Biochemical characterization of an α1,2-colitosyltransferase from Escherichia coli O55:H7

Article abstract of DOI:10.1093/glycob/cwv169

Colitose, also known as 3,6-dideoxy-l-galactose or 3-deoxy-l-fucose, is one of only five naturally occurring 3,6-dideoxyhexoses. Colitose was found in lipopolysaccharide of a number of infectious bacteria, including Escherichia coli O55 & O111 and Vibrio cholera O22 & O139. To date, no colitosyltransferase (ColT) has been characterized, probably due to the inaccessibility of the sugar donor, GDP-colitose. In this study, starting with chemically prepared colitose, 94.6 mg of GDP-colitose was prepared via a facile and efficient one-pot two-enzyme system involving an l-fucokinase/GDP-l-Fuc pyrophosphorylase and an inorganic pyrophosphatase (EcPpA). WbgN, a putative ColT from E. coli O55:H5 was then cloned, overexpressed, purified and biochemically characterized by using GDP-colitose as a sugar donor. Activity assay and structural identification of the synthetic product clearly demonstrated that wbgN encodes an α1,2-ColT. Biophysical study showed that WbgN does not require metal ion, and is highly active at pH 7.5-9.0. In addition, acceptor specificity study indicated that WbgN exclusively recognizes lacto-N-biose (Galβ1,3-GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP-l-Fuc (k_cat/K_m = 9.2 min^-1 mM^-1) as that toward GDP-colitose (k_cat/K_m = 12 min^-1 mM^-1). Finally, taking advantage of this, type 1 H-antigen was successfully synthesized in preparative scale.

Full text of DOI:10.1093/glycob/cwv169

Glycobiology Advance Access published December 23, 2015
1
Biochemical characterization of an 1,2-colitosyltransferase from Escherichia
coli O55:H7
Zhigang Wu†,§, Guohui Zhao†,§, Tiehai Li†, Jingyao Qu†, Wanyi Guan±, Jiajia Wang‡, Cheng Ma†,
Xu Li†, Wei Zhao‡, Peng G. Wang†,‡,*, and Lei Li†,*
†Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta,
GA 30303
‡State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University,
Tianjin 30071, China
±College of Life Science, Hebei Normal University, Shijiazhuang, Hebei 050024, China
*To whom correspondence should be addressed: Phone: +1-4044135607, Email: lli22@gsu.edu (L. Li), or
Phone: +1-4044135607, Email: pwang11@gsu.edu (P.G. Wang)
§Contributed Equally
The following Supplementary data is included:
1. DNA sequence of codon optimized WbgN gene
2. MS spectra of FKP and ColT catalyzed reactions
3. NMR spectra of GDP-colitose
4. NMR spectra of synthesized trisaccharide Col1,2-Gal1,3-GlcNAc
5. NMR spectra of synthesized Type 1 H-antigen
© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-
mail: journals.permissions@oup.com

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Abstract
Colitose, also known as 3,6-dideoxy-L-galactose or 3-deoxy-L-fucose, is one of the only five
naturally occurred 3,6-dideoxyhexoses. Colitose was found in lipopolysaccharide (LPS) of a number of
infectious bacteria, including Escherichia coli O55 & O111, Vibrio cholera O22 & O139, etc. To date, no
colitosyltransferase has been characterized, probably due to the inaccessibility of the sugar donor, GDP-
colitose. In this study, starting with chemically prepared colitose, 94.6 mg of GDP-colitose was prepared
via a facile and efficient one-pot two-enzyme system involving an L-fucokinase/GDP-L-Fuc
pyrophosphorylase (FKP) and an inorganic pyrophosphatase (EcPpA). WbgN, a putative
colitosyltransferase from E. coli O55:H5 was then cloned, overexpressed, purified and biochemically
characterized by using GDP-colitose as a sugar donor. Activity assay and structural identification of the
synthetic product clearly demonstrated that wbgN encodes an 1,2-colitosyltransferase (1,2-ColT).
Biophysical study showed that WbgN does not require metal ion, and is highly active at pH 7.5 to 9.0. In
addition, acceptor specificity study indicated that WbgN exclusively recognize lacto-N-biose (Gal1,3-
GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP-L-Fuc (kcat/Km
= 9.2 min-1 mM-1) as that toward GDP-colitose (kcat/Km = 12 min-1 mM-1). Finally, taking advantage of this,
type 1 H-antigen was successfully synthesized in preparative scale.
Keywords
Colitosyltransferase; GDP-colitose; Escherichia coli O55; H-antigen; Synthesis

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Introduction
Deoxysugars are widely found in natural products of plant and fungi, and secondary metabolites of
bacteria, which are common sources for antibiotics or anticancer agents, e.g., anthracyclines, avermectins,
enediynes, angucyclines, etc. A great number of bacterial lipopolysaccharide (LPS) and capsular
polysaccharides also contains diverse deoxysugars, for example, L-rhamnose (6-deoxy-L-mannose, found
in nearly one third of Escherichia coli LPS), 6-deoxy-L-talose, L-fucose (6-deoxy-L-galactose), D-
FucNAc (6-deoxy-GalNAc), etc. Deoxysugars are not only structural components of these bioactive
molecules, but in many cases are critical for the biological recognition of the mother compounds, serving
as ligands for cell-cell interaction or as targets for toxins, antibodies, and microorganisms (Weymouth-
Wilson, A.C. 1997).
Among the large number of monosaccharide residues found as components of O-antigen of bacteria
LPS, 3,6-dideoxyhexoses have drawn special attentions due to their highly immunogenic characteristics.
Of the eight possible stereoisomers of 3,6-dideoxyhexoses, only five exist naturally, including ascarylose,
abequose, paratose, tyvelose and colitose. In particular, colitose was found in a number of pathogenic
bacteria, for example, E. coli O55 & O111, Vibrio cholera O22 & O139, Salmonella enterica O35 & O50,
Yersinia pseudotuberculosis O6, O7 & O10, etc (Kenyon, J.J., De Castro, C., et al. 2011, Knirel, Y.A.,
Senchenkova, S.N., et al. 1998, Knirel, Y.A., Senchenkova, S.N., et al. 1996, Ovchinnikova, O.G.,
Kocharova, N.A., et al. 2007, Senchenkova, S.N., Shashkov, A.S., et al. 1997). In these cases, colitose
locates at the termini of O-antigens, serving as a special antigenic component of LPS (Luderitz, O., Staub,
A.M., et al. 1958, Luderitz, O., Westphal, O., et al. 1960, Westphal, O., Luderitz, O., et al. 1959). The
presence of colitose in pathogenic E. coli may enhance the lipophilic character of endotoxin LPS
(Medearis, D.N., Jr., Camitta, B.M., et al. 1968). Colitose was also considered an essential component of
specific ligands of certain LPS-binding lectins, e.g., houseshoe crab tachylectin-4 (specifically binds to E.
coli O111 LPS) (Saito, T., Hatada, M., et al. 1997). In addition, aerobic marine heterotrophic bacteria of

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the genera Alteromonas, Echinicola and Pseudoalteromonas also contain colitose in their O-antigens
(Gorshkova, N.M., Gorshkova, R.P., et al. 2001, Muldoon, J., Perepelov, A.V., et al. 2001, Silipo, A.,
Molinaro, A., et al. 2005, Tomshich, S.V., Kokoulin, M.S., et al. 2015). Given the emerging importance,
chemical synthesis had been performed to prepare colitose and colitose-containing oligosaccharides for
vaccine development (Calin, O., Eller, S., et al. 2013, Calin, O., Pragani, R., et al. 2012, Ruttens, B. and
Kovac, P. 2004, Senchenkova, S.N., Shashkov, A.S., et al. 1997, Turek, D., Sundgren, A., et al. 2006). In
contrast, there are limited studies on the biosynthesis of colitose-containing O-antigen, especially on
characterization of colitosyltransferases that are responsible for the transfer of colitose. Lack of efficient
approaches for the access of sufficient amounts of the sugar donor, GDP-colitose, has been a roadblock.
Different from the other four 3,6-dideoxyhexoses (ascarylose, abequose, paratose and tyvelose) that
are derived from CDP-glucose, the biosynthetic pathway of colitose (also known as 3,6-dideoxy-L-
galactose or 3-deoxy-L-fucose) begins from mannose-1-phosphate, and share a few enzyme-catalyzed
steps with that of GDP-L-galactose and GDP-L-Fuc. This is understandable when concerning structural
similarities between colitose and L-galactose/L-fucose (L-Fuc) (Fig. 1). Functional assignment and
biochemical characterization (Alam, J., Beyer, N., et al. 2004, Beyer, N., Alam, J., et al. 2003) of putative
enzymes in GDP-colitose biosynthesis gene clusters revealed a similar pathway as that of other 3,6-
dideoxyhexoses. As depicted in Figure 1, the biosynthesis of GDP-colitose begins with GDP-mannose
pyrophosphorylase (GMP, or ColE)-catalyzed pyrophosphorylation of mannose-1-phosphate to form
GDP-mannose, followed by GDP-mannose 4,6-dehydratase (GMD, or ColB)-catalyzed intramolecular
oxidoreduction of GDP-mannose to afford GDP-4-keto-6-deoxy-mannose (GKDM), an key intermediate
for biosynthesis of both GDP-colitose and GDP-L-Fuc. Subsequently, GKDM was converted into GDP-4-
keto-3,6-dideoxy-mannose (GKDDM) by GDP-4-keto-6-deoxy-mannose-3-dehydrase (ColD)-catalyzed
C-3 deoxygenation in the presence of L-glutamate and cofactor pyridoxal-5’-phosphate (PLP) (Alam, J.,
Beyer, N., et al. 2004). Finally, GDP-colitose is generated by GDP-colitose synthase (ColC), a
bifunctional enzyme catalyzing the C-5 epimerization of GKDDM and the subsequent C-4 keto reduction

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(Alam, J., Beyer, N., et al. 2004). The last step requires equal moles of NAD(P)H as that of GKDDM.
Given the involvement of multiple steps and cofactors, and the structural similarities between final
product and intermediates, one can imagine it must be complicated to produce and purify milligrams of
GDP-colitose via such a biosynthetic pathway (Heath, E.C. and Elbein, A.D. 1962, Isshiki, Y., Kondo, S.,
et al. 1996). A facile and efficient approach is thus in demand for large scale preparation of GDP-colitose.
E. coli O55:H7 belongs to enteropathogenic E. coli (EPEC), a group of E. coli isolates among the
major causes of acute and prolonged infantile diarrhea in developing countries (Ochoa, T.J. and Contreras,
C.A. 2011). Genomic analysis revealed the E. coli O55:H7 is closely related to the most notorious E. coli
isolate O157:H7, which accounts for majority of E. coli infections in North America every year, leading
to severe acute haemorrhagic diarrhea, complication hemolytic uremic syndrome (HUS), even death
(Sodha, S., Heiman, K., et al. 2015). The O-antigen repeating unit of E. coli O55:H7 contains five
monosaccharide residues, with a structure of Col1,2-Gal1,3-GlcNAc1,3-Gal1,3GalNAc (Fig. 2)
(Lindberg, B., Lindh, F., et al. 1981). The O-antigen biosynthesis gene cluster has been sequenced (Wang,
L., Huskic, S., et al. 2002), unveiling four putative glycosyltransferase genes (wbgM, wbgN, wbgO and
wbgP) for the assembly of the repeating unit (Fig. 2). Among these four genes, we have biochemically
demonstrated that wbgO encodes an 1,3-galactosyltransferase (Liu, X.W., Xia, C., et al. 2009). WbgN
shares 23% sequence identity and 47% sequence similarity with human FUT2 (1,2-fucosyltransferase),
and was thought to the colitosyltransferases (ColT). In this study, we cloned the wbgN gene,
overexpressed and purified the recombinant protein, and performed biochemical characterization using
chemoenzymatically prepared GDP-colitose as sugar donor.

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Results and Discussion
Chemoenzymatic synthesis of GDP-colitose
De novo biosynthetic pathways of GDP-L-Fuc and GDP-colitose share the same initial steps
including generation of intermediates mannose-1-phosphate, GDP-mannose and GKDM (Fig. 1). The
complexity of the pathways and lack of cost-effective approaches to access intermediates greatly
restricted large scale preparation of these valuable sugar donors. In efforts to develop facile and cost-
effective approaches for large scale synthesis of GDP-L-Fuc and derivatives, we and others exploited L-
fucokinase/GDP-L-Fuc pyrophosphorylase (FKP) (Wang, W., Hu, T., et al. 2009, Yi, W., Liu, X., et al.
2009, Zhao, G., Guan, W., et al. 2010), a bifunctional enzyme found in the fucosylation salvage pathway
of a human intestinal bacterium, Bacteroides fragilis 9343 (Coyne, M.J., Reinap, B., et al. 2005). As
depicted in Figure 3, the salvage pathway involves 1) L-fucokinase activity of FKP catalyzed formation
of L-Fuc-1-phosphate (L-Fuc-1-P) from L-Fuc in presence of ATP, and 2) GDP-L-Fuc pyrophosphorylase
activity of FKP catalyzed generation of GDP-L-Fuc and byproduct pyrophosphate in presence of GTP.
Using a one-pot two-enzyme system which contains FKP and an inorganic pyrophosphatase (drive
reaction forward by digesting byproduct pyrophosphate), GDP-L-Fuc and a number of C-5 modified
derivatives including GDP-L-galactose were successfully prepared in large scale (Wang, W., Hu, T., et al.
2009, Yi, W., Liu, X., et al. 2009). In another example, a C-2 modified derivative, GDP-2-deoxy-2-
fluoro-L-fucose (GDP-Fuc2F), was also efficiently synthesized (Rillahan, C.D., Antonopoulos, A., et al.
2012). These reports indicated that FKP is very promiscuous towards L-Fuc and derivatives.
To explore whether FKP could tolerate colitose (3-deoxy-L-fucose) as a substrate, we chemically
prepared colitose starting with L-Fuc as previously reported (Ruttens, B. and Kovac, P. 2004). Activity
assays of FKP towards colitose were carried out in presence of ATP (for fucokinase activity) or both ATP
and GTP (for pyrophosphorylase activity), with positive controls using L-Fuc as substrate instead of
colitose. As shown in Figure 4, after incubation with FKP, both spots corresponding to colitose and ATP

7
weakened, whereas two new spots appeared (Fig. 4, lane 5). Mass spectroscopy (MS) analysis of the
reaction mixture (Supplementary Data 2) showed two peaks at m/z 227.0290 and 426.0151,
corresponding to colitose-1-phoshate and ADP. In addition, when GTP was added to above reaction
mixture, the spot corresponding to colitose-1-phoshate disappeared, yielding a new spot (Fig. 4, lane 6) of
GDP-colitose, which is later confirmed by MS analysis with a m/z peak of 572.0705 [M-H]-. These
results clearly indicate that FKP can well accept colitose and efficiently catalyze the formation of GDP-
colitose. To confirm the structure, large scale synthesis of GDP-colitose was carried out using the one-pot
two-enzyme system as illustrated in Figure 3. After P2 purification, 94.6 mg of GDP-colitose was
obtained, with a total yield of 83%. The chemical structure was confirmed by high resolution MS and
NMR (see Supplementary Data 3 for spectra). Such a one-pot two-enzyme system provides a facile,
efficient and cost-affordable approach for the access of large scales of GDP-colitose.
wbgN encodes an 1,2-colitosyltransferase
According to sequence similarities, WbgN was classified into glycosyltransferase family 11
(www.cazy.org), a group of enzymes with known activities of 1,2-L-fucosyltransferase (1,2-FucT) and
1,3-L-fucosyltransferase (1,3-FucT). In addition, amino acid sequence of WbgN shares 37% identity
and 57% similarity with a recently identified 1,2-FucT, WbgL, which is involved in the biosynthesis of
E. coli O126 O-antigen (Engels, L. and Elling, L. 2014). Considering the structure similarities between O-
antigen of E. coli O55:H5 (contains Col1,2-Gal1,3-GlcNAc motif) and O126 (contains L-Fuc1,2-
Gal1,3-GlcNAc motif), it is assumed that wbgN encodes the 1,2-colitosyltransferase that is responsible
for transfer of colitose from GDP-colitose to the Gal residue of Gal1,3-GlcNAc via an 1,2-linkage. To
test this hypothesis, the wbgN gene was codon optimized (host E. coli), synthesized and cloned into
pQE80 L for expression. Even though induction was performed under a low temperature (16 °C) with
minimum amounts of inducers (0.1 mM IPTG), majority of WbgN proteins tend to precipitate (Fig. 5,
lane 4), further optimization to increase soluble form proteins failed. Such phenomena were found

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common among O-antigen synthesis enzymes (especially fucosyltransferases) (Engels, L. and Elling, L.
2014, Li, M., Liu, X.W., et al. 2008, Li, M., Shen, J., et al. 2008), which are thought membrane associated
and with low solubility (Raetz, C.R. and Whitfield, C. 2002). Nevertheless, 1.2 mg of soluble WbgN was
purified from 1 L of cell culture. As shown in Figure 5 (Lane 7), the N-terminal His-tagged WbgN has an
apparent MW of 30 kDa on SDS-PAGE, slightly lower than the calculated value (35 kDa). This is normal
for membrane associated hydrophobic proteins (Rath, A., Glibowicka, M., et al. 2009). Western blot
using an anti-His antibody as a primary antibody confirmed the result. Alternatively, fusion protein
strategy has been extensively used to increase the solubility of 1,2-FucTs (Engels, L. and Elling, L. 2014,
Li, M., Liu, X.W., et al. 2008, Li, M., Shen, J., et al. 2008), which usually resulted in greatly decreased
activity. Therefore, it was not applied in this study.
To reveal the function of WbgN, a panel of sugar nucleotides (GDP-colitose, UDP-Gal, UDP-
GlcNAc, and UDP-GalNAc) were tested using Lacto-N-biose (LNB, Gal1,3-GlcNAc) as an acceptor.
MS analysis of the reaction with GDP-colitose as donor found a new peak at m/z of 536.1933
(Supplementary Data 2), corresponding to the trisaccharide product Col1,2-Gal1,3-GlcNAc [M+Na].
No new peaks were found when using other sugar nucleotides as donors. Thus, the donor specificity study
indicated that wbgN encodes colitosyltransferase, consistent with bioinformatics analysis. To investigate
whether WbgN catalyzes the formation of the 1,2-linkage, large scale preparation of the trisaccharide
were performed. Briefly, a 5 mL reaction system was set up containing 30 mg of LNB (16 mM), 1.25
equiv. of GDP-colitose, WbgN (20 g) and 50 mM Tris-HCl (pH 8.0) buffer. After LNB was totally
converted (monitored by TLC), the trisaccharide product was isolated by gel filtration chromatography
with a total yield of 92% (37 mg), The structure of trisaccharide was confirmed by NMR spectroscopy
including 1H, 1H-1H-COSY, 13C, HSQC and HMBC (Supplementary Data 4). The -linkage of Col with
Gal was identified by the coupling constant (JH1-H2 = 4.8 Hz) of colitose. Moreover, HMBC revealed a
good correlation of the anomeric carbon (C-1) of colitose with H-2 of Gal, which indicated 1,2-linked
colitose with Gal. Collectively, these results provide solid evidences that wbgN encodes an 1,2-ColT

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that transfers colitose onto LNB via a 1,2-linkage. To the best of our knowledge, WbgN is the first
biochemically characterized colitosyltransferase.
Biophysical Properties of WbgN
Study on pH optimum with LNB as an acceptor revealed that WbgN is highly active with in a broad
pH spectrum ranging from pH 7.5 to 9.0, and exhibits around 60% activity at a higher basic condition (pH
10.2) (Fig. 6A). Sequence analysis showed that WbgN doesn’t contain a DXD motif, a conserved amino
acid sequence exists in all glycosyltransferase superfamily A (GT-A) members (Breton, C., Šnajdrová, L.,
et al. 2006). Such DXD motifs play a role in coordinating the binding of sugar nucleotides in the active
site with the requirement of certain divalent metal cation. This is consistent with activity assay results
showing metal ion is not necessary for WbgN. To further investigate the metal ion dependence, a panel of
divalent metal cations and EDTA were tested with positive (no additive) and negative (no enzyme)
controls. As shown in Figure 6B, WbgN exhibited highest activities in the presence or the absence of
EDTA, indicating that a metal ion is not required for the activity of WbgN. In contrast, the addition of
metal ions dramatically decreased its activity to 0- 40%. It is thus assumed that instead of GT-A, WbgN
belongs to GT-B superfamily, which is in accordance with similar fucosyltransferases WbgL (Engels, L.
and Elling, L. 2014), WbsJ (Li, M., Liu, X.W., et al. 2008), and WbiQ (Pettit, N., Styslinger, T., et al.
2010).
WbgN can efficiently use GDP-Fuc as a sugar donor
Concerning the sequence similarities between WbgN and a number of 1,2-FucT (e.g., WbgL from
E. coli O126, WbsJ from E. coli O128 and WbwK from E. coli O86), as well as the structural similarities
between sugar donors GDP-colitose and GDP-L-Fuc, it is of great interest to reveal whether WbgN could
recognize GDP-Fuc. To this end, reaction was conducted using GDP-Fuc as the sugar donor and LNB as
the acceptor. Briefly, a 50 L reaction was set up containing 50 mM Tris-HCl (pH 8.0), 5 mM acceptor

10
Gal1,3-GlcNAc, 5 mM of acceptor GDP-L-Fuc, and 50 g of recombinant WbgN. The reaction was
allowed to proceed at 37 °C for 2 h before subjecting to MS analysis. Two new peaks at m/z of 530.2071
and 552.1888 were found on MS profile, corresponding to the trisaccharide product L-Fuc1,2-Gal1,3-
GlcNAc [M+H]+, and [M+Na]+. In addition, MS peak corresponding to the acceptor LNB disappeared
(Supplementary Data 2). This result clearly indicated that WbgN can employ GDP-L-Fuc as a sugar
donor efficiently. To further investigate the donor preference of WbgN, kinetic characterization were
performed. As listed in Table 1, WbgN has a lower Km value toward GDP-colitose (0.76 ± 0.18 mM) than
that toward GDP-L-Fuc (1.3 ± 0.42 mM), implying that GDP-colitose has a higher enzyme affinity. In
contrast, a higher turnover rate was found toward GDP-L-Fuc (kcat = 12 ± 1.5 min-1) than that toward
GDP-colitose (kcat = 8.8 ± 0.8 min-1), indicating that WbgN-catalyzed reaction is more favorable towards
the trisaccharide product containing L-Fuc. Collectively, WbgN exhibits comparable activities toward
both sugar nucleotide donors, with a kcat/Km value of 12 min-1 mM-1 toward GDP-colitose, and that of 9.2
min-1 mM-1 toward GDP-L-Fuc.
Among all colitose-containing bacterial polysaccharide identified so far
(http://csdb.glycoscience.ru/bacterial/), none contains L-Fuc residue(s). This could be caused by 1) lack of
sugar donor GDP-L-Fuc, or/and 2) lack of corresponding FucT(s). Genetic analysis revealed that GDP-L-
Fuc synthase (GFS) encoding gene (fcl) is always missing or inactivated in gene clusters responsible for
the biosynthesis of these bacterial polysaccharides (Bastin, D.A. and Reeves, P.R. 1995, Cunneen, M.M.,
Pacinelli, E., et al. 2011, Kenyon, J.J., De Castro, C., et al. 2011, Wang, L., Huskic, S., et al. 2002, Wang,
L. and Reeves, P.R. 2000, Yamasaki, S., Shimizu, T., et al. 1999), supporting the first assumption. On the
other hand, our results clearly showed that, in E. coli O55:H7, ColT can also function as FucT (or
possibly is intrinsic FucT), excluding the second assumption. This phenomenon could be common among
other colitose-containing bacteria, as putative ColTs usually share significant sequence similarities with
FucTs (Bastin, D.A. and Reeves, P.R. 1995, Cunneen, M.M., Pacinelli, E., et al. 2011, Kenyon, J.J., De

11
Castro, C., et al. 2011, Wang, L., Huskic, S., et al. 2002, Wang, L. and Reeves, P.R. 2000, Yamasaki, S.,
Shimizu, T., et al. 1999). Biochemical characterizations are needed to confirm such hypothesis.
WbgN is specific for Lacto-N-biose
The purified WbgN was used to investigate its substrate specificity towards a group of mono- and
disaccharide acceptors. As shown in Table 2, WbgN is highly specific for LNB in the presence of either
GDP-colitose or GDP-L-Fuc, and cannot tolerate any other acceptors tested, including galacto-N-biose
(GNB, Gal1,3-GalNAc), LacNAc (Gal1,4-GlcNAc) and lactose. These results indicated that not only
the terminal Gal, but also the adjacent sugar residue and the way they coupled together are involved in the
enzyme-acceptor binding. Such a strict acceptor preference can be applied specifically for the preparation
of type 1 H-antigen (L-Fuc1,2-Gal1,3-GlcNAc), or complex glycans that contain the antigen. In a test
trial, 40 mg (95% yields) of type 1 H-antigen was synthesized in a WbgN-catalyzed reaction, starting with
LNB and GDP-L-Fuc (see Supplementary Data 5 for NMR characterization). Taken together, the
acceptor pattern shown in the assay is in agreement with the proposed function of WbgN in the
biosynthesis of the E. coli O55:H7 O-antigen.
Conclusion
In summary, taken advantage of relaxed substrate specificity of FKP, we have developed a facile and
cost-efficient system for enzymatic synthesis of rare sugar nucleotide GDP-colitose in preparative scale.
Using the synthesized GDP-colitose, we have biochemically characterized the first ColT (WbgN) from E.
coli O55:H7. WbgN was found high active in a pH range of 7.5-9.0, and a metal ion is not required for its
activity. Interestingly, WbgN exhibited similar activity towards both GDP-colitose and GDP-L-Fuc,
taking together with the fact that ColTs and FucTs share significant sequence similarities, it is possible
that many ColTs may have FucT activities. Acceptor substrate specificity study revealed that WbgN is

12
specific for Lacto-N-biose, which may found great use in the preparation of type 1 H-antigen-containing
complex glycans.
Materials and Methods
Materials
E. coli DH5 competent cells were purchased from Life Technologies (Carlsbad, CA), E. coli
BL21(DE3) competent cells and Nitrocellulose membrane were purchased from New England BioLabs
(Ipswich, MA). Vector pQE80L and Ni-NTA agarose were from Qiagen (Valencia, CA). Anti-His
monoclonal antibody (from mouse) was from Sigma (St. Louis, MO). HRP-linked anti-mouse IgG, and
ECL plus Western Blot detection reagents were purchased from GE healthcare (Piscataway, NJ). Fast
Digest restriction enzymes were purchased from Fermentas (Glen Burnie, MD). Shrimp alkaline
phosphatase was from New England Biolabs (Ipswich, MA). GDP-Fuc, Gal1,3-GlcNAc, Gal1,3-
GalNAc, Gal1,4-GlcNAc (LacNAc), FKP and EcPpA were prepare previously in in our group (Li, L.,
Liu, Y., et al. 2015, Li, L., Liu, Y., et al. 2013, Zhao, G., Guan, W., et al. 2010). All other chemicals and
solvents were purchased from Sigma-Aldrich.
Plasmid construction, protein expression and enzyme purification
Full length putative 1,2-L-colitosyltransferase gene (wbgN) from E. coli O55:H7 (GeneBank:
18266395) was codon optimized (target host, E. coli) and synthesized (Qinglan Bio, China) together with
5’-terminal BamH I restriction sites and 3’-terminal Hind III restriction sites (see Supplementary Data 1
for optimized gene sequence). The gene was then digested with BamHI and HindIII, and inserted into
pQE80L pre-treated with same restriction enzymes, yielding an expression plasmid pQE-WbgN. After
DNA sequencing, the plasmid was transformed into E. coli BL21(DE3) for overexpression.
E. coli BL21 (DE3) harboring the pQE-WbgN was grown at 37 °C in 1 L of LB medium (Lennox)
with 100 g/mL Ampicillin until OD600 reached 0.6-0.8. After cooling the culture on ice for 20 min,

13
isopropyl 1-thio--D-galactopyranoside (IPTG) was added to a final concentration of 0.1 mM. Expression
was allowed to proceed at 16 °C for 20 h. Cells were harvested by brief centrifugation and stored at -
20 °C until use.
To purify soluble recombinant enzymes, cells were resuspended in buffer A (20 mM Tris-HCl, pH
8.0, 0.3 M NaCl, 5 mM imidazole, 0.2% Triton X-100, 10% glycerol), and disrupted by a Microfluidics
M-110P Homogenizer. The lysate was cleared by centrifugation (15,000 × g, 30 min, 4 °C) and loaded
onto a 3 mL Ni-NTA gravity column preequilibrated with buffer B (20 mM Tris-HCl, pH 8.0, 0.3 M
NaCl, 5 mM imidazole, 10% glycerol). The column was washed with 200 mL of buffer B and 200 mL of
buffer C (20 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 25 mM imidazole, 10% glycerol). The protein was
finally eluted with buffer D (20 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 250 mM imidazole, 10% glycerol)
and desalted against buffer E (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 20% glycerol) for long term storage
at -20 °C.
Chemoenzymatic synthesis of GDP-colitose and purification
To test the fucokinase activity of FKP towards colitose, reactions were carried out in a 50 L system,
containing 100 mM of Tris-HCl (pH 8.0), 20 mM of colitose or L-Fuc (as positive control), 20 mM of
ATP, 20 mM of MgCl2, and 10 g of purified FKP. To test the GDP-L-Fuc pyrophosphorylase activity,
20 mM of GTP and 2 g of EcPpA were added to the above reaction mixtures. Reactions were incubated
at 37 °C for 1 h, and analyzed by MS and TLC (developing solvent: isopropanol/ammonium acetate (1
M)/Acetic acid = 7:3:0.1 (v/v/v)). The compounds on TLC plates were stained with anisaldehyde/acetic
acid/H2SO4/H2O = 7:3:10:27 (v/v/v/v), and visualized by brief heating. For large scale GDP-colitose
synthesis, a one-pot two-enzyme system similar as described before was used (Zhao, G., Guan, W., et al.
2010). Briefly, a reaction was carried out at 37 °C for 5 h in a total volume of 10 mL in 100 mM Tris-HCl
(pH 8.0) buffer, containing 20 mM colitose, 22 mM ATP, 22 mM GTP, 20 mM MgCl2, 20 g/mL FKP
and 2 g/mL EcPpA. The reaction was monitored by TLC. To purify GDP-colitose, the reaction was

14
quenched by the addition of equal volumes of cold ethanol. The mixture was placed on ice for 30 min,
and centrifuged to remove debris. The supernatant was then concentrated and loaded onto bio-gel P2 for
separation. The fractions contain pure GDP-colitose (detected by TLC) were then concentrated and
lyophilized for MS and NMR analysis. The fractions contain impure GDP-colitose were also collected
and treated with shrimp alkaline phosphatase (New England Biolabs) to digest ADP byproduct following
instructions (100 U, 30 °C for 5 h), and then subject to bio-gel P2 for further separation.
GDP-L-Colitose: 1H NMR (400MHz, D2O) δ: 8.00 (s, 1 H), 5.83 (d, J = 6.0 Hz,1 H), 4.82 (t, J = 8.0 Hz,
1 H), 4.43 (t, J = 4.0 Hz, 1 H), 4.25 (s,1 H), 4.10 (d, J = 4.4 Hz,1 H), 3.56 - 3.76 (m, 4 H), 2.07- 2.11 (m,
1 H), 1.56-1.63 (m, 1 H), 1.07 (d, J = 6.4 Hz, 3 H); 13C NMR (100MHz, D2O) δ:172.89, 158.97, 153.89,
140.84, 116.23, 100.34,100.28, 86.73, 83.83, 83.74, 74.70, 73.45, 70.42, 67.93, 36.24, 15.57; ESI HRMS:
[M-H]- calcd for 572.0946, found: 572.0941.
Activity assay of WbgN
Briefly, a 50 L reaction was set up containing 50 mM Tris-HCl (pH 8.0), 5 mM acceptor Gal1,3-
GlcNAc, 5 mM of acceptor GDP-colitose, and 20 g of recombinant WbgN. The reaction was performed
at 37 °C for 1 h, and quenched by adding equal volume of ice cold ethanol. After brief centrifugation, the
supernatant was analyzed by mass spectrometry.
Kinetics study
To study the kinetics of WbgN towards GDP-colitose and GDP-L-Fuc, reactions were performed at
37 °C for 10 min in a reaction buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM Gal1,3-GlcNAc, 5.25
M recombinant WbgN and varied concentrations of GDP-colitose or GDP-L-Fuc (0.25, 0.5, 0.75, 1.0,
2.0, and 4.0 mM). To study the kinetics of WbgN towards an acceptor Gal1,3-GlcNAc, reactions were
performed at 37 °C for 10 min in a reaction buffer containing 20 mM Tris-HCl (pH 8.0), 5 mM GDP-
colitose, 18 g recombinant WbgN and varied concentrations of Gal1,3-GlcNAc (0.25, 0.5, 0.75, 1.0,

15
2.0, and 4.0 mM). Reactions were quenched immediately by addition of equal volume of ice-cold ethanol
and subject to capillary electrophoresis (CE) analysis using Beckman Coulter P/ACE MDQ Capillary
electrophoresis to detect substrate GDP-colitose or GDP-Fuc and by-product GDP. CE conditions were
as follows: 75 m i.d. capillary, 25 KV/170 A, 5 s vacuum injection, with monitoring at 254 nm and 50
mM sodium tetraborate running buffer, pH 9.4. The parameters Km and Vmax were obtained by
Lineweaver-Burk plots of substrate concentration-initial velocity.
Biophysical property study
To study pH dependence of WbgN, reactions were carried out in a total volume of 50 L, containing
20 g of recombinant WbgN, 5 mM GDP-colitose, 5 mM Gal1,3-GlcNAc, and 50 mM of different
buffer (PBS, pH 6.5; Tris-HCl, pH 7.5; Tris-HCl, pH 7.5; Glycine-NaOH, pH 9.0; Glycine-NaOH, 10.2).
To study metal ion dependence, reactions were carried out in a total volume of 50 L, containing 50 mM
Tris-HCl, pH 8.0, 10 g of recombinant WbgN, 5 mM GDP-colitose, 5 mM Gal1,3-GlcNAc, and 10
mM of different additions (EDTA, None, MgCl2, MnCl2, CoCl2, CaCl2, ZnCl2 or CuCl2). A negative
control was also carried out containing EDTA but no acceptor Gal1,3-GalNAc. All reactions were
performed in triplicate. Reactions were incubated at 37 °C for 60 min, quenched and analyzed by CE as
described above.
Acceptor specificity study
Reactions were carried out in a total volume of 50 L, containing 50 mM Tris-HCl, pH 8.0, 10 g of
recombinant WbgN, 5 mM GDP-colitose, and 2.5 mM of different acceptors, including galactose (Gal),
Gal1,3GlcNAc, Gal1,4GlcNAc (LacNAc), Gal1,3GalNAc, and Gal1,4Glc (Lac). All reactions were
performed in triplicate. Reactions were incubated at 37 °C for 60 min, quenched and analyzed by CE as
described above.

16
Enzymatic synthesis of Col1,2-Gal1,3-GlcNAc and Type 1 H-antigen
In a 15 mL centrifuge tube, reactions were carried out in a total volume of 5 mL, containing 50 mM
Tris-HCl, pH 8.0, 300 g of recombinant WbgN, 20 mM GDP-colitose (or GDP-L-Fuc for the synthesis
of type 1 H-antigen), and 16 mM of Gal1,3-GlcNAc. The reactions were allowed to proceed at 37 °C
and monitored by TLC (developing solvent: isopropanol: NH4OH: H2O = 7: 3: 2, v/v/v). After Gal1,3-
GlcNAc was totally converted to products, reactions were quenched by the addition of equal volumes of
ice-cold ethanol, followed by centrifugation at 4 °C for 10 min (10,000 × g). The supernatant was ten
concentrated and subject for separation by P2 gel filtration. The fractions contain pure products were
pooled, lyophilized, and stored at -20 °C until chemical characterization. The fractions contain impure
products were also collected and treated with shrimp alkaline phosphatase (New England Biolabs) to
digest GDP byproduct following instructions (50 U, 30 °C for 5 h), and then subject to bio-gel P2 for
further separation. NMR and ESI HRMS data of trisaccharides (see Supplementary Data 4, 5 for
spectra):
Col1,2-Gal1,3-GlcNAc: 1H NMR (D2O, 400 MHz):δ 1.16 (t, J = 5.6 Hz 3 H, CH3), 1.76-1.87 (m, 1 H,
H′′-3), 1.93-1.96 (m, 1 H, H′′-3), 2.07 (s, 3 H, NHCOCH3), 3.52-3.57 (m, 1 H, H′-2 ), 3.63 (dd, J = 4.8 Hz,
J = 6.8 Hz,1 H, H′′-2), 3.70-3.71(m, 1 H, H-2), 3.73-3.76 (m, 2 H), 3.79-3.83 (m, 4 H), 3.90-3.93 (m, 2 H),
3.99-4.03 (m, 1 H), 4.15-4.28 (m, 2 H), 4.63-4.71 (m, 2.2 H, H′′-5, H′-1, H-1-), 5.12 ( m, 1.8 H, H-1-α,
H′′-1); 13C NMR (D2O, 100 MHz): δ 15.25 (CH3), 21.79 (NHCOCH3), 32.74 (C′′-3), 53.63, 60.43, 60.93,
63.03, 66.05, 68.29, 68.59, 69.01, 71.14 (C′′-2), 73.48 (C-2), 74.86, 74.98, 76.41 (C′-2), 90.68 (C-1-α),
95.46 (C-1-), 98.73 (C′′-1), 100.12 (C′-1), 173.55 (NHCOCH3); ESI HRMS: m/z calcd for
[C20H35NO14+Na]+, 536.1955, found: 536.1933
Fuc1,2-Gal1,3-GlcNAc: 1H NMR (D2O, 400 MHz): δ 1.23 (t, J = 5.6 Hz, 3 H, CH3), 2.08 (s, 3 H,
NHCOCH3), 3.52-3.65 (m, 2 H, H′-2, H′′-2), 3.68-3.90 (m, 9 H), 4.14-4.33 (m, 2 H), 4.63-4.70 (m, 2.3 H,
H′′-5, H′-1, H-1-), 5.12 (d, J = 2.8 Hz, 0.7 H, H-1-α), 5.20 ( d, J = 4.0 Hz, 1 H, H′′-1); 13C NMR (D2O,
100 MHz): δ 14.41 (CH3), 21.06 (NHCOCH3), 52.87, 59.69, 60.18, 60.23, 65.60, 67.22, 67.85, 68.27,

17
68.66, 70.41, 70.97, 72.68, 74.14, 75.76, 89.94 (C-1-α), 98.58 (C′′-1), 99.25 (C-1-), 99.34 (C′-1), 172.79
(NHCOCH3); ESI HRMS: m/z calcd for [C20H35NO15 +Na]+ 552.1904, found: 552.1888.
Supplementary Data
Supplementary data for this article is available online at http://glycob.oxfordjournals.org/.
Funding
This work was supported by National Institute of General Medical Sciences (U01GM116263 to P.G.
Wang & L. Li, R01GM085267 to P.G. Wang) and National Institute of Allergy and Infectious Diseases
(R01AI083754 to P.G. Wang).
Abbreviations
ADP, adenine 5’-diphosphate; ATP, adenine 5’-triphosphate; CDP, cytosine 5’-diphosphate; Gal,
galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Col, colitose;
GDP; guanosine 5'-diphosphate; GDP-L-Fuc, GDP-L-fucose; GNB, Galacto-N-biose; GTP, guanosine 5'-
triphosphate; L-Fuc, L-fucose; LNB, Lacto-N-biose.
Conflict of interest
None declared.

18
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Legends to Figures
Figure 1. De novo biosynthetic pathways of GDP-L-galactose, GDP-L-Fuc and GDP-colitose. GMP,
GDP-mannose pyrophosphorylase; GME, GDP-mannose 3,5-epimerase; GMD, GDP-mannose 4,6-
dehydratase; GFS, GDP-L-Fuc synthase; ColD, GDP-4-keto-6-deoxy-mannose-3-dehydratase; ColC,
GDP-colitose synthase; GKDM, GDP-4-keto-6-deoxy-mannose. GKDDM, GDP-4-keto-3,6-dideoxy-
mannose; PLP, pyridoxal-5’-phosphate.
Figure 2. Structure of E. coli O55:H7 O-antigen repeating unit. WbgN, putative 1,2-
colitosyltransferases. The repeating units are polymerized between the GalNAc and GlcNAc residue to
form O-antigen.
Figure 3. The salavage biosynthetic pathway of GDP-L-Fuc and chemoenzymatic synthesis of GDP-
colitose. FKP, L-fucokinase/GDP-L-Fuc pyrophosphorylase from Bacteroides fragilis 9343; EcPPA,
inorganic pyrophosphatase from E. coli O157.
Figure 4. TLC analysis of FKP catalyzed reactions. 1, ATP; 2, GTP; 3, ADP; 4, colitose; 5, FKP reaction
containing colitose, ATP and MgCl2; 6, 5 + GTP and EcPpA; 7, L-Fuc; 8, FKP reaction containing L-Fuc,
ATP and MgCl2; 9, 8 + GTP and EcPpA; 10, GDP-L-Fuc.
Figure 5. SDS-PAGE analysis of gene expression and purification (lanes 1-7) and western blot analysis
of purified WbgN (lane 8). 1, before IPTG induction; 2, after IPTG induction; 3, supernatant; 4,
precipitate; 5, wash; 6, flow through; 7, eluted enzyme; 8, western blot of purified WbgN using anti-His
tag antibody as first antibody.
Figure 6. Biophysical studies of recombinant WbgN (buffers were used in a concentration of 50 mM). (A)
pH dependent study, and (B) metal ion dependent study (metal ion were used in a concentration of 10
mM).

24
Tables
Table 1. Kinetic parameters of recombinant WbgN. LNB was used as an acceptor to study kinetics
towards a sugar donor, and GDP-colitose was used as a sugar donor to study kinetics towards the acceptor
LNB.
Substrate
Km (mM)
Vmax (m mg-1)
0.046 ± 0.004
0.062 ± 0.008
0.027 ± 0.003
kcat (min-1)
8.8 ± 0.8
12 ± 1.5
5.2 ± 0.6
kcat/Km (min-1mM-1)
GDP-colitose
GDP-L-Fuc
Gal1,3-GlcNAc
0.76 ± 0.18
1.3 ± 0.42
1.4 ± 0.34
12a
9.2a
3.7a
a kcat/Km values were calculated using the average values of Km and kcat.

25
Table 2. Relative acceptor specificity of WbgN. Activity of WbgN towards LNB in presence of GDP-
colitose was set as 100%.
Sugar Nucleotide Donor
Acceptor
GDP-colitose GDP-L-Fuc
Gal
<1%
<1%
100%
~1%
<1%
<1%
98%
~1%
GlcNAc
Gal1,3-GlcNAc (LNB)
Gal1,3-GalNAc (GNB)
Gal1,4-GlcNAc (LacNAc) <1%
Gal1,4-Gal (Lac)
<1%
<1%
<1%

26

27

28

29

30