Helicobacter hepaticus Hh0072 gene encodes a novel α1-3- fucosyltransferase belonging to CAZy GT11 family

Article abstract of DOI:10.1093/glycob/cwq068

Lewis x (Le^x) and sialyl Lewis x (SLe^x)-containing glycans play important roles in numerous physiological and pathological processes. The key enzyme for the final step formation of these Lewis antigens is α1-3-fucosyltransferase. Here we report molecular cloning and functional expression of a novel Helicobacter hepaticus α1-3- fucosyltransferase (HhFT1) which shows activity towards both non-sialylated and sialylated Type II oligosaccharide acceptor substrates. It is a promising catalyst for enzymatic and chemoenzymatic synthesis of Le^x, sialyl Le^x and their derivatives. Unlike all other α1-3/4- fucosyltransferases characterized so far which belong to Carbohydrate Active Enzyme (CAZy, http://www.cazy.org/) glycosyltransferase family GT10, the HhFT1 shares protein sequence homology with α1-2-fucosyltransferases and belongs to CAZy glycosyltransferase family GT11. The HhFT1 is thus the first α1-3-fucosyltransferase identified in the GT11 family.

Full text of DOI:10.1093/glycob/cwq068

Glycobiology vol. 20 no. 9 pp. 1077–1088, 2010
doi:10.1093/glycob/cwq068
Advance Access publication on May 6, 2010
Helicobacter hepaticus Hh0072 gene encodes a novel
α1-3-fucosyltransferase belonging to CAZy GT11 family
Lei Zhang3, Kam Lau3, Jiansong Cheng2,3, Hai Yu3,
Yanhong Li3, Go Sugiarto3, Shengshu Huang3, Li Ding3,
Vireak Thon3, Peng G Wang4, and Xi Chen1,3
3Department of Chemistry, University of California, One Shields Avenue,
Davis, CA 95616, USA, and 4Departments of Biochemistry and Chemistry,
Ohio State University, Columbus, OH 43210, USA
fucosyltransferase-catalyzed reaction, fucosyltransferases are
categorized into α1-2, α1-3 and/or α1-4, α1-6 and O-fucosyl-
transferases (Ma et al. 2006). Except for O-fucosyltransferases
(O-FucT) which catalyze the transfer of a fucose residue from
GDP-Fuc directly to the serine or threonine residue on proteins
(Ma et al. 2006), all others catalyze the transfer of a fucose
residue to galactose (Gal), N-acetylglucosamine (GlcNAc) or
another fucose (Fuc) residue (Marques et al. 1998) on oligo-
saccharides, polysaccharides or glycoconjugates. Furthermore,
based on their protein sequence similarity, except for an un-
usual α1-2FucT in Dictyostelium discoideum (van Der Wel
et al. 2001), all other α1-2FucTs are categorized into Carbo-
hydrate Active Enzyme (CAZy, http://www.cazy.org/)
(Cantarel et al. 2009) glycosyltransferase family GT11, while
all α1-3 and/or α1-4-FucTs characterized so far belong to
GT10 (Ma et al. 2006).
L-Fucose is usually a terminal monosaccharide in oligosac-
charides and the carbohydrate moieties of glycoconjugates.
Protein O-fucosylation on serine or threonine has also been
found in many plasma glycoproteins and is believed to be im-
portant for regulating protein functions including Notch
signaling (Okajima et al. 2008). Fucose-containing structures
in eukaryotes are believed to be involved in tissue develop-
ment, angiogenesis, fertilization, cell adhesion, inflammation
and tumor metastasis (Ma et al. 2006; Miyoshi et al. 2008).
On the other hand, fucose-containing lipopolysaccharides
(LPS) are expressed by some pathogenic bacteria including
Helicobacter pylori and Escherichia coli (Guo et al. 2005).
They have been suggested to be involved in molecular mimic-
ry, adhesion, colonization and modulating the host immune
response (Ma et al. 2006).
Received on April 3, 2010; revised on April 29, 2010; accepted on
April 29, 2010
Lewis x (Lex) and sialyl Lewis x (SLex)-containing glycans
play important roles in numerous physiological and path-
ological processes. The key enzyme for the final step
formation of these Lewis antigens is α1-3-fucosyltransfer-
ase. Here we report molecular cloning and functional
expression of a novel Helicobacter hepaticus α1-3-fucosyl-
transferase (HhFT1) which shows activity towards both
non-sialylated and sialylated Type II oligosaccharide ac-
ceptor substrates. It is a promising catalyst for enzymatic
and chemoenzymatic synthesis of Lex, sialyl Lex and their
derivatives. Unlike all other α1-3/4-fucosyltransferases
characterized so far which belong to Carbohydrate Active
Enzyme (CAZy, http://www.cazy.org/) glycosyltransfer-
ase family GT10, the HhFT1 shares protein sequence
homology with α1-2-fucosyltransferases and belongs to
CAZy glycosyltransferase family GT11. The HhFT1 is thus
the first α1-3-fucosyltransferase identified in the GT11
family.
Keywords: cloning/fucosyltransferase/Helicobacter
hepaticus/Lewis x/sialyl Lewis x
Lewis x (Lex) trisaccharide Galβ1-4(Fucα1-3)GlcNAc and
sialyl Lewis x (SLex) tetrasaccharide Neu5Acα2-3Galβ1-4
(Fucα1-3)GlcNAc are among the most important fucose-
containing oligosaccharides that play essential roles in numer-
ous physiological and pathological processes. For example,
Lex antigens expressed by human pathogenic bacterium H. py-
lori mimic the host cell antigens and mask the pathogenic
bacterium from the host immune detection (Chan et al. 1995;
Moran et al. 1996; Monteiro et al. 1998). Lex antigens on para-
site Schistosoma mansoni downregulate the host’s protective
immune response against the parasite (Wang et al. 2009). SLex
is the essential recognition component of E-, P-, and L-selectin
ligands (Kannagi 2002; Lowe 2003; Dube and Bertozzi 2005).
The interaction of SLex and selectins is believed to mediate lym-
phocyte homing, initiate leukocyte–endothelial cell adhesion in
acute and chronic inflammation (Kannagi 2002; Lowe 2003)
Introduction
Fucosyltransferases (EC 2.4.1.-) are enzymes that catalyze the
transfer of monosaccharide L-fucose from its activated sugar
nucleotide guanosine-5′-diphosphate β-L-fucose (GDP-Fuc)
to acceptors for the formation of fucosides (fucose-containing
oligosaccharides and glycoconjugates). Mechanistically, fu-
cosyltransferases are inverting glycosyltransferases (GTs)
as α-fucosylated products are formed from β-fucosylated
sugar nucleotide donor GDP-Fuc. Based on the types of ac-
ceptors and the regio-specificity of the fucosides formed by
1To whom correspondence should be addressed: Tel: +1-530-754-6037; Fax:
+1-530-752-8995; e-mail: chen@chem.ucdavis.edu
2Current address: College of Pharmacy, Nankai University, Tianjin 300071,
China
© The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org 1077

L Zhang et al.
and be involved in cancer migration (Ma et al. 2006). SLex is
considered a cancer marker for diagnosis and prognosis of
cancer metastasis (Kannagi 2004; Magnani 2004). It has been
a leading structure for developing anti-inflammatory reagent
and cancer vaccine (Simanek et al. 1998; Danishefsky and Allen
2000; Ouerfelli et al. 2005; Seeberger and Werz 2007).
fucosyltransferases. The cloning and characterization of the
second putative H. hepaticus fucosyltransferase (HhFT2) en-
coded by gene HH1776 are ongoing processes.
Results
Sequence comparison of HhFT1, HhFT2 and other bacterial
fucosyltransferases
Due to the importance of fucosides in biological systems and
their potential application in treating inflammation, bacterial and
viral infection and cancer, they have been attractive synthetic
targets. Chemical synthesis of fucosidic bond is challenged
by its acid lability; thus, fucosyltransferase-catalyzed enzy-
matic and chemoenzymatic approaches are considered
preferred alternatives. Although many fucosyltransferases
have been identified from humans, Caenorhabditis elegans,
plants and bacteria (Ma et al. 2006), only H. pylori fucosyl-
transferases and human recombinant α1-3-fucosyltransferase
have been used for the preparative-scale synthesis of Lex
(Lubineau et al. 1997, 1998; Wang et al. 2009) and SLex
(Ichikawa et al. 1992; Bowman et al. 2001; Belot et al.
2002; Pratt and Bertozzi 2004). As the expression levels
of reported H. pylori and human α1-3-fucosyltransferases
are not high, there is a need for obtaining active and soluble
α1-3-fucosyltransferases with high expression level, prefera-
bly in E. coli expression hosts, to allow large-scale enzymatic
synthesis of fucosides.
H. pylori and Helicobacter hepaticus are two well-studied
Gram-negative Helicobacter species. Unlike H. pylori, which
is a gastric Helicobacter species, H. hepaticus is an enterohe-
patic Helicobacter species which colonizes the intestinal tracts
and livers of mice to cause chronic hepatic inflammation, liver
cancer and inflammatory bowel disease (Fox et al. 1994; Cahill
et al. 1997). The association of H. hepaticus infection with liv-
er tumors in mice provides a valuable animal model for
studying mechanisms of liver cancers caused by bacterial in-
fection (Rogers and Fox 2004). Like its close relative H.
pylori, which can also cause chronic inflammation and carci-
noma in the host (Suerbaum and Michetti 2002), H. hepaticus
exhibits phase variation in genes encoding putative fucosyl-
transferases and other glycosyltransferases, which may
contribute to LPS modification and antigenic mimicry (Wang
et al. 2000) for efficient immune evasion of hosts (Suerbaum et
al. 2003). Nevertheless, unlike fucosyltransferases from H. py-
lori (Chan et al. 1995; Monteiro et al. 1998; Ma et al. 2003,
2005; Rabbani et al 2005; Sanabria-Valentin et al. 2007; Sun
et al. 2007, Wang et al. 2009), which have been well studied,
no fucosyltransferase from H. hepaticus has been functionally
characterized so far. Analysis of H. hepaticus ATCC51449 ge-
nome (Suerbaum et al. 2003) identified two genes (HH0072
and HH1776) encoding putative fucosyltransferases in addition
to two putative truncated α1-2-fucosyltransferase mutants (en-
coded by HH0069 and HH0070). We report herein the cloning,
functional expression and characterization of the first fucosyl-
transferase (HhFT1) from H. hepaticus encoded by gene
HH0072 as a novel α1-3-fucosyltransferase which can use
both sialylated (Neu5Acα2-3Galβ1-4GlcNAc) and non-sialy-
lated (Galβ1-4GlcNAc) type II glycans as acceptors for the
synthesis of sialyl Lewis x [Neu5Acα2-3Galβ1-4(Fucα1-3)
GlcNAc] and Lewis x [Galβ1-4(Fucα1-3)GlcNAc] antigens.
It is the first α1-3-fucosyltransferase identified in CAZy glyco-
syltransferase family GT11, which contains, so far, only α1-2-
Proteins encoded by HH0072 (GenBank accession number
AAP76669) and HH1776 (GenBank accession number
AAP78373) in carcinogenic bacterium H. hepaticus ATCC
51449 genome are annotated as conserved hypothetical pro-
teins predicted to be a fucosyltransferase and an α1-3-
fucosyltransferase, respectively. Here, we name them HhFT1
and HhFT2, respectively. Based on protein sequence homolo-
gy, HhFT1 encoded by HH0072 is classified into CAZy
glycosyltransferase family GT11 which contains all α1-2-fu-
cosyltransferases characterized so far except for an unusual
α1-2FucT in D. discoideum (van Der Wel et al. 2001).
HhFT2 encoded by HH1776 is classified into CAZy glyco-
syltransferase family GT10 which contains all α1-3/4-
fucosyltransferases characterized so far. Nevertheless, they
are quite divergent from other characterized bacterial fucosyl-
transferases (Figure 1). The protein which has the closest
sequence homology to HhFT1 is an E. coli O128:B12 α1-
2-fucosyltransferase WbsJ (GenBank accession number
AAO37698) which is a part of O-antigen biosynthesis clus-
ter (Shao et al. 2003). They share 27% identity and 50%
similarity. Sequence alignment (Figure 2) indicates that
HhFT1 has three conserved motifs shared by GT11 family
fucosyltransferases including motif II which is likely a part
of the GDP-Fuc binding domain (Shao et al. 2003). The
protein which has the closest sequence homology to HhFT2
is a H. pylori α1-4-fucosyltransferse (GenBank accession
number AAR88243) (Rabbani et al. 2005), and they share
35% identity and 51% similarity. Sequence-based protein
solubility prediction (Smialowski et al. 2007) indicates that
HhFT1 has a better solubility score than all other possible
fucosyltransferases except for a putative protein from Geo-
bacter lovleyi SZ (GenBank accession number ACD96461).
Unlike α1-3FucTs from H. pylori (Ge et al. 1997; Lin et al.
2006), HhFT1 does not have C-terminal tandem repeats,
and it is a shorter protein (320 amino acid residues) com-
pared to the full-length Hp1-3FT (478 amino acid residues)
(Lin et al. 2006).
Cloning, expression and purification of recombinant protein
Full-length H. hepaticus HH0072 synthetic gene with codons
optimized for E. coli expression was initially cloned into
pET15b vector as an N-His6-tagged recombinant protein
(His6-HhFT1), but a low amount of soluble protein was ob-
tained in cell lysate (less than 1 mg/L cell culture, data not
shown). In order to increase the amount of soluble recombinant
protein, pMAL-c4X vector (Kapust and Waugh 1999) was
chosen to express HhFT1 as a maltose binding protein
(MBP)-fusion protein with the MBP tag at the N-terminus. A
His6-tag was also introduced at the C-terminus to allow easy
purification of the recombinant protein by Ni2+-affinity col-
umns. The gene and protein sequences of the resulting fusion
protein MBP–HhFT1–His6 are shown in Figure 3. The codon-
1078

Helicobacter hepaticus α1-3-fucosyltransferase
Fig. 1. Phylogenetic analysis of identified (labeled with unfilled diamonds) and putative (labeled with unfilled circles) FucTs from bacteria. Neighbor-Joining (NJ)
method was used to create this unrooted tree. Bootstrap values (expressed as percentages of 1000 replications) greater than 50% are indicated above the branches.
The protein identification numbers are followed by predicted class probability of insolubility (Smialowski et al. 2007) and species from which the genes were
cloned. HP, H. pylori; EC, E. coli; HH, Helicobacter hepaticus; UN, unknown.
optimized HhFT1 gene contains 27% adenine, 18% cytosine,
22% guanine and 33% thymine as compared to the original se-
quence containing 29% adenine, 19% cytosine, 21% guanine
and 31% thymine (GenBank accession no. AE017125). Ex-
pression of the recombinant protein MBP–HhFT1–His6 in E.
coli BL21 (DE3) followed by Ni2+-column purification re-
sulted in a yield of 45 mg L−1 cell culture. This expression
level is the highest among all reported recombinant fucosyl-
transferases. As shown in Figure 4, the purified protein
exhibited a molecular mass of about 75 kDa in sodium dode-
cylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
which is close to the calculated molecular weight of 80 kDa.
towards type II Galβ1-4GlcNAcβProN3 substrate, and no
product was observed when type I disaccharide Galβ1-
3GlcNAcβProN3 was used as the potential acceptor substrate.
Further TLC tests indicated that α2-3-sialylated type II oligo-
saccharide with N-acetylneuraminic acid (Neu5Ac) as the
sialic acid form (Neu5Acα2-3Galβ1-4GlcNAcβProN3) was
also an acceptor for the enzyme, suggesting that this novel re-
combinant enzyme is most likely an α1-3-fucosyltransferase in
spite of its protein sequence homology to α1-2-fucosyltrans-
ferases in CAZy glycosyltransferase family GT11.
Confirming the α1-3-fucosyltransferase activity of MBP–
HhFT1–His6 by one-pot two-enzyme synthesis of Galβ1-4
(Fuc1-3)GlcNAcβProN3
Acceptor substrates of MBP–HhFT1–His6
Using GDP-fucose as a donor, both type I (Galβ1-3GlcNAcβ-
ProN3) and type II (Galβ1-4GlcNAcβProN3) disaccharides
were tested as potential acceptor substrates for the recombinant
MBP–HhFT1–His6. Thin-layer chromatography (TLC) analy-
ses indicated that the recombinant enzyme was reactive
In order to confirm the α1-3-fucosyltransferase activity of
MBP–HhFT1–His6, a preparative-scale synthesis of Lewis x
trisaccharide Galβ1-4(Fucα1-3)GlcNAcβProN3 from type II
disaccharide Galβ1-4GlcNAcβProN3 was carried out using a
one-pot two-enzyme reaction (Figure 5) containing the recombi-
Fig. 2. Alignment of HhFT1 and E. coli O128:B12 α1-2-fucosyltransferase WbsJ (GenBank accession number AAO37698).
1079

L Zhang et al.
1081 GCCCTGAAAGACGCGCAGACTAATTCGAGCTCGAACAACAACAACAATAACAATAACAAC
361 A L K D A Q T
N
S
S
S
N
N
N
N
N
N
N
N
N
1141 AACCTCGGGATCGAGGGAAGGATTTCAGAATTCAAAGATGATCTGGTCATATTGCATCCG
381
N
L
G
I
E
G
R
I
S
E
F
K
D
D
L
V
I
L
H
P
1201 GATGGGGGCATTGCAAGTCAGATAGCTTTTGTCGCCCTTGGATTAGCGTTTGAGCAAAAA
401
D
G
G
I
A
S
Q
I
A
F
V
A
L
G
L
A
F
E
Q
K
1261 GGAGCTAAAGTAAAATATGATCTGAGCTGGTTTGCAGAGGGAGCCAAAGGCTTTTGGAAT
421
G
A
K
V
K
Y
D
L
S
W
F
A
E
G
A
K
G
F
W
N
1321 CCTAGTAATGGGTATGATAAGGTTTATGACATCACTTGGGATATTTCAAAGGCTTTTCCT
441
P
S
N
G
Y
D
K
V
Y
D
I
T
W
D
I
S
K
A
F
P
1381 GCACTTCATATTGAAATCGCCAATGAGGAAGAGATTGAGCGTTATAAAAGTAAGTATTTA
461
A
L
H
I
E
I
A
N
E
E
E
I
E
R
Y
K
S
K
Y
L
1441 ATCGATAATGATCGTGTGATAGATTATGCGCCTCCGCTGTATTGTTATGGATATAAAGGT
481
I
D
N
D
R
V
I
D
Y
A
P
P
L
Y
C
Y
G
Y
K
G
1501 CGTATTTTTCATTATCTTTATGCTCCTTTTTTCGCACAATCTTTTGCTCCGAAAGAGGCC
501
R
I
F
H
Y
L
Y
A
P
F
F
A
Q
S
F
A
P
K
E
A
1561 CAGGATTCTCATACACCGTTTGCAGCGTTACTTCAGGAAATAGAATCTAGTCCGAGTCCG
521
Q
D
S
H
T
P
F
A
A
L
L
Q
E
I
E
S
S
P
S
P
1621 TGCGGCGTCCATATTCGTCGCGGAGATTTGTCTCAGCCTCATATTGTATATGGTAATCCT
541
C
G
V
H
I
R
R
G
D
L
S
Q
P
H
I
V
Y
G
N
P
1681 ACAAGTAATGAGTATTTTGCAAAAAGTATTGAGTTGATGTGTCTTCTTCATCCTCAATCT
561
T
S
N
E
Y
F
A
K
S
I
E
L
M
C
L
L
H
P
Q
S
1741 TCTTTTTATTTATTTTCTGATGATTTAGCTTTCGTAAAAGAACAAATCGTGCCTTTGTTA
581
S
F
Y
L
F
S
D
D
L
A
F
V
K
E
Q
I
V
P
L
L
1801 AAGGGTAAGACGTACCGTATATGCGATGTCAATAACCCTTCACAAGGTTATCTTGATCTT
601
K
G
K
T
Y
R
I
C
D
V
N
N
P
S
Q
G
Y
L
D
L
1861 TATCTTTTAAGCCGTTGCCGCAATATCATAGGTTCTCAGGGTAGTATGGGCGAGTTTGCA
621
Y
L
L
S
R
C
R
N
I
I
G
S
Q
G
S
M
G
E
F
A
1921 AAAGTGTTGAGCCCGCATAATCCGTTATTGATTACTCCTCGTTACCGTAATATTTTTAAA
641
K
V
L
S
P
H
N
P
L
L
I
T
P
R
Y
R
N
I
F
K
1981 GAGGTAGAGAATGTCATGTGCGTGAATTGGGGGGAAAGCGTGCAACATCCTCCGCTTGTT
661
E
V
E
N
V
M
C
V
N
W
G
E
S
V
Q
H
P
P
L
V
2041 TGTTCTGCACCGCCGCCTCTTGTATCACAACTGAAGCGTAATGCTCCTTTAAATTCTCGT
681
C
S
A
P
P
P
L
V
S
Q
L
K
R
N
A
P
L
N
S
R
2101 TTATATAAAGAGAAAGATAATGCCAGTGCACACCACCACCACCACCACTGA
701 A H H H H H H
L
Y
K
E
K
D
N
A
S
&
Fig. 3. Gene and protein sequences of the codon-optimized MBP–HhFT1–His6. Only the C-terminal sequence of MBP is shown (in italics). The multiple cloning
sites of pMAL-c4X vector are underlined. The six histidine residues introduced at the C-terminus during cloning are in bold.
nant fucosyltransferase MBP–HhFT1–His6 and a recombinant
bifunctional L-fucokinase/GDP-fucose pyrophosphorylase
(FKP) from Bacteroides fragilis strain NCTC9343 (Yi et al.
2009). The presence of the bifunctional FKP allows the synthe-
sis of GDP-fucose, the sugar nucleotide donor for MBP–
HhFT1–His6, from simple starting materials such as L-fucose,
adenosine 5′-triphosphate (ATP) and guanosine-5′-triphosphate
(GTP) (Yi et al. 2009). Lewis x (Lex) trisaccharide Galβ1-4
(Fucα1-3)GlcNAcβProN3 was obtained in a yield of 63% with
the supplement of additional ATP and GTP periodically (see
Materials and methods for details). Nuclear magnetic resonance
(NMR) and high-resolution mass spectrometry (HRMS) studies
confirmed the structure of the trisaccharide product. As shown in
Table I, the 13C NMR chemical shifts of the purified product
agree well with previously reported data for Lewis x trisaccha-
ride (Wang et al. 2009). More specifically, comparing the
chemical shifts of the disaccharide starting material and the tri-
saccharide product indicates that significant changes occur at
C-3 (a downfield shift of 2.35 ppm from 72.70 ppm in the di-
saccharide starting material to 75.05 ppm in the trisaccharide
product) and C-4 (an upfield shift of 3.22 ppm from 78.69 ppm
in the disaccharide starting material to 75.47 ppm in the trisac-
charide product) of the GlcNAc residue, confirming that
fucosylation takes place at C-3 of the GlcNAc residue. High-
1080

Helicobacter hepaticus α1-3-fucosyltransferase
Table I. 13C NMR chemical shifts assignment of LacNAcβProN3 (Galβ1-
4GlcNAcβProN3) and LexβProN3 [Galβ1-4(Fuc1-3)GlcNAcβProN3]
1
2
3
4
5
Chemical shift (ppm)
Residue
βDGlcNAc
Carbon atom
C
LacNAcβProN3
LexβProN3
100 kD
75 kD
1
2
3
4
5
6
C=O
CH3
1
2
3
4
5
6
1
2
101.27
55.26
72.70
78.69
74.91
60.26
174.61
22.37
103.07
71.14
72.54
68.72
75.52
61.19
101.09
55.94
75.05
75.47
73.49
59.88
174.40
22.36
101.96
71.16
72.58
68.47
75.10
61.63
98.78
67.82
69.33
72.03
66.85
15.43
67.32
28.24
47.88
50 kD
37 kD
βDGal(1-4)
αLFuc(1-3)
ProN3
Fig. 4. SDS-PAGE analysis of MBP–HhFT1–His6 expression and
purification. Lanes: 1, protein standards; 2, whole cell extraction before
induction; 3, whole cell extraction after induction; 4, cell lysate after induction;
5, Ni2+-column purified protein.
3
4
5
CH3
OCH2CH2CH2N3
OCH2CH2CH2N3
OCH2CH2CH2N3
67.29
28.29
47.96
resolution mass spectrometry spectrum (ESI) obtained shows
the desired m/z for molecular ion [M]+. of 612.2425 (calculated
612.2490 for C23H40N4O15).
pH Profile of MBP–HhFT1–His6
acceptor Neu5Acα2-3Galβ1-4GlcNAcβPro2AA was used
(Figure 6). The fucosyltransferase activity was optimum in a
relatively broad pH range of 6.0–8.0. Medium activity was ob-
served at pH 5.5 and in a pH range of 8.5–9.0. No or
minimum activity was observed when the pH fell below 5.0
or higher than 9.5. This pH profile is similar to that reported
recently for an E. coli O128:B12 α1-2-fucosyltransferase
WbsJ, which is also active in a wide range of pH values
(5.5–8.5) (Li et al. 2008).
High-performance liquid chromatography (HPLC)-based pH
profile study for the fucosyltransferase was carried out using
fluorophore 2-anthranilic acid (2AA)-labeled non-sialylated
and sialylated type II oligosaccharides (Galβ1-4GlcNAcβ-
Pro2AA and Neu5Acα2-3Galβ1-4GlcNAcβPro2AA
respectively) as acceptors. A similar pH profile pattern was
seen for the MBP–HhFT1–His6 with these two acceptors, al-
though the overall activity was higher when the sialylated
Pi
O-
PpA
PPi
O
P
GTP
ATP
ADP
OH
OH
OH
O
FKP
FKP
OGDP
OH
O
O
O
OH
GDP
Fucokinase
GDP-Fucose
OH
OH
OH
pyrophosphorylase
HO
HO
HO
HhFucT1
OH
O
HO
HO
OH
O
OH
O
HO
HO
OH
O
O
OR
O
HO
OH
OR
O
OH
NHAc
NHAc
Galβ1-4GlcNAcβOR
O
OH
OH
HO
Galβ1-4(Fucα1-3)GlcNAcβOR
Fig. 5. Schematic illustration for the one-pot three-enzyme preparative-scale synthesis of Lewis x trisaccharide Galβ1-4(Fucα1-3)GlcNAcβProN3 from fucose,
ATP, GTP and type II disaccharide Galβ1-4GlcNAcβProN3 (LacNAcβProN3). Enzymes used: FKP, a recombinant bifunctional L-fucokinase/GDP-fucose
pyrophosphorylase from B. fragilis strain NCTC9343; HhFT1, H. hepaticus α1-3-fucosyltransferase 1; PpA, a recombinant inorganic pyrophosphatase from P.
multocida.
1081

L Zhang et al.
25
20
15
10
5
glycan Neu5Acα2-3LacNAcβPro2AA (kcat/Km = 0.63) is a lit-
tle better than the non-sialylated glycan LacNAcβPro2AA
(kcat/Km = 0.43) due to a smaller Km and a better kcat for the
sialylated acceptor (Table II). This is different from a C-termi-
nal truncated α1-3-fucosyltransferase (HpFTΔ45) cloned from
H. pylori (NCTC 11639) (Lin et al. 2006) which prefers N-
acetyllactosamine (LacNAc) over Neu5Acα2-3LacNAc as an
acceptor. Nevertheless, the Km value of LacNAcβPro2AA
(45 mM) for MBP–HhFT1–His6 is about 50-fold higher than
that (0.71–0.89 mM) of LacNAc for truncated α1-3-fucosyl-
transferases cloned from H. pylori (NCTC 11639) (Lin et al.
2006), indicating a much weaker binding of the type II glycan
to the HhFT1 than the H. pylori FucT.
GDP-fucose hydrolysis activity of the MBP–HhFT1–His6
In the absence of a glycan acceptor, water can be considered as
an acceptor for the fucosyltransferase activity of the enzyme.
This leads to the GDP-fucose hydrolysis. Significant GDP-
fucose hydrolysis activity was observed for MBP–HhFT1–
His6 with an optimal activity at pH 8.0 (Figure 8). At pH 7.0,
the hydrolysis of GDP-fucose (kcat/Km = 3.7) catalyzed by the
enzyme is about 3-fold more efficient than the fucosyltransfer-
ase activity of the enzyme when either non-sialylated (kcat/Km =
1.0) or sialylated type II glycans (kcat/Km = 1.1) was used as an
acceptor (Table II).
0
4
5
6
7
8
9
10
11
12
pH
Fig. 6. The pH profile of MBP–HhFT1–His6 when LacNAcβPro2AA (filled
diamonds) or Neu5Acα2-3LacNAcβPro2AA (dashed line with unfilled
diamonds) was used as an acceptor substrate. Buffers used: MES (pH 5.0–6.5),
HEPES (pH 7.0–8.0), Tris-HCl (pH 8.5–9.0), N-cyclohexyl-2-hydroxyl-3-
aminopropanesulfonic acid (CAPSO) (pH 10), and N-cyclohexyl-3-
aminopropanesulfonic acid (CAPS) (pH 10.0–11.0).
Fucosidase activity studies
Effects of metal ions, EDTA and dithiothreitol on the
No fucosidase activity was observed for the MBP–HhFT1–
His6 when LexβProN3 was used as a substrate for TLC assays
or when 4-methylumbelliferyl β-Lewis x (LexβMU) and
Neu5Acα2-3LexβMU were used in HPLC-based assays.
fucosyltransferase activity of MBP–HhFT1–His6
The effects of dithiothreitol (DTT), metal ions Mg2+ and Mn2+,
and a chelating agent EDTA on the fucosyltransferase activity
of MBP–HhFT1–His6 were examined at pH 7.0. As shown in
Figure 7, a divalent metal ion is required for the fucosyltrans-
ferase activity of the enzyme as the enzyme activity decreased
dramatically in the absence of metal ions. This is consistent
with the requirement of the divalent metal ion by most glyco-
syltransferases using nucleotide diphosphate sugars as donor
substrates. The enzyme activity increases with the increase of
the metal ion concentration up to 50 mM. The divalent metal
ions may facilitate the glycosidic bond cleavage in the donor
byneutralizing the negative chargesontheGDPproduct. Indeed,
protein sequence analysis of HhFT1 indicates a metal-binding
D92ND94 motif. It is also noticed that the enzyme activity did
not disappear completely without the addition of metal ions
or in the presence of a metal chelator EDTA. The reason is
unclear, but it is possible that positively charged amino acid
residues in the protein may contribute to stabilize the GDP
formed (Sun et al. 2007).
Product inhibition studies using GDP or LexβProN3 as an
inhibitor
No product inhibition effect was observed for GDP or Lexβ-
ProN3 by HPLC-based fluorescent assays.
35
30
25
20
15
10
5
There are seven cysteine residues in the HhFT1 protein se-
quence, so the effect of DTT on the fucosyltransferase
activity of the enzyme was studied. The addition of DTT did
not change the enzyme activity significantly, indicating that di-
sulfide formation is not required for the fucosyltransferase
activity of MBP–HhFT1–His6.
0
MgCl2
DTT
MnCl2
Kinetics
Kinetic studies carried out for the fucosyltransferase activity of
MBP–HhFT1–His6 using sialylated and non-sialylated type II
acceptors indicate that both glycans are similarly good accep-
tors for the enzyme although the efficiency for sialylated
Fig. 7. Effects of metal ions (Mg2+ and Mn2+), EDTA, and DTT (in the
presence of 20 mM Mg2+) on the fucosyltransferase activity of MBP–HhFT1–
His6 when LacNAcβPro2AA (white columns) or Neu5Acα2-
3LacNAcβPro2AA (black columns) was used as an acceptor substrate.
1082

Helicobacter hepaticus α1-3-fucosyltransferase
Table II. Kinetic parameters of recombinant MBP–HhFT1–His6
Substrates
Km (mM)
kcat (min-1)
kcat/Km (mM-1 min-1)
Fucosyltransferase
LacNAcβPro2AA
GDP-fucose
Neu5Acα2-3LacNAcβPro2AA
GDP-fucose
45
2.4 0.4
40
3.3 0.6
13
2
19
2.3 0.1
25
3.6 0.2
48
1
0.43
1.0
0.63
1.1
1
1
GDP-Fucose hydrolysis
GDP-fucose
2
2
3.7
Discussion
the enzyme, experimental confirmation is necessary to charac-
terize the function of the protein.
In this study, we report the molecular cloning, functional expres-
sion and characterization of a novel H. hepaticus α1-3-
fucosyltransferase. Based on its protein sequence homology,
HhFT1 is classified into glycosyltransferase family 11 (GT11)
in the Carbohydrate Active enZyme database (CAZy, http://
www.cazy.org/) (Campbell et al. 1997; Coutinho et al. 2003).
All members of GT11 family that have been functionally deter-
mined so far are α1-2 fucosyltransferases. These include WbsJ
from E. coli O128:B12 (Li et al. 2008), a potential α1-2FT
WbwK from E. coli O86:B7 (Guo et al. 2005), HpFT2 from
H. pylori (Wang et al. 1999), CE2FT-1 and CE2FT-2 (Zheng
et al. 2008) from C. elegans, FUT1 and FUT2 from human
(Larsen et al. 1990; Kelly et al. 1995; Rouquier et al. 1995; Kudo
et al. 1996; Koda et al. 1997), mouse (Lin et al. 2000; Domino
et al. 2001), rat (Piau et al. 1994; Bureau et al. 2001), bovine
(Barreaud et al. 2000; Saunier et al. 2001), and primates
(Apoil et al. 2000), as well as Sec1 from bovine (Barreaud et
al. 2000; Saunier et al. 2001), mouse (Lin et al. 2000; Domino
et al. 2001) and primates (Borges et al. 2008). The enzyme has
the highest sequence homology to HhFT1 is WbsJ, an E. coli
O128:B12 α1-2-fucosyltransferase which is a part of O-antigen
biosynthesis cluster (Shao et al. 2003). It shares 27% identity
and 50% similarity to HhFT1.
All fucosyltransferases are inverting glycosyltransferases
and are classified into eight glycosyltransferase (GT) families
in CAZy website: GT10, GT11, GT23, GT3, GT56, GT65,
GT68 and GT74 (Campbell et al. 1997; Coutinho et al.
2003; Cantarel et al. 2009). The only known fucosyltransferase
crystal structures are for H. pylori α1-3FT in the GT10 family
(Sun et al. 2007) and Bradyrhizobium sp. WM9 α1-6FT NodZ
(EC 2.4.1.-) (Brzezinski et al. 2007) and human GlcNAc α1-
6FT (FUT8) (EC 2.4.1.68) (Ihara et al. 2007) in the GT23 fam-
ily. All of these fucosyltransferases have a glycosyltransferase
B (GT-B) fold containing two separated Rossmann domains
(Breton et al. 2006). Protein x-ray crystal structures have not
been reported for any member of the GT11 family. The good
solubility and expression level (45 mg L−1 cell culture) of
MBP–HhFT1–His6 present an excellent opportunity for struc-
tural characterization of GT11 fucosyltransferases.
Similar to H. pylori, the hypothetic genes involved in LPS
synthesis of H. hepaticus ATCC 51449 are scattered through-
out the whole genome. Homologues of HP0826 (β1-4-
galactosyltransferase gene), HP0360 (galE), HP0326 (neuA)
as well as GDP-fucose biosynthetic genes rfbM (HP0043),
rfbD (HP0044) and wbcJ (HP0045), which are involved in
the synthesis of Lewis antigen in H. pylori 26695 (Moran
2008), are also harbored by H. hepaticus ATCC 51449
(HH0323, HH0380, HH0900, HH0675, HH0172 and
HH0173, respectively). Analysis of H. hepaticus ATCC
51449 genome has identified two CMP-Neu5Ac pathway
genes, neuB (HH0908) and neuC (HH0082) (Daines et al.
2000), although no sialyltransferase has been discovered by
searching the whole H. hepaticus ATCC 51449 genome using
known sialyltransferase sequences from uniproKB database
(http://www.uniprot.org/). A putative sialidase (HH1672) is al-
so found in the genome of H. hepaticus ATCC 51449.
In comparison, all α1-3 and/or α1-4-fucosyltransferases
characterized so far are listed in GT10 family. HhFT1 thus re-
presents the first α1-3-fucosyltransferase that belongs to the
CAZy GT11 family. This also indicates that, while protein se-
quence homology can be used as a guide to predict the type of
50
40
30
20
10
0
GDP-fucose can be efficiently hydrolyzed by MBP–
HhFT1–His6, which competes with the fucosyltransferase ac-
tivity of the enzyme, leading to low yields due to the
consumption of the donor substrate by water (Figure 9). This
can be overcome by adding additional GDP-fucose periodical-
ly during the reaction process. In situ generation of GDP-
H2O
Fucose
GDP
Lex
GDP-Fucose
HhFucT1
LacNAc
5
6
7
8
9
10
11
pH
Fig. 8. The pH profile of GDP-fucose hydrolysis catalyzed by MBP–HhFT1–
His6. Buffers used: MES, pH 6.0; HEPES, pH 7.0, 8.0; Tris-HCl, pH 9.0; and
CAPS, pH 10.0.
Fig. 9. Schematic illustration of water as a competing acceptor for the
fucosyltransferase activity of MBP–HhFT1–His6 causing hydrolysis of GDP-
fucose.
1083

L Zhang et al.
fucose from GTP and ATP using a bifunctional FKP from B.
fragilis strain NCTC9343 (Yi et al. 2009) also helps to improve
the yield. As most glycosyltransferases can catalyze the hydro-
lysis of the corresponding sugar nucleotide donor substrates,
periodical addition of sugar nucleotides and in situ generation
of sugar nucleotides can be used as general strategies to im-
prove the yields of glycosyltransferase-catalyzed reactions.
These will be extremely useful approaches when weak accep-
tors are used and water competes significantly for the same
donor.
tagged with C-His6-tagged protein in pMAL-c4X vector, the
forward primer used was 5′-GATCGAATTCAAAGAT-
GATCTGGTCATATTGCAT-3′ (EcoRI restriction site is
underlined), and the reverse primer used was 5′-AA-
GAAGCTTTCAGTGGTGGTGGTGGTGGTGTGCACTGG-
CATTATCTTTCTCTT-3′ (HindIII restriction site is underlined,
and the sequence that encodes the hexahistidine tag is itali-
cized). Polymerase chain reactions (PCRs) for amplifying the
target gene were performed in a 50-µL reaction mixture contain-
ing plasmid DNA (10 ng), forward and reverse primers (0.2 µM
each), 1× Herculase buffer, dNTP mixture (0.2 mM), and 5 U
(1 µL) of Herculase-enhanced DNA polymerase. The reaction
mixture was subjected to 30 cycles of amplification at an an-
nealing temperature of 55°C. The resulting PCR product was
purified and double digested with NdeI and BamHI or EcoRI
and HindIII restriction enzymes. The purified and digested
PCR product was ligated with the predigested pET15b or
pMAL-c4X vector and transformed into electrocompetent E.
coli DH5α cells. Selected clones were grown for minipreps
and characterized by restriction mapping. DNA sequencing
was performed by the Davis Sequencing Facility in the Uni-
versity of California-Davis.
Materials and methods
Materials
E. coli electrocompetent DH5α and chemically competent
BL21 (DE3) cells were from Invitrogen (Carlsbad, CA,
USA). Vector plasmid pET15b was purchased from Novagen
(EMD Biosciences Inc. Madison, WI, USA). QIAprep spin
miniprep kit and QIAquick gel extraction kit were from Qiagen
(Valencia, CA, USA). Herculase-enhanced DNA polymerase
was from Stratagene (La Jolla, CA, USA). T4 DNA ligase
and 1 kb DNA ladder were from Promega (Madison, WI,
USA). NdeI, BamHI, EcoRI, HindIII restriction enzymes and
vector plasmid pMAL-c4X were from New England Biolabs
(Beverly, MA, USA). Ni2+-NTA agarose (nickel-nitrilotriacetic
acid agarose) was from 5 PRIME (Gaithersburg, MD, USA).
Bicinchoninic acid (BCA) protein assay kit was from Pierce
Biotechnology Inc. (Rockford, IL).
Overexpression
Positive plasmids were selected and transformed into E. coli
BL21 (DE3) chemical competent cells. The plasmid-bearing
E. coli strains were cultured in LB-rich medium (10 g L−1 tryp-
tone, 5 g L−1 yeast extract and 10 g L−1 NaCl) supplemented
with ampicillin (100 µg mL−1). Overexpression of the target
protein was achieved by inducing the E. coli culture with
0.1 mM of isopropyl-1-thio-β-D-galactopyranoside (IPTG)
when OD600 reached 0.8 followed by incubating at 20°C for
20 h with vigorous shaking at 250 rpm in a C25KC incubator
shaker (New Brunswick Scientific, Edison, NJ).
Chemical synthesis of GDP-fucose
GDP-fucose was synthesized by following a reported proce-
dure (Timmons and Jakeman 2007). Detailed procedures are
presented in the supporting information.
Synthesis of LacNAcβPro2AA and Neu5Acα2-
3LacNAcβPro2AA as acceptors for HhFT1
Purification
Fluorescent LacNAcβPro2AA was synthesized from Lac-
NAcβProNH2 (obtained by reduction of azidoproyl LacNAc,
LacNAcβProN3) (Chokhawala et al. 2008) and 2-(methoxycar-
bonyl) succinanilic acid NHS ester (2AA-OSu). Its α2-3-
sialylated form Neu5Acα2-3LacNAcβPro2AA was synthe-
sized from LacNAcβPro2AA using a one-pot two-enzyme
sialylation reaction (Yu et al. 2009) catalyzed by a Neisseria
meningitidis CMP-sialic acid synthetase (NmCSS) (Yu et al.
2004) and a multifunctional Pasteurella multocida α2-3-sialyl-
transferase (PmST1) (Yu et al. 2005). Detailed procedures are
presented in the supporting information.
His6-tagged target proteins were purified from cell lysate. To
obtain the cell lysate, cell pellet harvested by centrifugation
at 4000 rpm for 2 h was resuspended in lysis buffer (pH
8.0, 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl
containing 0.1% Triton X-100) (20 mL L−1 cell culture). Lyso-
zyme (50 µg mL−1) and DNaseI (3 µg mL−1) were then
added, and the mixture was incubated at 37°C for 60 min
with vigorous shaking. Cell lysate was obtained by centrifuga-
tion at 12,000 rpm for 30 min as the supernatant. Purification
of His-tagged proteins from the lysate was achieved using a
Ni2+-resin column. The column was pre-equilibrated with
10 column volumes of binding buffer (5 mM imidazole,
0.5 M NaCl, 50 mM Tris-HCl, pH 7.5) before the lysate was
loaded. After washing with 8 column volumes of binding buffer
and 10 column volumes of washing buffer (40 mM imidazole,
0.5 M NaCl, 50 mM Tris-HCl, pH 7.5), the protein was eluted
with an elute buffer (200 mM imidazole, 0.5 M NaCl, 50 mM
Tris-HCl, pH 7.5). The fractions containing the purified en-
zymes were collected and stored at 4°C.
Cloning
Full-length H. hepaticus HH0072 synthetic gene with codons
optimized for E. coli expression was customer synthesized by
Biomatik (Wilmington, DE, USA) and provided in a pGH vec-
tor. It was cloned as an N-His6-tagged or an N-MBP-tagged with
a C-His6-tagged fusion protein. The primers used for cloning the
N-His6-tagged protein in pET15b vector were: forward primer
5′-GATCCATATGAAAGATGATCTGGTCATATTGCAT-3′
(NdeI restriction site is underlined) and reverse primer 5′-
AAGGGATCCTCATGCACTGGCATTATCTTTCTCTT-3′
(BamHI restriction site is underlined). To clone the N-MBP-
Quantification of purified protein
The concentration of purified enzyme was obtained in a 96-well
plate using a BCA Protein Assay Kit (Pierce Biotechnology,
1084

Helicobacter hepaticus α1-3-fucosyltransferase
Rockford, IL) with bovine serum albumin as a protein stan-
dard. The absorbance of samples was measured at 562 nm by
a BioTek SynergyTM HT Multi-Mode Microplate Reader.
Premier C18 column (250 × 4.6 mm i.d., 5 µm particle size,
Shimadzu) protected with a C18 guard column cartridge was
used. The mobile phase was 18% acetonitrile when LacNAcβ-
Pro2AA was used as an acceptor or 6.9% acetonitrile with
6.9% methanol when Neu5Acα2-3LacNAcβPro2AA was used
as an acceptor. Fluorescence of the products LacNAcProβ2AA
and Neu5Acα2-3LacNAcβPro2AA and the acceptor substrates
were detected by excitation at 315 nm and emission at 400 nm.
All assays were carried out in duplicate.
Sodium dodecylsulfate-polyacrylamide gel electrophoresis
SDS-PAGE was performed in a 12% Tris-glycine gel using a
Bio-Rad Mini-protein III cell gel electrophoresis unit (Bio-
Rad) at DC = 150 V. Bio-Rad Precision Plus Protein Standards
(10–250 kDa) were used as molecular weight standards. Gels
were stained with Coomassie Blue.
Effects of metal Ions, EDTA and DTT
EDTA (5 and 10 mM), different concentrations (5, 10, 20, and
50 mM) of MgCl2 or MnCl2, and various concentrations of
DTT (0.2, 1, and 5 mM) with 20 mM MgCl2 were used in
HEPE buffer (pH 7.0, 100 mM) to analyze their effects on
the fucosyltransferase activity of the recombinant enzyme
(9.0 µg). A reaction without EDTA, DTT and metal ions was
used as a control. The concentrations of the substrates and oth-
er reaction conditions were the same as described above for the
pH profile assays.
TLC analysis of the substrate specificity
LacNAcβProN3 (Galβ1-4GlcNAcβProN3, type II, 10 mM) or
Galβ1-3GlcNAcβProN3 (type I, 10 mM) was used as a po-
tential acceptor in the presence of GDP-fucose (10 mM) in a
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
buffer (pH 7.0, 100 mM) to analyze the acceptor substrate
specificity of the fucosyltransferase activity of the recombi-
nant enzyme (9.0 µg) at 37°C. The crude reaction mixture
was analyzed by TLC.
Kinetics by HPLC assay
One-pot two-enzyme synthesis of Galβ1-4(Fucα1-3)
GlcNAcβProN3 using MBP–HhFT1–His6
The assays were carried out in a total volume of 10 µL in
HEPES buffer (100 mM, pH 7.0) containing MgCl2
(20 mM), GDP-fucose, acceptor substrate (LacNAcβPro2AA
or Neu5Acα2-3LacNAcβPro2AA) and the recombinant pro-
tein (9.0 µg). Reactions were allowed to proceed for 20 min
at 37°C. Apparent kinetic parameters were obtained by varying
the GDP-fucose concentration from 2.0 to 50.0 mM (2.0, 3.0,
5.0, 10.0, 20.0, and 50.0 mM) and a fixed concentration of
LacNAcβPro2AA or Neu5Acα2-3LacNAcβPro2AA
(5 mM); or a fixed concentration of GDP-fucose (10 mM)
and varied concentrations of LacNAcβPro2AA or Neu5A-
cα2-3LacNAcβPro2AA (1, 1.5, 2.5, 5.0, 10.0, and
25.0 mM). Apparent kinetic parameters were obtained by fit-
ting the data (the average values of duplicate assay results) into
the Michaelis–Menten equation using Grafit 5.0.
HhFT1 acceptor Galβ1-4GlcNAcβProN3 (30 mg), L-fucose
(21 mg, 2 equivalents), ATP (71 mg, 2 equivalents), GTP
(67 mg, 2 equivalents) and MgCl2 (45 mg, 22 mM) were dis-
solved in H2O. A stock solution of 2-(N-morpholino)
ethanesulfonic acid (MES) buffer (0.5 M, pH 6.2, 1 mL) was
added. After the addition of a recombinant FKP from B. fragi-
lis strain NCTC9343 (Yi et al. 2009) (2.3 mg, FKP is a
bifunctional L-fucokinase/GDP-fucose pyrophosphorylase
which catalyzes the synthesis of GDP-fucose from L-fucose,
ATP and GTP via a fucose-1-phosphate (Fuc-1-P) intermedi-
ate) and MBP–HhFT1–His6 (0.9 mg), water was added to
bring the volume of the reaction mixture to 10 mL. The reac-
tion was carried out by incubating the solution in an incubator
shaker for 72 h at 37 °C. The supplements of additional 0.5
equivalents of ATP (18 mg) and GTP (17 mg) were added pe-
riodically in every 12 h (2.5 equivalent totals). The reaction
was quenched by adding cold ethanol (10 mL) and was then
centrifuged to remove precipitates. A BioGel P-2 filtration
and a silica gel column (EtOAc:MeOH:H2O, 7:2:1) were used
to purify the product to afford Galβ1-4(Fucα1-3)GlcNAcβ-
ProN3 (25 mg, 63 %).
pH profile of GDP-fucose hydrolysis by capillary
electrophoresis assays
Assays were performed in a total volume of 10 µL in a buffer
(200 mM) with pH varying from 6.0 to 10.0 containing GDP-
fucose (10 mM), MgCl2 (20 mM) and the recombinant enzyme
(9.0 µg). Reactions were allowed to proceed for 30 min at
37°C before being quenched by adding ice-cold water
(90 µL) to make 10-fold dilutions. The samples were then
kept on ice until aliquots of 6 µL were withdrawn and ana-
lyzed by a Beckman P/ACE MDQ capillary electrophoresis
system (60 cm × 75 µm i.d.) with a PDA detector. The ratio
of the absorbance for GDP-fucose and GDP at 254 nm was
determined at different concentrations (2.5, 5 and 10 mM).
All assays were carried out in duplicate.
pH profile by HPLC
Assays were performed in a total volume of 10 µL in a buffer
(200 mM) with pH varying from 5.0 to 11.0 containing GDP-
fucose (12 mM) with LacNAcβPro2AA (10 mM) or GDP-fu-
cose (10 mM) with Neu5Acα2-3LacNAcβPro2AA (5 mM)
and the recombinant enzyme (9.0 µg). Reactions were allowed
to proceed for 60 min (when LacNAcβPro2AA was used as an
acceptor) or 30 min (when Neu5Acα2-3LacNAcβPro2AA was
used as an acceptor) at 37°C before being quenched by adding
ice-cold 12% acetonitrile (1990 µL) to make 200-fold dilu-
tions. The samples were then kept on ice until aliquots of
5 µL were injected and analyzed by a Shimadzu LC-2010A
system equipped with a membrane online degasser, a temper-
ature control unit and a fluorescence detector. A reverse-phase
Kinetics of GDP-fucose hydrolysis by capillary electrophoresis
assays
The enzymatic assays were carried out in a total volume of
10 µL in HEPES buffer (100 mM, pH 7.0) containing MgCl2
(20 mM), GDP-fucose and the recombinant protein (9.0 µg).
Reactions were allowed to proceed for 20 min at 37°C. Appar-
1085

L Zhang et al.
ent kinetic parameters were obtained by varying the GDP-fu-
cose concentration from 2.0 to 50.0 mM (2.0, 3.0, 5.0, 10.0,
20.0 and 50.0 mM). Apparent kinetic parameters were ob-
tained by fitting the data (the average values of duplicate
assay results) into the Michaelis–Menten equation using Grafit
5.0.
Neu5Ac, N-acetylneuraminic acid; NMR, nuclear magnetic res-
onance; PCR, polymerase chain reactions; SDS-PAGE, sodium
dodecylsulfate-polyacrylamide gel electrophoresis; SLex, sialyl
Lewis x; TLC, thin-layer chromatography; Tris, tris(hydroxy-
methyl)aminomethane.
References
Fucosidase activity studies
Assays were carried out in a total volume of 10 µL in HEPES
buffer (100 mM, pH 7.0) containing MgCl2 (20 mM), Lex-
βProN3 (10 mM) and various amounts of the recombinant
protein (9.0, 18.0 or 27.0 µg). After 1 h reaction, the crude re-
action mixture was analyzed by TLC. In addition, both
LexβMU and Neu5Acα2-3LexβMU (5 mM) were used as sub-
strates to test the fucosidase activity of the recombinant
enzyme in a buffer (200 mM) containing MgCl2 (20 mM) with
pH varying from 5.0 to 8.0.
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Funding
This work was supported by the Alfred P. Sloan Research Fel-
lowship and Camille Dreyfus Teacher-Scholarship (to X.
Chen) and NIH R01HD061935 (to P.G. Wang).
Supplementary data
Supplementary data for this article is available online at http://
glycob.oxfordjournals.org/.
Conflict of interest statement
None declared.
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ATP, adenosine 5′-triphosphate; BCA, bicinchoninic acid;
CAPS, N-cyclohexyl-3-aminopropanesulfonic acid; CAPSO,
N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid;
CAZy, Carbohydrate Active Enzyme; DTT, dithiothreitol;
FKP, L-fucokinase/GDP-fucose pyrophosphorylase; FT, fuco-
syltransferase; Fuc, fucose; Gal, galactose; GDP-Fuc,
guanosine-5′-diphosphate L-fucose; GlcNAc, N-acetylglucosa-
mine; GT, glycosyltransferase; GTP, guanosine-5′-triphosphate;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
HhFT1, Helicobacter hepaticus α1-3-fucosyltransferase;
HhFT2, H. hepaticus fucosyltransferase; HPLC, high-
performance liquid chromatography; LacNAc, N-acetyllactosa-
mine; Lex, Lewis x; LPS, lipopolysaccharides; MBP, maltose
binding protein; MES, 2-(N-morpholino)ethanesulfonic acid;
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