Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP)

Article abstract of DOI:10.1039/c2cc17577k

A promiscuous UDP-sugar pyrophosphorylase (BLUSP) was cloned from Bifidobacterium longum strain ATCC55813 and used efficiently with a Pasteurella multocida inorganic pyrophosphatase (PmPpA) with or without a monosaccharide 1-kinase for one-pot multienzyme synthesis of UDP-galactose, UDP-glucose, UDP-mannose, and their derivatives. Further chemical diversification of a UDP-mannose derivative resulted in the formation of UDP-N-acetylmannosamine. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc17577k

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COMMUNICATION
Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous
UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP)w
a
a
Musleh M. Muthana,z Jingyao Qu,z Yanhong Li,a Lei Zhang,a Hai Yu,a Li Ding,ab
Hamed Malekana and Xi Chen*a
Received 5th December 2011, Accepted 9th January 2012
DOI: 10.1039/c2cc17577k
A promiscuous UDP-sugar pyrophosphorylase (BLUSP) was cloned
from Bifidobacterium longum strain ATCC55813 and used efficiently
with a Pasteurella multocida inorganic pyrophosphatase (PmPpA)
with or without a monosaccharide 1-kinase for one-pot multienzyme
synthesis of UDP-galactose, UDP-glucose, UDP-mannose, and their
derivatives. Further chemical diversification of a UDP-mannose
derivative resulted in the formation of UDP-N-acetylmannosamine.
the less common access to UTP:galactose-1-phosphate uridylyl-
transferases or UDP-Gal pyrophosphorylases (EC 2.7.7.10) for
direct formation of UDP-Gal from Gal-1-phosphate and UTP.
For example, UDP-Gal used in galactosyltransferase-catalyzed
enzymatic synthesis of galactosides has been more frequently
obtained from UDP-Glc by reactions catalyzed by UDP-Gal
4-epimerases13–16 or UDP-glucose:galactose-1-phosphate uridylyl-
transferases (EC 2.7.7.12, GalT or GalPUT) in the Leloir
pathway.17,18
Carbohydrates are widespread in nature and play pivotal roles
in biological systems. The key enzymes for the formation of
glycosidic bonds in carbohydrates are glycosyltransferases.
Most glycosyltransferases require monosaccharide nucleotides as
the common activated donor substrates. Among monosaccharide
nucleotides used by mammalian glycosyltransferases, many are
uridine 50-diphosphate (UDP)-monosaccharides such as UDP-
glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-glucuronic
acid (UDP-GlcA), UDP-N-acetylglucosamine (UDP-GlcNAc),
UDP-N-acetylgalactosamine (UDP-GalNAc), and UDP-xylose
(UDP-Xyl).1 In addition, UDP-mannose (UDP-Man) has been
isolated from Mycobacterium smegmatis and proposed to be an
intermediate in the biosynthesis of mycobacterial polysaccharides.2
Furthermore, UDP-N-acetylmannosamine (UDP-ManNAc) and
UDP-N-acetylmannosaminuronic acid (UDP-ManNAcA) have
been used by some bacteria for producing capsular polysaccharides
containing ManNAc3 or ManNAcA4,5 residues or forming
ManNAcb1-4GlcNAc-PP-undecaprenol (lipid II) for the bio-
synthesis of cell wall teichoic acids of Gram-positive bacteria.6–10
The simplest biosynthetic route for obtaining monosaccharide
nucleotides such as UDP-monosaccharides usually involves the
formation of a monosaccharide-1-phosphate catalyzed by a
monosaccharide-1-phosphate kinase followed by the formation of
monosaccharide nucleotides catalyzed by a nucleotidyltransferase
(or pyrophosphorylase).11,12 However, the simplest route has
not been applied routinely for the formation of UDP-Gal due to
Nevertheless, UDP-galactose pyrophosphorylase activity
was identified from yeast Saccharomyces fragilis,19 pigeon liver,
and mammalian livers.20 The enzyme was purified from bovine
liver20 and Gram-positive bacterium Bifidobacterium bifidum.21
Recently, promiscuous UDP-sugar pyrophosphorylases (USPs)
(EC 2.7.7.64) that can use various monosaccharide 1-phosphates
in the presence of UTP for direct synthesis of UDP-mono-
saccharides including UDP-Glc, UDP-Gal, UDP-GlcA, etc.
were cloned from plants such as pea (Pisum sativum L.) sprouts
(PsUSP)22 and Arabidopsis thaliana (AtUSP).23 Enzymes which
share sequence homology to plant USPs were also cloned from
Leishmania major and Trypanosoma cruzi, two trypanosomatid
protozoan parasites, and were shown to have good activity
towards Gal-1-P and Glc-1-P and weaker activity towards
xylose-1-phosphate and GlcA-1-P.24,25 A USP with broad
substrate specificity and optimal activity at 99 1C was also
cloned from a hyperthermophile archaea Pyrococcus furiosus
DSM 3638 for which Glc-1-P, Man-1-P, Gal-1-P, Fuc-1-P,
GlcNH2-1-P, GalNH2-1-P, and GlcNAc-1-P were all shown
to be tolerable substrates, and both UTP and dTTP could be
used as nucleotide triphosphate substrates by the enzyme.26
Nevertheless, none of these enzymes has been used in preparative-
scale or large-scale synthesis of sugar nucleotides and non-natural
derivatives of monosaccharide-1-P have not been tested as
substrates for USPs.
Here we report the cloning of a promiscuous USP from a
probiotic Bifidobacterium longum strain ATCC55813 and its
application in an efficient one-pot three-enzyme system for
preparative-scale synthesis of UDP-monosaccharides and
their derivatives from simple monosaccharides or derivatives
(except for UDP-Glc which was synthesized from Glc-1-P in
a one-pot two-enzyme system as discussed below). These
compounds will be tested as potential donor substrates for
various glycosyltransferases.
a Department of Chemistry, University of California,
One Shields Avenue, Davis, California 95616, USA.
E-mail: chen@chem.ucdavis.edu; Fax: +1 530-752-8995;
Tel: +1 530-754-6037
b College of Science, Northwest A&F University, Yangling,
Shaanxi, China
w Electronic supplementary information (ESI) available: Experimental
details for cloning, expression, and purification of BLUSP, chemical and
enzymatic synthesis, NMR and HRMS data. See DOI: 10.1039/c2cc17577k
z These authors contributed equally.
c
2728 Chem. Commun., 2012, 48, 2728–2730
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Table 1 Synthesis of UDP-monosaccharides using the one-pot three-
enzyme system shown in Scheme 1. ND, not detected
Substrate
Kinase
Product
Yield (%)
86
EcGalK or SpGalK
Scheme 1 One-pot three-enzyme system for the synthesis of UDP-
monosaccharides and derivatives. Enzymes used: NahK_ATCC15697,
Bifidobacterium infantis strain ATCC15697 N-acetylhexosamine 1-kinase;28
SpGalK, Streptococcus pneumoniae TIGR4 galactokinase;27 EcGalK,
E. coli galactokinase;17 BLUSP, Bifidobacterium longum UDP-sugar
pyrophosphorylase; PmPpA, Pasteurella multocida inorganic
pyrophosphatase.29
EcGalK or SpGalK
ND
ND
ND
In general, three enzymes were used in one-pot to synthesize
UDP-monosaccharides including UDP-Gal, UDP-Glc, UDP-
Man, and their derivatives. As shown in Scheme 1, the first
enzyme was a monosaccharide 1-kinase selected from a galacto-
kinase cloned from Escherichia coli K-12 (EcGalK),17 a galacto-
kinase cloned from Streptococcus pneumoniae TIGR4 (SpGalK),27
or an N-acetylhexosamine 1-kinase cloned from Bifidobacterium
infantis strain ATCC15697 (NahK_ATCC15697).28 The
second enzyme was a promiscuous USP that we cloned from
Bifidobacterium longum ATCC55813 (BLUSP) (see ESIw for
details about cloning, expression, and purification). It catalyzes
the reversible formation of UDP-monosaccharide and pyropho-
sphate from UTP and monosaccharide-1-phosphate (UTP +
monosaccharide-1-phosphate " UDP-monosaccharide + PPi)
with tolerance of some substrate modifications. The third
enzyme was an inorganic pyrophosphatase that we cloned from
Pasteurella multocida strain P-1059 (ATCC15742) (PmPpA)29
for hydrolyzing the pyrophosphate by-product formed by
BLUSP to drive the reaction towards the formation of
UDP-monosaccharides and derivatives.
EcGalK
SpGalK
SpGalK
None
ND
99
NahK
NahK
NahK
56
43
61
Prior to applying the one-pot three-enzyme system shown in
Scheme 1 to the preparative-scale synthesis of the UDP-sugars,
galactose (1) and its C2-derivatives (2–5), glucose and its
C2-derivatives (7–10), as well as mannose (11) and its C2-derivatives
(12–15) (Table 1) were tested as substrates for various mono-
saccharide-1-kinases including SpGalK, EcGalK, and NahK
in small-scale reactions containing Tris–HCl buffer (100 mM,
pH 8.0), monosaccharide or a derivative (15 mM), MgCl2
(10 mM), ATP (18 mM), and a kinase (0.2 mg mLꢀ1). The
reactions were carried out at 37 1C for 1 h, 4 h, and 24 h and
analyzed by thin layer chromatography (TLC) and capillary
electrophoresis assays. We were able to identify at least one of
these kinases for each monosaccharide that gave a yield higher than
58% for the formation of the corresponding monosaccharide-1-
phosphate. The observed results from thin-layer chromatography
(TLC) and capillary electrophoresis (CE) (Table S1 and Fig. S4,
ESIw) confirmed the previously reported activities of NahK,
SpGalK, and EcGalK toward their respective substrates except for
mannosamine (13), a NahK substrate which was not previously
tested.27,28,30
NahK
ND
NahK
NahK
60
92
As the one-pot three-enzyme system was not able to synthesize
UDP-Glc from Glc efficiently in small-scale assays (o20% yield
was observed), inexpensive and commercially available Glc-1-P (6)
was used in a two-enzyme system containing BLUSP and
PmPpA for the high-efficient synthesis of UDP-Glc (21) in
99% yield.
NahK
NahK
ND
90
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 2728–2730 2729

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Table 1 (continued )
Substrate Kinase
are excellent probes for testing the donor substrate specificity of
diverse glycosyltransferases.
Product
Yield (%)
ND
This work was supported by an NIH grant R01HD065122
and an NSF grant CHE-1012511. X. Chen is a Camille Dreyfus
Teacher-Scholar and a UC-Davis Chancellor’s Fellow. The
authors would like to thank Professor Min Chen of Shandong
University in China and Professor Peng George Wang of
Georgia State University for providing the plasmid of SpGalK.
NahK
Notes and references
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Scheme 2 Synthesis of UDP-ManNAc (30) from UDP-ManN3 (29)
in 79% yield via the formation of UDP-ManNH2 (28) by catalytic
hydrogenation followed by acetylation.
The synthesis of all other UDP-sugars listed in Table 1 was
carried out using the one-pot three-enzyme system shown in
Scheme 1. As shown in Table 1, the one-pot three-enzyme
system provided excellent yields for the formation of UDP-Gal
(16, 86%), UDP-ManF (27, 92%), and UDP-ManN3 (29, 90%)
from the corresponding monosaccharides Gal (1), ManF (12),
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UDP-Glc including UDP-2-deoxyGlc (22), UDP-GlcNH2
(23), and UDP-GlcN3 (24) were obtained from 2-deoxyGlc
(7), glucosamine (GlcNH2, 8) and GlcN3 (9) in 56%, 43%, and
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corresponding monosaccharides (10, 13, and 15) using the
one-pot three-enzyme system. These were most likely due to
the substrate restriction of BLUSP instead of the kinases used.
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not directly available from ManNH2 (13) and ManNAc (15),
respectively, via the one-pot three-enzyme system shown in
Scheme 1, they can be readily prepared via simple chemical
modification reactions from UDP-ManN3 (29) obtained from
the one-pot three-enzyme system. As shown in Scheme 2, a simple
one-step catalytic hydrogenation of UDP-ManN3 (29) produced
UDP-ManNH2 (28). Acetylation of the amino group in UDP-
ManNH2 (28) provided an easy access of UDP-ManNAc (30).
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In conclusion, taking advantage of the substrate promiscuity of
several monosaccharide-1-phosphate kinases and a Bifidobacterium
longum UDP-sugar pyrophosphorylase (BLUSP), we developed
an efficient one-pot multienzyme system to quickly obtain
structurally defined UDP-Gal, UDP-Glc, UDP-Man, and their
derivatives in preparative scales. With additional simple chemical
modifications of the UDP-ManN3 produced by the one-pot
three-enzyme reaction, biosynthetically useful UDP-ManNAc
was readily obtained. The acquired UDP-sugar and derivatives
c
2730 Chem. Commun., 2012, 48, 2728–2730
This journal is The Royal Society of Chemistry 2012