Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP)

Article abstract of DOI:10.1016/j.bmcl.2013.04.090

Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP.

Full text of DOI:10.1016/j.bmcl.2013.04.090

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3764–3768
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar
pyrophosphorylase from Arabidopsis thaliana (AtUSP)
Jun Liu a,b, Yang Zou a,b, Wanyi Guan c, Yafei Zhai a,b, Mengyang Xue a,b, Lan Jin b, Xueer Zhao a,b
,
Junkai Dong b, Wenjun Wang d, Jie Shen d, Peng George Wang a, Min Chen a,b,
⇑
a The State Key Laboratory of Microbial Technology and School of Life Sciences, Jinan, Shandong 250100, China
b National Glycoengineering Research Center, Jinan, Shandong 250100, China
c College of Life Science, Hebei Normal University, Shijiazhuang, Hebei 050016, China
d College of Pharmacy Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metab-
olism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important
for the characterization of glycosyltransferases. Given that limited methods are available for preparation
of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient
one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides.
In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP)
was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophos-
phatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives
of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the
nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP
exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%,
respectively. These results provide abundant information for better understanding of the relationship
between substrate specificity and structural features of AtUSP.
Received 27 March 2013
Revised 25 April 2013
Accepted 30 April 2013
Available online 9 May 2013
Keywords:
UDP-sugar pyrophosphorylase
Galactokinase
One-pot
Nucleotide sugars
Ó 2013 Elsevier Ltd. All rights reserved.
Nucleotide sugars such as UDP-galactose and UDP-glucose are
critical to the survival of all organisms.1 They are activated forms
of monosaccharides and key intermediates of carbohydrate metab-
olism in both prokaryotes and eukaryotes. In primary metabolism,
as the activated donor substrates of Leloir-type glycosyltransfer-
ases, nucleotide sugars play an important role in protein glycosyla-
tion, the biosynthesis of glycogen, and even the synthesis of cell
wall.2–4 In secondary metabolism, nucleotide sugars are used to
synthesize different glycosylated natural products or their deriva-
tives which act as the precursors of many front-line drugs.5–8 Be-
sides, some nucleotide sugars were immediate activators of P2Y14
receptor which promotes the rearrangement of cytoskeleton,
change of cell shape, and enhancement of cell migration.9 With in-
creased attention to biological functions of sugar chains and glyco-
syltransferases,10,11 the availability of nucleotide sugars is
particularly important to such research.
approaches are more powerful and in some cases a single transfor-
mation can substitute several chemical reactions.13,14
In higher plants, sugar nucleotides can be synthesized by both
de novo and salvage pathways.15 For example, UDP-galactose can
be synthesized by epimerization of UDP-glucose in de novo path-
way. However the low yield and difficulty in separating UDP-gal-
actose from UDP-glucose limit the application of this method.
The simplest route for synthesizing UDP-sugars involves the
sequential action of monosaccharide kinases and pyrophosphoryl-
ases in the salvage pathway.16 Thus finding highly active and pro-
miscuous monosaccharide kinases and nucleotide sugar
pyrophosphorylases becomes a key factor to obtain structurally di-
verse nucleotide sugars with high yield. Different sourced UDP-gal-
actose pyrophosphorylases had been identified such as UDP-sugar
pyrophosphorylases (USPs) from pea (Pisum sativum L.) sprouts,
Leishmania major and Trypanosoma cruzi.17–19 However, the
1-phosphorylation forms of monosaccharides were not easily ob-
tained in vitro. And few non-natural derivatives of monosaccha-
ride-1-P as substrates for USPs have been tested. Recently, Chen
et al. cloned a promiscuous UDP-sugar pyrophosphorylase (BLUSP)
from Bifidobacterium longum strain ATCC55813 and used it to-
gether with a monosaccharide 1-kinase to set up a one-pot mul-
ti-enzyme system for efficient synthesis of eight UDP-sugars.20
UDP-sugars can be synthesized by chemical or enzymatic ap-
proaches. Recently, various chemical approaches have been re-
ported.12 Compared with chemical methods, enzymatic
⇑
Corresponding author. Tel.: +86 531 8836 6078; fax: +86 531 8836 3002.
E-mail address: chenmin@sdu.edu.cn (M. Chen).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.090

J. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3764–3768
3765
This is the first time to use the USP in preparative scale synthesis.
And more importantly, non-natural derivatives of monosaccha-
ride-1-P were tested as substrates for USPs.
UDP-Glc (2) in our research. This may be attributed to the less
SpGalK kinase activity for Glc.23 Besides, AtUSP was reported to
have no activity toward L-Fuc (
ever, we synthesize UDP- -6-deoxy-Gal (4, 29%) by our one-pot
system from D-6-deoxy-Gal. This suggests that AtUSP could utilize
-6-deoxy-Gal-1-P. Less than 5% UDP-2-N3-Gal (6) was synthe-
L
-6-deoxy-Gal) by Yoichi,15 how-
UDP-sugar pyrophosphorylase from A. thaliana (AtUSP) exhibits
housekeeping function in nucleotide sugar metabolism.21 It was
characterized with different natural monosaccharide-1-P sub-
strates.15,22 The results indicated that AtUSP had relatively broader
substrate tolerance compared with other UDP-sugar pyrophospho-
rylases and its highest activity occurred to GlcA-1-P, Glc-1-P and
Gal-1-P. Given that limited methods are available for the prepara-
tion of nucleotide sugars, especially their useful non-natural deriv-
atives, we introduced herein an efficient one-step three-enzyme
catalytic system using AtUSP for the synthesis of nucleotide sugars
from monosaccharides.
In this study, we report the cloning of UDP-sugar pyrophos-
phorylase from A. thaliana (AtUSP) and its application in a one-
pot three enzyme system to synthesize four UDP-monosaccharides
from simple monosaccharides or their derivatives. Besides, the
nucleotide triphosphate substrate specificity of AtUSP was system-
atically annotated.
In brief, three enzymes were used in the one-pot system to syn-
thesize UDP-monosaccharides including UDP-Gal, UDP-Glc, UDP-
6-deoxy-Gal and UDP-4-N3-Gal. As shown in Figure 1, the first en-
zyme was a galactokinase cloned from S. pneumoniae TIGR4
(SpGalK)23 to phosphorylate the monosaccharide at C1 position.
The second one was a promiscuous USP cloned from A. thaliana
which catalyzed the reversible formation of UDP-monosaccharide
and pyrophosphate from UTP and monosaccharide-1-phosphate.
The third one was a commercially available inorganic pyrophos-
phatase from Saccharomyces cerevisiae (PPase) for the hydrolysis
of pyrophosphate formed by AtUSP to drive the reaction towards
the formation of UDP-monosaccharides.
To achieve a more efficient reaction for UDP-monosaccharide
preparation, the optimal reaction conditions were investigated in
our research including the optimal pH and temperature, as well
as the concentration of Mg2+. AtUSP was firstly overexpressed and
purified. As shown in Fig. S1, the recombinant AtUSP had an appar-
ent molecular weight of 70 kDa. The soluble AtUSP protein expres-
sion level was about 30 mg/L. The optimal reaction conditions were
explored according to the supporting information. At last, the one-
pot system has a highest reaction conversion at 45 °C, pH 8.0 and
10 mM MgCl2 with ATP, UTP and Gal as substrates.
D
D
sized in the system to the extent that it can be only detected by
MS. If substituting SpGalU (a UTP-glucose-1-phosphate uridylyl-
transferase from S. pneumoniae TIGR4) for AtUSP in the one-pot
system, the reaction conversion for UDP-Gal and UDP-Glc is almost
the same in the AtUSP system or SpGalU system.24 But great differ-
ence was displayed in the synthesis of 3, 4, 5, 6, and 7. For 3, 4, 6
and 7, the SpGalU system got a higher conversion than the AtUSP
system. On the contrary, the AtUSP system showed higher yield
for 5 than SpGalU system. Considering the structural diversity of
the tested monosaccharides, the AtUSP may have better tolerance
for C4-derivatives of Gal-1-P (5) while the SpGalU for C2- and
C6-derivatives of Gal-1-P (3, 4, 6 and 7).
To investigate the uridylyltransferase activity of AtUSP on dif-
ferent nucleotide substrates (NTPs) or deoxynucleotide triphos-
phates (dNTPs), Glc-1-P and nine NTPs or dNTPs were tested as
initial substrates (Fig. 2). The 20
lL reaction mixture containing
50 mM Tirs–HCl, pH 8.0, 10 mM Glc-1-P, 10 mM MgCl2, 0.4
l
M
AtUSP and 10 mM different NTPs or dNTPs (Table 2) was incubated
at 45 °C for 1 h. The reactions were finally detected by thin-layer
chromatography (TLC, Fig. S6) and capillary electrophoresis (CE).
Both dUDP-Gal and dTDP-Glc were synthesized in large scale and
purified by Bio-Gel P2 gel filtration chromatography. Identification
of the purified dUDP-Gal and dTDP-Glc was based on mass spec-
trometry (Fig. S7) and NMR spectrometry (Fig. S8). As shown in
Table 2, AtUSP presented considerable yields toward UTP, dUTP
and dTTP with a conversion of 87%, 85% and 84%, respectively. Sur-
prisingly, among the nine NTPs or dNTPs tested, the UDP-N-acety-
lgalactosamine pyrophosphorylase from Homo sapiens (AGX1)
could also recognize only UTP, dUTP and dTTP.25 Amino acid se-
quence alignment showed that the identity of the two pyrophos-
phorylase was less than 26%. But BLAST analysis of each
pyrophosphorylase suggested that both enzymes belong to the
Glyco_tansf_GTA_type superfamily and had the same conserved
domains. According to previous reports,25–28 the hydrogen bonds
constructed between the exocyclic oxygen O2 and O4 of the uracil
base and Gly110, Gly222 and Gln196 of the AGX1 are crucial for
nucleotide-binding and enzyme catalysis. To understand the
molecular-level interactions at the binding site of the AtUSP sub-
strates, the primary sequence of AtUSP was blast with other pyro-
phosphorylase. It appears that three amino acid residues (Gly137,
Gln224 and Gly253) in AtUSP interact with nucleotides and are
well conserved. The three amino acid residues may act with the
uracil base to form hydrogen bonds.
Based on the optimum conditions, eight monosaccharides listed
in Table 1 were tested as initial substrates to synthesize UDP-
monosaccharides using the one-pot three-enzyme system. Four
UDP-monosaccharides were successfully synthesized from their
corresponding monosaccharides including Gal, Glc, 6-deoxy-Gal
and 4-N3-Gal. As shown in Table 1, the one-pot three-enzyme sys-
tem provided considerable reaction conversion for the formation of
UDP-Gal (1, 95%) and UDP-4-N3-Gal (5, 43%). AtUSP showed high
activity to Glc-1-P, but we got just 28% conversion for the
Kinetic parameters of AtUSP toward UTP, dUTP and dTTP were
determined with Glc-1-P as the sugar donor. Although there was
2Pi
PPase
R3
R3
R3
R4
O
R4
O
R4
O
UTP
PPi
ADP
ATP
Mg2+
Mg2+
R2
HO
R2
HO
R2
HO
SpGalK
AtUSP
OH
R1
R1
OPO3
R1
2-
OUDP
R1=OH, AcHN, N3; R2=H, OH; R3=OH, H, N3; R4=OH, H
Figure 1. One-pot three enzymes synthesis of UDP-sugars. SpGalK, S. pneumoniae TIGR4 galactokinase; AtUSP, A. thaliana UDP-sugar pyrophosphorylase; PPase, inorganic
pyrophosphatase.

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J. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3764–3768
Table 1
Synthesis of UDP-sugars using the one-pot three enzymes system shown in Figure 1
Entry Producs
Reaction conversiona (%)
SpGalU
AtUSP
95
OH
OH
O
1
UDP-Gal
90
HO
HO
HO
OUDP
OH
O
2
3
4
5
6
UDP-Glc
28
26
32
68
<5b
78
HO
HO
OUDP
OH
O
OH
OH
N3
UDP-GalNAc
HO
HO
HO
HO
HO
ND
29
AcNH
OUDP
O
UDP-6-deoxy-Gal (UDP-Fuc)
UDP-4-N3-Gal
OH
OUDP
OUDP
OUDP
OH
O
43
HO
OH
OH
OH
O
UDP-2-N3-Gal
<5b
N3
OH
O
7
8
UDP-2-Deo-Gal
UDP-Man
NDc
ND
15
OUDP
HO
HO
HO
O
NTd
HO
OUDP
a
b
c
The ratio of products to monosaccharide.
Only detected by MS.
No products detected by TLC and CE.
Not test.
d
OH
O
HO
HO
O
P
O-
O
O
P
O-
HO
O
O
OH
R
X
-O
O
P O
O-
O
AtUSP
PPi
O
R
O
O P O P
OH OH
O
HO
HO
O
HO
2-
OPO3
OH
OH
X
X=OH or H; R=Nucleobase
Figure 2. The uridylyltransferase reaction of AtUSP towards NTPs or dNTPs. NTPs, nucleotide triphosphates; dNTPs, deoxynucleotide triphosphates.
little difference in the conversion percentage between the three
NTPs or dNTPs, kinetic parameters for them differed greatly. As
shown in Table 3, the Vmax for UTP was the greatest of the three
NTPs or dNTPs. It was 1.5 times and 8 times of that for dUTP and
dTTP, respectively. But the Km for UTP was threefold lower than
for dTTP. The kcat/Km for UTP, dUTP and dTTP were 120.54 mM
À1 sÀ1, 58.65 mMÀ1 sÀ1 and 3.34 mMÀ1 sÀ1, respectively.
UDP-sugars are the direct substrates of Leloir-type glycosyl-
transferase to synthesize oligosaccharides involved in adhesion
and signaling molecules. The glycan structures on these molecules

J. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3764–3768
3767
Table 2
NTPs or dNTPs substrates specificity study for AtSUP
Entry
Name
Structure
Conversiona (%)
O
NH
N
O
O
O
O
11
UTP
87
HO
HO
P
O
O
P
O
O
P
O
O
O
OH
OH
OH
OH OH
O
NH
N
O
O
O
O
12
13
14
15
dUTP
85
P
P
P
O
OH
OH
OH
OH
O
H3C
NH
N
O
O
O
O
P
dTTP
84
HO
HO
P
O
O
P
O
O
O
OH
OH
OH
OH
NH2
N
N
N
ATP
NDb
ND
ND
O
O
O
P
N
P
P
O
O
O
OH
OH
OH
OH OH
H3C
NH
N
N
N
N
dm6ATP
O
O
O
HO
HO
HO
P
O
O
O
P
O
O
O
P
O
O
O
O
OH
OH
OH
OH
O
N
N
N
NH
NH
O
O
O
NH2
16
GTP
P
P
P
O
OH
OH
OH
OH
OH
O
N
N
N
O
O
O
NH2
17
dGTP
ND
P
P
P
O
OH
OH
OH
OH
(continued on next page)

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J. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3764–3768
Table 2 (continued)
Entry
Name
CTP
Structure
Conversiona (%)
O
N
N
NH
N
O
O
O
NH2
18
ND
HO
HO
P
O
O
P
O
O
P
O
O
O
OH
OH
OH
OH
NH2
N
N
O
O
O
O
19
dCTP
ND
P
P
P
O
OH
OH
OH
OH OH
a
The ratio of products to NTPs or dNTPs.
Not detected by TLC and CE.
b
References and notes
Table 3
Kinetic parameters of purified AtUSP towards UTP, dUTP and dTTP
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and successfully synthesize dUDP-Gal and dTDP-Glc in mg scale.
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This work was supported by Significant New Drugs Develop-
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grant Project of Chinese Ministry of Education (No.313033).
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.
090.
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