroxyl was involved in a glycosidic linkage.24
The assignment of the anomeric carbon of terminus galactose with
d value of 103.6 shows that the anomeric configuration is b, by
comparison with the published data.24 The determination of the
configuration was also supported by the 1H NMR, with the d value
of 4.45 and coupling constant as 7.7 Hz of H-1.25
Expression and purification of WbgO with His6-tag failed using
either plasmid pET-15b or pET-22b. On the contrary, expression
and purification with pGEX-4T-1 as a GST-fused protein succeeded.
The fusion protein GST-WbgO was produced with an N-terminus
GST and was purified to homogeneity in one-step GST affinity chro-
matography (Fig. 2). The purified GST-WbgO showed a molecular
weight between 50 and 64 kDa on SDS–PAGE which was consistent
withthetheoreticalvalue(56.894 kDa). Totally1.6 mgof GST-WbgO
can be purified from 1-L batch fermentation with a specific activity
of 67.5 mU mgꢀ1 with GlcNAc as the acceptor. One unit of GST-
WbgO was defined as the amount of the enzyme that catalyzes the
glycosidic bonds in the pentasaccharide, four are catalyzed by spe-
cific glycosyltransferases during the assembly of the O-antigen
repeating units. The last glycosidic bond is formed when the
repeating units are polymerized into O-antigen.
The O-antigen biosynthesis gene cluster of E. coli O55:H7 has
been sequenced and four postulated glycosyltransferases are pro-
posed to be responsible for the assembly of the O-antigen repeat-
ing unit, which are WbgO, WbgP, WbgM and WbgN.22 The WbgN
protein belongs to GT family 11,23 which should be a colitosyl-
transferase, however the activities of other three glycosyltransfer-
ases are unpredictable relying only on bioinformatic methods. In
this study, we distinguished the b1,3-GalT among these glycosyl-
transferases and further characterized this enzyme. Not only does
this study enrich the basic knowledge for bacterial GlcNAc b1,3-
GalT, but it also provides an alternative enzymatic route for the
synthesis of Galb1,3GlcNAc containing oligosaccharides.
2. Results and discussion
To identify the b1,3-GalT among the three glycosyltransferases
from E. coli O55:H7, we cloned wbgO, wbgP, and wbgM genes into
a glutathione S-transferase (GST) fusing expression vector, pGEX-
4T-1. The recombinant enzymes were expressed in E. coli and puri-
1
2
3
4
formation of 1 lmol of the sugar product per minute. Cleavage of
kDa
148
GST tag from the fusion protein with thrombin was unsuccessful.
The cleavage efficiency was low and the resultant WbgO showed
undetectable activity. Like many other glycosyltransferases from
bacteria,26,27 even without transmembrane regions, WbgO may be
associated with the membrane. It is also possible that WbgO forms
protein complex with other enzymes involved in O-antigen biosyn-
thesis. Without those associations in physical environment, the sta-
bility and activity may not be able to maintain. Apparently the fusion
with GST stabilized WbgO and assisted its activity under artificial
conditions. Therefore, GST-WbgO was used for biochemical charac-
terization and enzymatic synthesis in this study.
98
64
50
36
Figure 2. SDS–PAGE analysis of purified GST-fused WbgO, WbgP and WbgM. Lanes:
1, protein standards (the molecular weights are indicated in kDa on the left side); 2,
GST-WbgO; 3, GST-WbgP; 4, GST-WbgM.
The effects of pH buffer systems and divalent metal ions on the
activity of WbgO were then investigated. As shown in Figure 3A,
OH
OH
UDP-Gal UDP
OH
OH
O
O
HO
HO
O
O
NO2
O
NHAc
1
NO2
O
HO
HO
GST-WbgO
NHAc
HO
2
Scheme 1. Synthesis of p-nitrophenyl lacto-N-bioside with recombinant WbgO.

4912
X. Liu et al. / Bioorg. Med. Chem. 17 (2009) 4910–4915
fer systems with the HEPES buffer system being the most efficient.
A
The enzyme displayed comparable activity in MES buffer compared
with that in HEPES buffer at the same pH value. In contrast, the
enzyme presented lower activity in the Tris–HCl buffer than that
in HEPES buffer under the overlapping pH range (pH 7.5 and 8.0).
Sodium citrate buffer was found to exert a strong inhibitory effect
on the activity of the enzyme as indicated by the distinct lower
activity in this buffer at pH 6.5 than that in the MES buffer at the
same pH value.
The activity of GST-WbgO was found to be absolutely depen-
dent on specific divalent metal ions (Fig. 3B). Both MnCl2 and
MgCl2 significantly supported its activity at the concentration of
5 mM. The activity with MgCl2 was about 90% of that with MnCl2.
None of the other divalent metal cations (Ca2+, Cu2+, Co2+, Ni2+ and
Zn2+) were efficient cofactors for this enzyme. In addition, the en-
zyme showed no activity in the absence of metal ion or in the pres-
ence of EDTA. The dependence of a metal ion for its activity
indicates that WbgO belongs to the GT-A superfamily.28 The exis-
tence of a DXD motif which is responsible for coordinating the
divalent metal ion to bind to the sugar nucleotide is a common
structure feature of the GT-A superfamily. Consistent with this fea-
ture, a D99S100D101 motif was found present in WbgO. This motif is
conserved among homologous glycosyltransferases as identified by
a BLAST search from the NCBI database (Fig. 4).29
A series of acceptors including monosaccharides (GlcNAc, Gal-
NAc, glucose and galactose) and oligosaccharides (lacto-N-triose,
globotetraose, isoglobotetraose and GlcNAcb1,3Galb1,4Glc) were
used to explore the acceptor specificity of GST-WbgO. The result
(Table 2) suggests that GlcNAc and oligosaccharides with GlcNAc
at the non-reducing end (GlcNAc-R) are the predominant accep-
tors. Furthermore, the trisaccharide lacto-N-triose was preferred
by the enzyme with a twofold increase of enzymatic activity over
GlcNAc. Lacto-N-triose is the best among all the acceptors tested
in our assay, illuminating the potential of WbgO as a catalyst for
the synthesis of biologically important oligosaccharides. GalNAc
and oligosaccharides with GalNAc at the non-reducing end are also
tolerated by the enzyme displaying relative activities 16–40% that
of the monosaccharide GlcNAc. This phenomenon suggests a lim-
ited influence of the configuration of the 4-hydroxyl group on
acceptor substrate binding. On the other hand, glucose and galact-
ose do not serve as substrates for this enzyme, which indicates the
70
60
50
so
MEES
HE
o
d
d
i
S
i
u
u
m
m ci
i
t
t
r
r
a
a
t
te
e
40
E
PP
EE
SS
Trriiss--HHCCll
30
20
10
0
5.0
6.0
7.0
8.0
9.0
pH
B
60
50
40
30
20
10
0
4
2
2
2
4
2
2
Figure 3. The effects of pH and divalent metal ions on the b1,3-galactosyltrans-
ferase activity of GST-WbgO. (A) The pH profile; (B) Effects of divalent metal ions.
the activity assay of GST-WbgO under different pH conditions
demonstrates that the enzyme catalyzes the galactosyl-transfer-
ring reaction from UDP-galactose (UDP-Gal) to an acceptor under
neutral condition (pH 6–8) with optimal activity at pH 7.0. There
was sharp decline in enzymatic activity below pH 6.0 or above
pH 8.0. In addition, GST-WbgO showed preference for certain buf-
Figure 4. Amino acid sequence alignment of segments from different b1,3-GalTs. The DXD conserved motif was indicated by a box. P.l.: Photorhabdus luminescens subsp.
laumondii TTO1 (Genbank accession NO.: NP_931977); E.c.: Escherichia coli (ABE98422); WbiP: Escherichia coli O127:H6 (YP_002329684); WbgO: Escherichia coli O55:H7
(AF461121); S.e.: Salmonella enterica subsp. salamae serovar Greenside (AAV34525); L.n.: Lutiella nitroferrum 2002 (ZP_03696917); C.M.p.: Candidatus Methanosphaerula
palustris E1-9c (YP_002467214); P.a.: Prosthecochloris aestuarii DSM 271 (YP_002015057); Ps: Pseudovibrio sp. JE062 (YP_002680025).

X. Liu et al. / Bioorg. Med. Chem. 17 (2009) 4910–4915
4913
Table 2
OH
O
OH
O
HO
HO
Acceptor substrate specificity of purified GST-WbgO
O
Acceptorsa
Relative activity (%)
3
O
HO
OH
OH
GlcNAc
100
73
221
43
33
34
GlcNAcb-O-pNP
Lacto-N-triose-b-Obnb
GalNAc
Globotetrose-N3
Isoglobotetrose-OMe
GalNAcb1,3Galb1,4Glc
Glucose, galactose
UDP-GlcNAc
LgtA
UDP
OH
O
OH
O
OH
O
HO
O
16
NDc
HO
O
4
O
HO
HO
a
The reactions were performed in a total volume of 50
l
L containing 50 mM
NHAc
OH
OH
UDP-Gal
UDP
HEPES buffer (pH 7.0), 5 mM MnCl2, 0.3 mM UDP-Gal, 0.3
(10,000 cpm), 2 mM acceptor and 4 g (35 pmol) enzymes. The reactions were
lM
UDP-[6-3H]Gal
l
incubated at 37 °C for 1 h. The acceptor was omitted in the control reaction.
GST-WbgO
b
Glycan sequences: lacto-N-triose-b-OBn, GlcNAcb1,3Galb1,4Glcb-OBn; globo-
tetrose-N3, GalNAcb1,3Gal
3Gal 1,3Galb1,4Glcb-OMe.
ND: not detectable.
a1,4Galb1,4Glcb-N3; isoglobotetrose-OMe, GalNAcb1,
a
c
OH
O
OH
HO
OH
O
OH
O HO
HO
HO
O
O
O
NHAc
O
5
O
HO
OH
OH
OH
vital role of the 2-acetamido group for the acceptor binding of
WbgO.
Scheme 2. Synthesis of benzyl b-lacto-N-tetraoside with recombinant LgtA and
The kinetic parameters of WbgO were determined for both the
donor (UDP-Gal) and some acceptors (GlcNAc, GalNAc and lacto-N-
triose). The result is found in Table 3. The apparent Km value for
UDP-Gal is 3.4 mM which is comparable with galactosyltransferas-
es from mammalians.13,30 The difference of kinetic parameters
among different acceptors is in line with the result from the accep-
tor substrate specificity study. The Km value for lacto-N-triose
(0.055 mM) is about one-sixth of that for both GlcNAc
(0.323 mM) and GalNAc (0.327 mM), and the increased binding
affinity for lacto-N-triose can explain the relative activities be-
tween the three acceptors. The activity difference between GlcNAc
WbgO.
and GalNAc appears mainly due to differences in Vmax
.
To demonstrate the synthetical application of WbgO, LNT-b-
OBn (compound 5) was synthesized from lactose-b-OBn (com-
pound 3) in two steps with a GlcNAc-transferase from Neisseria
meningitidis (LgtA)31 and GST-WbgO (Scheme 2). At first, the syn-
thesis of lacto-N-triose-b-OBn (Compound 4) from lactoside 3 with
LgtA was carried out with a yield of 86% (19 mg). Then 8 mg resul-
tant trisaccharide 4 was used to synthesize tetrasaccharide 5.
Totally 8.7 mg product was obtained after purification by Bio-Gel
P-2 column and lyophilization with a yield of 87%. The product
was confirmed by ESI-MS and NMR analysis. It is worth to note that
the conversion of 4 to 5 was completed from the TLC detection
(Fig. 5). The decrease of yield should be resulted from the loss dur-
ing purification. The yield can be improved as the synthesis scale is
enlarged. Since the Galb1,3GlcNAcb-R motif is the basic structure
for type I oligosaccharides,1 WbgO provides an alternative syn-
thetic method for these oligosaccharides. In addition, WbgO also
displays remarkable galactosyl-transferring activity towards
GalNAc containing oligosaccharides (globotetraose and isoglobo-
tetraose) compared with LgtD, which has been previously used
for the synthesis of these oligosaccharides.32 The remarkable activ-
ity toward both oligosaccharides with GlcNAc at the non-reducing
end and those with GalNAc at the non-reducing end makes WgbO a
more attractive tool for biotechnological applications.
1
2
3
4
5
Figure 5. TLC detection of GST-WbgO catalyzed lacto-N-tetraoside synthesis
reaction. (1) UDP-Gal; (2) lacto-N-triose-b-OBn 4; (3) 4 h reaction; (4) 8 h reaction
and (5) 12 h reaction. Arrow indicates the tetrasaccharide product 5.
3. Conclusion
We identified and characterized a b1,3-galactosyltransferase
(WbgO) from E. coli O55:H7, which uses GlcNAc and oligosaccharides
with GlcNAc at the non-reducing end as its preferred acceptors. This
enzyme was overexpressed in E. coli BL21 (DE3) with a GST-fused at
its N-terminus and purified by one-step affinity chromatography.
WbgO was classified into glycosyltransferase family 2 (GT2),
one of the largest GT families. There are many bacterial b1,3-galac-
tosyltransferases identified in this family but few of them use Glc-
NAc-OR as the predominant acceptor. This is the second GlcNAc
b1,3-galactosyltransferase identified in bacteria however it is the
first at which has been biochemically characterized. Further more,
we demonstrated the potential applications of WbgO in the enzy-
matic synthesis of important oligosaccharides.
4. Experimental
4.1. Generation of glycosyltransferase expression constructs
Table 3
Apparent kinetic parameters for the b1,3-galactosyltransferase activity of GST-WbgO
The wbgO, wbgP and wbgM genes were amplified by PCR from
chromosomal DNA of E. coli O55:H7 using primers listed in Table
4. The PCR fragments were digested with BamH I/Xho I restriction
endonucleases and then ligated into the plasmid pGEX-4T-1 (GE
Healthcare Life Sciences, Piscataway, NJ, USA). The confirmed con-
structs were subsequently transformed into E. coli BL21 (DE3) for
protein expression.
Substrates
Km
(mM)
Vmax
kcat
kcat/Km
(pmol minꢀ1
)
(minꢀ1
)
(minꢀ1 mMꢀ1
)
UDP-galactose
GlcNAc
Lacto-N-triose
GalNAc
3.40
490
179
176
120
13.9
2.55
2.50
1.71
4.09
7.89
16.7
5.23
0.323
0.055
0.327

4914
X. Liu et al. / Bioorg. Med. Chem. 17 (2009) 4910–4915
Table 4
average values from duplicated assays were used to obtain the
Lineweaver–Burk plots. The apparent Km, Vmax and kcat values were
calculated accordingly.
Primers used for PCR cloning of wbgO, wbgP and wbgMa
Genes
wbgO
Primer sequences (50 to 30)
1
2
1
2
1
2
gcctGGATCCatgataatcgatgaagctgb
4.5. Synthesis of lacto-series glycoside 2, 4 and 5
cggaattCTCGAGtcactttatgtatttacagt
gcctGGATCCatgattgtgaaaacaataagtg
cggaattCTCGAGttataacttccggataaaaaccac
gcctGGATCCatggtaaaaattctgcatgtgcacc
cggaattCTCGAGtcaattatttaaatataacgactc
wbgP
The synthesis of lacto-N-biose-b-O-pNP 2 was performed in a
2.5 mL system containing 17 mg GlcNAcb-O-pNP 1, 35 mg UDP-
Gal, 5 mM MnCl2 and 1.2 mg GST-WbgO in 50 mM HEPES buffer
(pH 7.0). The reaction was incubated at room temperature for 48 h.
For lacto-N-triose-b-OBn 4 synthesis, 15 mg lactose-b-OBn 3,
24 mg UDP-GlcNAc and 5 mM MnCl2 were added into 50 mM Tris–
HCl buffer (pH 7.5) with 1 U of LgtA, a b1,3-GlcNAc-transferase from
Neisseriameningitidis.31 Thereactionwas allowedto proceedfor 12 h
at room temperature. LgtA was overexpressed with vector pET15bin
E. coli BL21 (DE3) and purified as previously described.31
WbgO catalyzed LNT-b-OBn 5 synthesis reaction was performed
in 50 mM HEPES buffer (pH 7.0) with 8 mg lacto-N-triose-b-OBn 4,
11.5 mg UDP-Gal, 5 mM MnCl2, and 0.8 mg GST-WbgO (total vol-
ume: 1.2 mL). The reaction was incubated at 37 °C for 12 h.
All the reactions were eventually heated in boiling water for
5 min. The resultant saccharides were purified by Dowex 1X8-
400 anion exchange resin and then Bio-Gel P-2 column (Bio-Rad
Laboratories, Hercules, CA, USA). All the reactions were monitored
by TLC as previously described.33
wbgM
a
Primer series 1 are forward primers and series 2 are reverse primers.
A BamH I restriction site (in capital) is in the forward primers and an Xho I site
b
(in capital) is in the reverse primers.
4.2. Expression and purification of glycosyltransferases
E. coli BL21 (DE3) strains harboring the recombinant plasmids
were grown in 1 L of LB medium at 37 °C. When OD600 reached
0.8, isopropyl-b-D-thiogalactopyranoside was added to a final con-
centration of 0.2 mM for induction. Protein expression proceeded
at 25 °C for 12 h. Cells were harvested by centrifugation at 5000g
at 4 °C for 15 min. Batch purification of GST-fused proteins with
Glutathione Sepharose 4B slurry (GE Healthcare Life Sciences)
was performed following the manufacturer’s instructions. Protein
expression and purification were analyzed by 12% SDS–PAGE.
Protein concentration was quantified by the Bradford assay using
Bio-Rad Protein Assay reagents (Bio-Rad, Hercules, CA, USA) with
standard solutions of bovine serum albumin.
4.6. Analysis of oligosaccharide products
4.3. Activity assays for glycosyltransferases
Products were characterized by 1D (1H and 13C) and 2D (COSY
and HMQC) NMR spectra and high resolution mass. The NMR spec-
tra were recorded on a Bruker Avance DRX500 NMR Spectrometer.
The ESI-MS was performed on a Bruker MicrOTOF mass spectrom-
eter. The oligosaccharide products were repeatedly dissolved in
D2O and lyophilized before the NMR spectra were recorded at
303 K in a 5 mm tube. All the electronic spectra are available in
the Supplementary data.
To determine the GalT activity of recombinant GST-fused
WbgO, WbgP and WbgM, the activity assays were performed in a
total volume of 50
0.3 mM UDP-Gal, 0.3
acceptor (GlcNAc or GalNAc) and 10
g protein) at room temperature overnight. For GST-fused WbgO
characterization, reactions were performed in a total volume of
50 L containing 50 mM HEPES buffer (pH 7.0), 5 mM MnCl2,
0.3 mM UDP-Gal, 0.3
UDP-[6-3H]Gal (10,000 cpm), 2 mM
acceptor and 4 g (70 pmol) enzymes. The reactions were incu-
bated at 37 °C for 1 h. The acceptor was omitted in the control
reaction. The reactions were quenched by adding 150 L of
l
L with 50 mM Tris–HCl (pH 7.5), 5 mM MnCl2,
UDP-[6-3H]Gal (10,000 cpm), 2 mM
L purified protein (about
lM
l
5
l
l
4.6.1. Lacto-N-biose-b-O-pNP
lM
1H NMR (500 MHz, D2O): d 8.21 (d, J = 9.3 Hz, 2H, PhNO2), 7.16 (d,
J = 9.3 Hz, 2H, PhNO2), 5.33 (8.5 Hz, 1H, H-1), 4.45 (d, J = 7.7 Hz, 1H,
H-1), 4.15 (dd, J = 10.4, 8.5 Hz, H-2), 3.93 (dd, J = 12.5, 2.1 Hz, 1H,
H-6a), 3.92 (dd, J = 10.3, 8.2, 1H, H-3), 3.89 (d, J = 3.4 Hz, 1H, H-4),
3.79 (dd, J = 12.5, 5.2 Hz, 1H, H-5), 3.75–3.68 (m, 4H, H-6b, H-5, H-
6), 3.66 (m, 1H, H-4), 3.62 (dd, J = 10.0, 3.3 Hz, 1H, H-3), 3.52 (dd,
J = 9.9, 7.7 Hz, 1H, H-2), 1.98 (s, 3H, NHCOCH3); 13C NMR
(125 MHz, D2O): d 175.0, 161.7, 142.7, 126.1, 116.6, 103.6, 98.4,
81.9, 75.8, 75.4, 72.5, 70.7, 68.6, 68.4, 61.1, 60.5, 54.4, 22.2; HRMS
(ESI): C20H28N2O13Na [M+Na]+, calcd 527.1484, found 527.1476.
l
l
10 mM EDTA. Dowex 1X8-400 anion exchange resin (Sigma–Al-
drich, St. Louis, MO) was then added as a water suspension
(0.8 mL, v/v = 1:1). After centrifugation, the supernatant (0.5 mL)
was collected in a 20-mL plastic vial to which 10 mL of Scintiverse
BD (Fisher Scientific, Pittsburgh, PA, USA) was added. The incorpo-
rated radioactivity was quantified with a Beckmann LS-3801 liquid
scintillation counter (Beckman Instruments, Fullerton, CA, USA).
Reactions under different pH conditions were performed in dif-
ferent buffer systems (sodium citrate, pH 5.0, 5.5, 6.0 and 6.5; MES,
pH 6.0, 6.5 and 7.0; HEPES, pH 7.0, 7.5 and 8.0; Tris–HCl, pH 7.5,
8.0, 9.0 and 9.5) at the concentration of 200 mM. To determine
the dependence of WbgO activity on metal ions, GlcNAc was used
as acceptor and the activities were assayed with different metal ion
salts (MnCl2, MgCl2, CaCl2, CuCl2, CoCl2, NiSO4 and ZnSO4) at the
concentration of 5 mM. The effect of EDTA was tested in the pres-
ence of 5 mM MnCl2 and 6 mM EDTA. Other conditions are as same
as those described above for GST-fused WbgO characterization.
4.6.2. Lacto-N-triose-b-OBn
1H NMR (500 MHz, D2O): d 7.45–7.36 (m, 5H, Ph), 4.90 (d,
J = 11.6 Hz, 1H, PhCH2), 4.73 (d, J = 11.6 Hz, 1H, PhCH2), 4.65 (d,
J = 8.5 Hz, 1H, H-1), 4.51 (d, J = 8.0 Hz, 1H, H-1), 4.39 (d,
J = 7.9 Hz, 1H, H-1), 4.10 (d, J = 2.8 Hz, 1H), 3.94 (dd, J = 12.3,
1.9 Hz, 1H), 3.85 (d, J = 12.6, 1.9 Hz, 1H), 3.78–3.66 (m, 8H), 3.62–
3.58 (m, 2H), 3.57–3.51 (m, 2H), 3.43 (t, J = 9.9 Hz, 1H), 3.41 (m,
1H), 3.31 (t, J = 8.3 Hz, 1H), 2.00 (s, 3H); 13C NMR (125 MHz,
D2O): d 175.0, 136.6, 128.79, 128.76, 128.55, 128.52, 103.0, 102.8,
101.1, 82.0, 78.4, 75.7, 74.9, 74.8, 74.5, 73.6, 72.8, 71.7, 71.6,
70.0, 69.7, 69.6, 68.4, 61.0, 60.5, 60.4, 60.2, 59.6, 55.7, 22.2; HRMS
(ESI): C27H41NO16Na [M+Na]+, calcd 658.2318, found 658.2381.
4.4. Kinetics determination
The apparent kinetics parameters were estimated by activity as-
says with varied acceptor concentration (0.1, 0.2, 0.5, 1 and 2 mM)
along with fixed UDP-Gal concentration (1 mM) or varied UDP-Gal
concentration (0.1, 0.2, 0.5, 1 and 2 mM) with 20 mM GlcNAc. The
4.6.3. Lacto-N-tetraose-b-OBn
1H NMR (500 MHz, D2O): d 7.46–7.37 (m, 5H, Ph), 4.91 (d,
J = 11.7 Hz, 1H, PhCH2), 4.73 (d, J = 11.7 Hz, 1H, PhCH2), 4.52 (d,
J = 8.0 Hz, 1H, H-1), 4.41 (d, J = 7.7 Hz, 1H, H-1), 4.40 (d,

X. Liu et al. / Bioorg. Med. Chem. 17 (2009) 4910–4915
4915
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