Highly efficient chemoenzymatic synthesis of β1-4-linked galactosides with promiscuous bacterial β1-4-galactosyltransferases

Article abstract of DOI:10.1039/c0cc01381a

Two bacterial β1-4-galactosyltransferases, NmLgtB and Hp1-4GalT, exhibit promiscuous and complementary acceptor substrate specificity. They have been used in an efficient one-pot multienzyme system to synthesize LacNAc, lactose, and their derivatives including those containing negatively charged 6-O-sulfated GlcNAc and C2-substituted GlcNAc or Glc, from monosaccharide derivatives and inexpensive Glc-1-P.

Full text of DOI:10.1039/c0cc01381a

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Highly efficient chemoenzymatic synthesis of b1–4-linked galactosides
with promiscuous bacterial b1–4-galactosyltransferasesw
Kam Lau, Vireak Thon, Hai Yu, Li Ding, Yi Chen, Musleh M. Muthana, Denton Wong,
Ronald Huang and Xi Chen*
Received 13th May 2010, Accepted 7th June 2010
DOI: 10.1039/c0cc01381a
Two bacterial b1–4-galactosyltransferases, NmLgtB and
Hp1–4GalT, exhibit promiscuous and complementary acceptor
substrate specificity. They have been used in an efficient one-pot
multienzyme system to synthesize LacNAc, lactose, and their
derivatives including those containing negatively charged
6-O-sulfated GlcNAc and C2-substituted GlcNAc or Glc, from
monosaccharide derivatives and inexpensive Glc-1-P.
galactose residue to the terminal GlcNAc, Glc, and their
modified derivatives on oligosaccharides and glycoconjugates.
b1–4-Galactosyltransferases (b1–4GalTs) are enzymes that
catalyze the transfer of galactose (Gal) from sugar nucleotide
UDP-Gal to N-acetylglucosamine (GlcNAc) or glucose (Glc)
for the formation of b1–4-galactosyl linkage in LacNAc and
lactose. Bovine b1–4-galactosyltransferase T1 (b1–4GalT1) is
one of the most well studied mammalian glycosyltransferases.11
It uses GlcNAc as an acceptor in the absence of a-lactalbumin
(LA), a calcium binding protein. In the presence of a-lactalbumin,
the formation of b1–4GalT1-LA complex or lactose synthase
alters the acceptor specificity of b1–4GalT1 from GlcNAc to
glucose.12 The protein has been applied in the synthesis of
LacNAc-containing structures.10a Acceptor substrate specificity
assays using derivatives of methyl 2-acetamido-2-deoxy-b-D-
glucopyranoside (GlcNAcbOMe) in which one of the hydrogen
in the CH3 at the C-2 N-acetyl group and the hydrogen in the
–OH at different positions of GlcNAc was systematically
substituting with a methyl group indicated that 3-methyl ether
was a much poorer acceptor substrate but 6-methyl ether can
be well tolerated by the enzyme.13 The enzyme can tolerate
GlcNAc with an a1–6-linked L-fucose or an a2–6-linked
methyl ester of N-acetylneuraminic acid (Neu5Ac) but not
free Neu5Ac as acceptors.13 GlcNAc derivatives with different
C2 N-acyl groups from propionyl to dodecanoyl and other
N-acyl groups have been shown to be tolerable acceptors.14
N-Acetyl-D-muramic acid, the 3-O-lactyl ether of GlcNAc,
was also shown to be an acceptor for the enzyme.13,15
b1–4-Linked galactosides such as Galb1–4GlcNAc (N-acetyl-
lactosamine or LacNAc) and Galb1–4Glc (lactose or Lac) are
common glycan motifs in nature. The former has been
found broadly in oligosaccharides, N- and O-linked glyco-
proteins, and glycolipids; while the latter has been found in
oligosaccharides and glycolipids. Quite often, naturally
occurring modifications can take place on the Gal and/or
GlcNAc after the formation of oligosaccharides and glyco-
conjugates,1 resulting in diverse carbohydrate post-glycosylational
modifications (PGMs).2 Common natural modifications are
6-O-sulfation or 3-O-sulfation on the Gal,3 and/or 6-O-sulfation
on the GlcNAc.4 For example, keratan sulfate (KS)
[3Galb1–4GlcNAcb1–]n is a poly-O-sulfated polysaccharide
with numerous 6-O-sulfation on some of the Gal and GlcNAc
residues.5 6-O-Sulfation on Gal or GlcNAc is also common in
Lewis x and sialyl Lewis x structures6 and they have been
shown to affect the binding affinity of the related carbohydrate-
binding proteins (e.g. Selectins7 and Siglecs8). In addition,
N-sulfation can sometimes take place on the N-deacetylated
GlcNAc to give N-sulfo-glucosamine (GlcNS)-containing
structures.9 Although enzymatic synthesis of Galb1–4GlcNAc
has been successfully achieved using b1–4-galactosyltransferases
with or without the generation or regeneration of sugar
nucleotide donor UDP-Gal,10 the investigation of the enzymatic
systems in synthesizing structures with modified galactosyl-
transferase acceptors, especially those with naturally occurring
modifications including N- and O-sulfation, has been limited.
Here, we report the promiscuous acceptor substrate specificity
of two bacterial b1–4-galactosyltransferases and their application
in efficient one-pot multienzyme chemoenzymatic synthesis of
b1–4-linked galactosides containing sulfated GlcNAc. The method
can be used as a general approach to add a b1–4-linked
Many bacteria express b1–4GalT for the formation of
capsular polysaccharides (CPS) and lipopolysaccharides
(LPS).16 In fact, b1–4GalTs encoded by the lgtB gene of
Neisseria meningitidis and Neisseria gonorrhoeae have been
cloned and expressed in Escherichia coli.16a,b A b1–4GalT
encoded by HP0826 gene of Helicobacter pylori (Hp1–4GalT)
has also been identified.16c,17 The LgtB from Neisseria meningitidis
serogroup B strain and the Hp1–4GalT were shown to use
para-nitrophenyl-b-D-mannopyranoside (ManbpNP) as an
acceptor for the formation of Galb1–4ManbpNP. The
Hp1–4GalT can also catalyze the efficient synthesis of a
thio-linked LacNAc derivative Galb1-S-4GlcNAcbpNP.18
Nonetheless, bacterial b1–4GalTs have not been used in
synthesizing LacNAc and lactose derivatives containing
naturally existing negatively charged O- or N-sulfated
GlcNAc. Here, we report the cloning of two b1–4GalTs, from
Neisseria meniningitidis serogroup B strain MC58 genomic
DNA (ATCC BAA-335D) (GenBank accession number
AE002098) and Helicobacter pylori strain J99 genomic DNA
(ATCC 700824D) (GenBank accession number AB035971)
Department of Chemistry, University of California,
One Shields Avenue, Davis, California 95616, USA.
E-mail: chen@chem.ucdavis.edu; Fax: (+1 530) 754-6037;
Tel: (+1 530) 752-8995
w Electronic supplementary information (ESI) available: Experimental
details for cloning and characterization of enzymes, chemical and
enzymatic synthesis, NMR and HRMS data. See DOI: 10.1039/
c0cc01381a
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respectively, and their application in one-pot multienzyme
synthesis of LacNAc and Lac derivatives with natural and
non-natural modifications.
was used to produce UDP-glucose (UDP-Glc) which was
epimerized by an E. coli UDP-galactose-4-epimerase (GalE)21
to form UDP-Gal which was then used by the NmLgtB or
Hp1–4GalT to synthesize disaccharide products. A C-terminal
His6-tagged inorganic pyrophosphatase (PmPpA-His6) cloned
from Pasteurella multocida (See Supporting Information) was
used to hydrolyze the pyrophosphate formed in GalU-
catalyzed reaction to improve the yield of the synthesis.
Both NmLgtB and Hp1–4GalT exhibited substrate
promiscuity in preparative-scale synthesis of b1–4-linked
galactosides using the one-pot four-enzyme system shown in
Scheme 1. More interestingly, they showed complementary
acceptor substrate specificities. As shown in Table 1, both
NmLgtB and Hp1–4GalT can use free GlcNAc (1) and its
corresponding b-glycosides (2 and 4) as acceptor substrates
(64–85% yields). Interestingly, GlcNAc a-glycoside (3) was a
substrate for NmLgtB, but not for Hp1–4GalT. On the other
hand, 6-O-sulfated GlcNAc (5) and its b-glycoside (6) were
acceptable substrates for Hp1–4GalT, but not for NmLgtB.
In contrast to that reported,18 however, Glc (7) and b-D-
glucoside (GlcbOBn 8) as well as N-modified GlcNAc derivatives
(9–11) including N-sulfated glucosamine (10) were not acceptors
for Hp1–4GalT. On the contrary, they (7–11) were perfectly
good acceptors for NmLgtB. The Galb1–4-linked disaccharides
(18–22) were synthesized in 68–83% yields. The N-TFA group
and the N3 group in disaccharide products 20 and 22, respectively,
can be readily converted to a free amine, allowing further
derivatization to generate more diverse N-substituted GlcNAc
analogs.22 Precautions were taken in characterizing the structures
of the products using NMR. Different from LgtC (an Neisseria
meningitidis a1–4-galactosyltransferase)-catalyzed reactions
which resulted in altered regio-selectivity when different
acceptors were used,23 all of the glycosidic linkages formed
by the NmLgtB and Hp1–4GalT here were confirmed to be
b1–4-type.
In summary, NmLgtB can use Glc, both a- and b-linked
GlcNAc, and N-modified but not 6-O-sulfated GlcNAc as
acceptors. Quite interestingly, while Hp1–4GalT can not use
Glc, a-linked GlcNAc, and N-modified GlcNAc as acceptors,
it can use 6-O-sulfated GlcNAc such as GlcNAc6S (5) and
GlcNAc6SbProN3 (6) as suitable acceptor substrates. Therefore,
NmLgtB and Hp1–4GalT complement each other in efficient
synthesis of a broad range of LacNAc, Lac, and their derivatives
with charged and non-charged substitutions, many of which
are naturally existing glycans with post-glycosylational
modification (PGMs).2 The promiscuous bacterial enzymes
and the one-pot multi-enzyme approach allow the access to
diverse structural modified oligosaccharides from monosaccharide
derivatives which are readily accessible by simple chemical
synthesis (e.g., 6-O-sulfated GlcNAc 5–6 and N-sulfated
glucosamine 10 were obtained by one-step selective sulfation
from the corresponding unprotected glycans, see Supporting
Information). We show here once again the power of promiscuous
bacterial carbohydrate-biosynthetic enzymes in novel synthesis
of diverse and complex carbohydrates.
Neisseria meniningitidis b1–4GalT (NmLgtB) was cloned
into pET15b vector and expressed in Escherichia coli BL21
(DE3) cells as N-terminal His6-tagged fusion proteins. Under
common expression conditions (20 1C for 20 h with shaking at
250 rpm in LB medium containing 10 g/L tryptone, 5 g/L yeast
extract, and 10 g/L NaCl), only a low amount (0.7–0.8 mg/L
culture) of soluble active enzyme was obtained. The expression
level was improved 15-fold to 12.5 mg/L culture under osmotic
stress condition using LB medium supplemented with 0.2%
glucose and a higher concentration of NaCl (total NaCl
concentration was 0.5 M).19 NmLgtB was not stable when
stored at 4 1C and was degraded into fragments in 1–2 days.
Adding 10% glycerol in the storage buffer and storing at
À20 1C improved its stability significantly. Under these
conditions, the enzyme remained stable with no activity lost
for up to 14 months. Helicobacter pylori b1–4GalT
(Hp1–4GalT) was initially cloned as N- and C-His6-tagged
fusion proteins but the expression level was low (o0.4 mg/L
culture) for either constructs. It was subsequently cloned into
pMAL-c4X vector and expressed in Escherichia coli Origami B
(DE3) cells as an N-maltose binding protein (MBP)-tagged
and C-His6-tagged fusion protein. The C-His6-tag was
introduced during cloning to facilitate easy purification of
the protein using Ni2+-affinity columns. The resulted
Hp1–4GalT was routinely expressed at a level of B5 mg/L
culture in LB medium by incubating at 25 1C for 18 h with
shaking at 250 rpm after induction with 0.3 mM of isopropyl-
1-thio-b-D-galactopyranoside (IPTG).
Biochemical characterization of NmLgtB and Hp1–4GalT
using 4-methylumbelliferyl-b-D-GlcNAc (GlcNAcbMU) and
4-methylumbelliferryl b-D-Glc (GlcbMU) indicates that
while Hp1–4GalT specifically recognizes GlcNAc-terminated
glycans as acceptors, NmLgtB can use both GlcNAc- and
Glc-terminated glycans as acceptor substrates. Unlike bovine
b1–4GalT1, the tolerance of Glc as an acceptor substrate by
NmLgtB does not require the presence of a-lactalbumin.
To decrease the cost and increase the efficiency for
b1–4GalT-catalyzed preparative-scale synthesis of LacNAc,
Lac, and their derivatives with various modifications, a simple
one-pot multienzyme system (Scheme 1) was used to generate
UDP-Gal (the sugar nucleotide donor of b1–4GalTs) in situ
from inexpensive glucose-1-phosphate (Glc-1-P). In this
system, an E. coli K-12 Glc-1-P uridylyltransferase (GalU)20
Scheme 1 One-pot multienzyme synthesis of b1–4-linked galactosides
with in situ generation of UDP-Gal. GalU, glucose-1-phosphate
uridylyltransferase (or UDP-glucose pyrophosphorylase); GalE,
UDP-galactose C4-epimerase; PpA, Pasteurella multocida inorganic
pyrophosphatase. NmLgtB, Neiserria meningitidis b1–4-galactosylatrans-
ferase; Hp1–4GalT, Helicobacter pylori b1–4-galactosylatransferase.
This work was supported by NSF CAREER Award
CHE-0548235, NIH grants R01GM076360 and U01CA128442.
X.C. is an Alfred P. Sloan Research Fellow, a Camille Dreyfus
Teacher-Scholar, and a UC-Davis Chancellor’s Fellow.
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Table 1 One-pot four-enzyme synthesis of galactosides from Glc-1-P,
UTP, and glucose or N-acetyl glucosamine derivatives using GalU,
PmPpA, GalE, and NmLgtB or Hp1–4GalT
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This journal is The Royal Society of Chemistry 2010
6068 | Chem. Commun., 2010, 46, 6066–6068