Update on Nucleotide Sugar Synthesis
UDP-Sugar Pyrophosphorylase: A New Old
Mechanism for Sugar Activation1[W]
Leszek A. Kleczkowski*, Daniel Decker, and Malgorzata Wilczynska
Department of Plant Physiology, Umeå Plant Science Centre (L.A.K., D.D.), and Department of Medical
Biochemistry (M.W.), Umeå University, 90187 Umea, Sweden
Simple sugars (e.g. Glc, Gal) are the building blocks
of disaccharides (e.g. Suc) and polysaccharides (e.g.
cellulose, hemicellulose). To produce disaccharides
and polysaccharides, a given simple sugar needs to
be “activated.” This activation involves the addition of
a nucleoside-diphosphate group to a sugar, resulting
in the formation of a nucleotide sugar. Such an activated sugar can then be used by a variety of glucosyltransferases to link the sugar residue with an
appropriate receptor molecule. The latter can be a
carbohydrate but also a protein, a lipid, or any other
molecule that can be glycosylated (Drickamer and
Taylor, 2006). Among nucleotide sugars, those with a
uridyl group (UDP-sugars) are most prominent, and
they serve as precursors to primary metabolites (e.g.
Suc in plants), storage compounds (e.g. glycogen in
animals and yeast), structural components (e.g. cellulose in plants and bacteria; hemicellulose and pectin
in plants), as well as glycoproteins and glycolipids
(Feingold and Barber, 1990, Kleczkowski et al., 2010).
UDP-sugars are the main precursors for biomass production in plants (Kotake et al., 2010).
UDP-sugar pyrophosphorylase (USPase; EC 2.7.7.64)
catalyzes a reversible transfer of the uridyl group
from UTP to sugar-1-phosphate, producing UDPsugar and pyrophosphate (PPi). The enzyme was unequivocally identified and characterized only a few
years ago (Kotake et al., 2004), but the USPase-like
activities have been reported for at least 50 years now.
In those early studies, USPase-like activities were
observed in a wide range of organisms, from bacteria
to plants to animals, with reports on “UDP-Ara/UDPXyl pyrophosphorylase(s)” (Ginsburg et al., 1956),
“UDP-Gal pyrophosphorylase” (Chacko et al., 1972;
Lee et al., 1978; Smart and Pharr, 1981; Studer-Feusi
et al., 1999), “UDP-GlcA pyrophosphorylase” (Hondo
et al., 1983), and “UDP-Ara pyrophosphorylase”
(Feingold and Barber, 1990). The UDP-Gal pyrophosphorylase activities were especially significant, since
they implied an alternative to the Leloir pathway
believed to be the major, if not the only, mechanism
1
This work was supported by the Swedish Research Council
(grant to L.A.K.).
* Corresponding author; e-mail leszek.kleczkowski@plantphys.
umu.se.
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.111.174706
of Gal to Glc conversion (Smart and Pharr, 1981; Frey,
1996). It seems likely now that at least some of those
pyrophosphorylase activities correspond to the same
protein, namely USPase.
The work on USPase has been hampered by its
overlapping specificity with some other pyrophosphorylases. The activity with Glc-1-P, which usually
represents the most specific substrate for USPase,
overlaps with activities of at least three other UTPdependent pyrophosphorylases, namely UDP-Glc pyrophosphorylase (UGPase; EC 2.7.7.9), which exists as
distinct UGPase-A and UGPase-B types, and UDP-Nacetyl-glucosamine pyrophosphorylase (UAGPase;
EC 2.7.7.23), known in animals as AGX. These proteins
share low or very low identity at the amino acid level
(below 20%; Kleczkowski et al., 2010). Phylogenetic
analyses revealed that USPases, UGPases (either A or
B type), and UAGPases should all be categorized into
distinct groups (Geisler et al., 2004; Litterer et al.,
2006a, 2006b; Okazaki et al., 2009; Kleczkowski et al.,
2010).
In this review, we focus mainly on recent studies on
USPase, with emphasis on its substrate specificity,
structure, and role in metabolism. Recent work with
purified USPases (Kotake et al., 2004, 2007; Litterer
et al., 2006a, 2006b; Dai et al., 2006; Gronwald et al.,
2008; Damerow et al., 2010; Yang and Bar-Peled, 2010),
studies on transgenic plants with altered USPase contents (Schnurr et al., 2006; Kotake et al., 2007), and
resolution of the crystal structure of protozoan USPase
(Dickmanns et al., 2011) have reignited the interest in
USPase and provided new clues to the possible roles of
this multisubstrate-utilizing enzyme.
EVOLUTION OF USPASE AND
GENE ORGANIZATION
In Figure 1, we present a comprehensive phylogenetic tree for USPases, based on amino acid sequence
identity of the derived proteins. Most literature data
on USPases concern proteins from plants and protozoans (Leishmania major and Trypanosoma), and there is
usually about 30% to 35% identity between USPases
from these two groups. In most cases, genomes contain
a single gene (USP) for USPase. The only exception so
far is the genome of Physcomitrella, a moss, which has
three putative USP genes. One of those genes encodes
a protein that has about 60% identity to Arabidopsis
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Kleczkowski et al.
Figure 1. Evolutionary tree of USPase proteins. Publicly available ClustalW2 (http://
www.ebi.ac.uk/Tools/msa/clustalw2/) was
used to analyze amino acid sequences
acquired at the National Center for Biotechnology Information, and the resulting
tree was subsequently visualized using
Treeview version 1.6.6 and Adobe Photoshop CS3 Extended. The bar represents 0.1
substitutions per amino acid.
USPase (31% to Leishmania USPase), whereas proteins
derived from two other genes have 40% and 42%
identity to the plant protein. USPase proteins from
different plants share at least 60% identity at their
amino acid sequences. Surprisingly, based on amino
acid sequence comparisons, we have also found putative bacterial USPases (from Lentisphaera and Coralio
species) with at least 38% and 29% identity to
corresponding proteins from Arabidopsis (Arabidopsis
thaliana) and Leishmania, respectively. This fits earlier
reports (Lee et al., 1978, 1980) on a bacterial pyrophosphorylase capable of pyrophosphorolysis of both
UDP-Glc and UDP-Gal.
In the phylogenetic tree (Fig. 1), the lowest identity
was for Volvox USPase (24% and 31% identity to the
Leishmania and Arabidopsis proteins, respectively). It
is quite possible that USPases with even lower identities exist, but at the moment, based on analyses of
protein sequences derived from cDNAs and/or genomic clones, we cannot distinguish them from possible UGPases, UAGPases, or other related proteins. It
is also possible that there are USPases that evolved
independently in some lineages (no amino acid identity at all to “common” USPases), as is the case for
bacterial UGPases, which are not related by their
amino acid sequence to plant or animal UGPase but
carry out the same reaction (Kleczkowski et al., 2004).
For instance, based on assays of extracts from skin
fibroblasts, humans may contain separate UDP-Gal
pyrophosphorylase and UGPase proteins (Chacko
et al., 1972), the former possibly analogous to USPase.
However, using plant and Leishmania USPase amino
acid sequences as references, we found no USPase-like
protein(s) coded by the human genome nor in any
other mammalian/animal cDNA/genome databases.
Plant USP genes contain large number of introns
(e.g. 17 for Arabidopsis USP), similar to other genes
involved in UDP-Glc synthesis (Kleczkowski et al.,
2010). The junctions between introns and exons are
usually conserved, but the lengths of some introns
differ for USP from different plant species. On the
other hand, Leishmania USP gene (LmjF17.1160) has no
introns, probably reflecting a general intron scarcity in
the parasite genome (Lynn and McMaster, 2008).
GENE EXPRESSION
Using transgenic Arabidopsis plants with a reporter
gene linked to the USP promoter, the gene was found
to be expressed throughout various tissues and organs, suggesting a housekeeping function for USPase
in nucleotide sugar metabolism (Litterer et al., 2006b;
Kotake et al., 2007). The most predominant expression
was in cauline leaves, vascular tissues, stem, epidermis, flowers, and, especially, pollen (Litterer et al.,
2006b; Schnurr et al., 2006; Kotake et al., 2007). The
USP gene was strongly up-regulated in Arabidopsis
mutants deficient in UGPase-A activity, probably
reflecting a compensatory mechanism (Meng et al.,
2009b).
Analyses of microarray databases, using resources
at http://www.bar.utoronto.ca/ and http://www.
popgenie.org/, confirmed the strong expression of
USP in the pollen of Arabidopsis and, generally, in
flowers of different species. For instance, in aspen
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Update on USPase
(Populus spp.; a tree), USP was strongly expressed in
both female and male flowers as well as in developing
xylem and senescing leaves, but its expression was
low or very low in old roots, mature leaves, and in
early stages of programmed cell death in wood fibers.
Coexpression analyses of USP in relation to other
genes involved in UDP-Glc synthesis/metabolism
(genes coding for UGPases and Suc synthase [SuSy])
in Arabidopsis revealed that USP significantly coexpressed with genes coding for SuSy-1 and UGPase-B
(Kleczkowski et al., 2010). Coexpression with a SuSy
gene can, perhaps, be explained by the fact that the
two enzymes, at least theoretically, may work in concert (i.e. USPase using UDP-Glc produced by SuSy
during Suc hydrolysis). The involvement of the reverse
USPase reaction, using UDP-sugar and PPi as substrates, would be expected under energy-demanding
conditions (e.g. anoxia), when carbon is metabolized
via PPi-dependent energy transfer (Igamberdiev and
Kleczkowski, 2009, 2011).
ENZYMATIC PROPERTIES AND
SUBSTRATE SPECIFICITY
USPase, generally, has broad substrate specificity,
efficiently using a variety of sugar-1-phosphates with
UTP (forward reaction) and the corresponding UDPsugars with PPi (reverse reaction) as substrates. The
sugar-1-phosphate substrates include Glc-1-P, Gal-1-P,
GlcA-1-P, Xyl-1-P, and Ara-1-P (Fig. 2). On the other
hand, several other enzymes that are also involved in
UDP-sugar formation (Fig. 2) are usually specific for a
given substrate/product. The ability to produce a
variety of UDP-sugars places USPase at the very center
Figure 2. The central role of USPase in UDP-sugar production. Green
boxes represent products of USPase, whereas gray boxes refer to other
enzymes producing UDP-sugars. MUR4, UDP-Xyl 4-epimerase;
UDPG-DH, UDP-Glc dehydrogenase; UGDase, UDP-glucuronate decarboxylase.
of mechanisms that provide UDP-sugars for glycosylation reactions.
The USPase reaction is freely reversible, with slight
preference for the pyrophosphorolytic direction, with an
equilibrium constant (Keq) value of 0.2, as determined
for purified pea (Pisum sativum) USPase (Kotake et al.,
2004). A Keq of 0.5 was obtained for pollen UDP-GlcA
pyrophosphorylase from Typha latifolia (Hondo et al.,
1983), an enzyme likely corresponding to USPase. Similar Keq values were obtained for other pyrophosphorylases, such as AGPase and UGPase-A (Kleczkowski,
2000; Meng et al., 2008), suggesting that Keq values are
relatively constant across the pyrophosphorylase family
of enzymes, even though the proteins share little or no
homology at the amino acid level. The catalytic activity
of USPase is initiated by binding of UTP- or UDPsugar prior to the binding of sugar-1-phosphate or PPi,
respectively, as shown both for pea and Leishmania
USPases (Kotake et al., 2004; Damerow et al., 2010).
Binding of the first substrate results in a conformational change to accommodate the second substrate.
This so-called Ordered Bi Bi mechanism has been
demonstrated also for unrelated pyrophosphorylases
(Elling, 1996, Kleczkowski, 2000).
In Figure 3, we present relative UTP- and sugar-1phosphate-dependent activities of purified USPases
from a variety of species. In those experiments,
USPases were either purified from plant extracts
(pea) or overexpressed in Escherichia coli and purified
as recombinant proteins (for Arabidopsis, soybean
[Glycine max], Leishmania, and Trypanosoma enzymes).
In most cases, the activities with Glc-1-P, Gal-1-P, and
GlcA-1-P were higher than with Xyl-1-P and Ara-1-P.
On the other hand, the enzyme had low (below 7%) or
no activity with GalA-1-P, Man-1-P, N-acetyl-GlcA-1-P,
Fuc-1-P, inositol-1-P, and Glc-6-P (Kotake et al., 2004,
2007; Litterer et al., 2006a, 2006b; Damerow et al.,
2010). For all USPases, the Km values for UTP were low
(0.03–0.19 mM), regardless of the nature of the second
substrate, whereas Km values for sugar-1-phosphate
were in the range of 0.13 to 2.54 mM (Supplemental
Table S1). With some exceptions, USPase had lower
Km values for Glc-1-P, Gal-1-P, and GlcA-1-P than for
Ara-1-P and Xyl-1-P. This suggests that the enzyme
has higher affinity for hexose-1-phosphate than for
pentose-1-phosphate as a substrate. In the reverse
reaction (pyrophosphorolysis direction), the reported
Km values were in the range of 0.03 to 0.72 mM and 0.13
to 1.01 mM for a given UDP-sugar and PPi, respectively
(Supplemental Table S1).
Interestingly, a novel plant UGPase (so-called
UGPase-B), a chloroplastic enzyme involved in sulfolipid formation, has also been shown to react with
Gal-1-P in addition to its reaction with Glc-1-P. However, the Gal-1-P-dependent activity was 7-fold lower
than that with Glc-1-P (Okazaki et al., 2009), and it
is unknown whether the formation of UDP-Gal via
UGPase-B occurs in vivo. Since plastid membranes
have high content of galactolipids (Kobayashi et al.,
2007), which are rare in other types of cell membranes,
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Kleczkowski et al.
Figure 3. Relative UTP-dependent activities with
different sugar-1-phosphates of purified USPases
from pea (Kotake et al., 2004), Arabidopsis A
(Litterer et al., 2006b), Arabidopsis B (Kotake
et al., 2007), soybean (Litterer et al., 2006a),
Leishmania (Damerow et al., 2010), and T. cruzi
(Yang and Bar-Peled, 2010). Only activities with
substrates showing over 7% activity in comparison with the most active substrate are shown.
it is tempting to speculate that the UDP-Gal required
for the galactolipid synthesis is indeed produced
by UGPase-B. The enzyme has about 22% identity to
plant USPase, and it does not occur in animals
(Okazaki et al., 2009; Kleczkowski et al., 2010).
No major regulatory mechanisms controlling USPase
activity have been described. USPase is probably regulated simply by substrate availability (Kleczkowski
et al., 2010) and having activity appropriate for the given
sugar-1-phosphate serving as substrate (Fig. 3). Studies
with purified soybean and Arabidopsis USPases using
products and alternative substrates/products as possible inhibitors have revealed relatively small inhibition (Litterer et al., 2006a, 2006b; Schnurr et al., 2006;
Gronwald et al., 2008).
IS USPASE PRESENT IN ANIMALS?
It seems surprising that, based on amino acid sequence comparisons (Fig. 1), animals have no USPase,
which otherwise exists in bacteria, plants, and protozoans. One of the reasons could be the presence of the
Leloir pathway enzymes that, in animals, convert Gal
to UDP-Glc. This mechanism exists also in plants
(Main et al., 1983; Studer-Feusi et al., 1999), but its
activity is low, or scarce, depending on plant organ/
tissue. In the Leloir pathway, the conversion from Gal
to UDP-Glc occurs via three enzymes: Gal kinase (EC
2.7.1.6), Gal-1-P uridyltransferase (GALT; EC 2.7.7.12),
and UDP-Glc epimerase (UGE; EC 5.1.3.2; Frey, 1996;
Fig. 2). GALT is central to the Leloir pathway and
carries out the reaction of Gal-1-P + UDP-Glc 4 Glc-1P + UDP-Gal. In humans, genetic deficiency of the
enzyme results in the inability to metabolize Gal and
causes the disease galactosemia (Wang et al., 1998).
In mice, knockouts lacking the GALT protein allowed the identification of UDP-Gal pyrophosphorylase as an alternative route of the conversion of Gal-1-P
to UDP-Gal (Wehrli et al., 2007). This activity may
belong to a yet unknown mouse USPase or, more
likely, to UGPase-A, which has a small residual activity with some UDP-sugars in addition to its main
activity with UDP-Glc. Purified human liver UGPaseA, in contrast to plant UGPase-A (Meng et al., 2008),
was reported to react with several UDP-sugars, but the
respective activities with UDP-Gal, UDP-Xyl, or UDPMan were only up to 2% of those when UDP-Glc
served as a substrate (Knop and Hansen, 1970). The
ratio of activity with UDP-Glc and UDP-Gal was
constant throughout purification of the enzyme, suggesting that human liver does not contain a UDP-Gal
pyrophosphorylase activity that is separate from
UGPase-A (Knop and Hansen, 1970). A UDP-Gal
pyrophosphorylase activity reported for liver extracts
(Abraham and Howell, 1969) likely corresponds to
the nonspecific activity of human UGPase-A. Moreover, in a yeast mutant lacking GALT and unable to
grow on Gal in the medium, overexpression with
human UGPase-A rescued growth (Lai and Elsas,
2000), suggesting that human UGPase-A may fully
complement the lack of GALT in yeast. In humans,
however, genetic deficiency of the GALT enzyme does
result in galactosemia (Wang et al., 1998), indicating
that human UGPase-A is unable to compensate for
the loss of GALT during Gal metabolism.
SUBCELLULAR LOCATION
USPase is most likely localized in the cytosol, but
other locations cannot be excluded. Detailed analyses
of USPase purified from Arabidopsis revealed the
presence of two isoforms slightly differing in molecular masses, possibly arising via posttranslational
modification(s) or alternative splicing (Gronwald et al.,
2008). Each of these processes can contribute to altered location, as found for some other enzymes involved in NDP-sugar formation: barley (Hordeum
vulgare) AGPase isoforms targeted to different compartments upon alternative splicing (Kleczkowski,
1996) or phosphorylated and nonphosphorylated isoforms of maize (Zea mays) SuSy, which are cytosolic
and plasmalemma bound, respectively (Hardin et al.,
2006; Kleczkowski et al., 2010).
There is also a body of evidence for UDP-Glcdependent pyrophosphorylase activity associated with
cellular membranes (Becker et al., 1995; Kleczkowski
et al., 2004). It is unknown whether this activity belongs to USPase or reflects the involvement of other
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Update on USPase
UDP-Glc-utilizing/producing pyrophosphorylases (i.e.
UGPases or UAGPase). In plants, the synthesis of both
cellulose and callose occurs via the plasmalemmabound cellulose synthase and callose synthase complexes, respectively. These enzymes are using UDP-Glc
as substrate, and the presence of a membrane-bound
pyrophosphorylase producing UDP-Glc would facilitate an efficient transfer of the Glc molecule to the cell
wall components. Also, since the synthesis of hemicelluloses and pectins as well as protein N-glycosylation
reactions are occurring in endoplasmic reticulum and
Golgi bodies (Gibeaut, 2000), UDP-sugars used in these
processes must be produced there or transported
from cytosol through the endoplasmic reticulum and/
or Golgi membrane by specific transporters (Bakker
et al., 2009; Reyes et al., 2010). A putative membrane
association of some USPase activity may be for the sake
of providing UDP-sugars directly to UDP-sugar translocators.
PROTEIN STRUCTURE
The only USPase to be crystallized is that from
Leishmania (Dickmanns et al., 2011). This protein has an
overall kidney-shaped structure and is composed of
three distinct domains: a large central domain and
N- and C-terminal domains (Fig. 4). The central domain contains a classic Rossmann fold (mixed b-sheet
surrounded by a-helices) that is characteristic of pro-
teins that use nucleotides as substrates. The b-sheet of
the Rossmann fold is a structural basis for the active
site of the enzyme. This general structural blueprint is
shared by other UDP-Glc-producing pyrophosphorylases (UGPase-A, UGPase-B, and UAGPase; Peneff
et al., 2001; Geisler et al., 2004; Roeben et al., 2006;
Maruyama et al., 2007; McCoy et al., 2007; Steiner et al.,
2007; Okazaki et al., 2009; Kleczkowski et al., 2010;
Yang et al., 2010). Differences concern mostly details of
the C-terminal domain, which, in USPase, is composed
of two parts: a distorted b-sheet resembling that in
the C-terminal domain of human AGX (UAGPase),
and a left-handed b-helix that is characteristic of the
C-terminal domain of eukaryotic UGPase-A. The distorted b-sheet of USPase contains a loop similar to
the so called “I-loop” of AGX. For the latter protein,
the loop was shown to facilitate the formation of an
inactive dimer from active monomers (Peneff et al.,
2001). Oligomerization as a regulatory mechanism has
also been described for plant UGPase-A, but there the
molecular determinants of oligomerization differed
from those of AGX (Martz et al., 2002; McCoy et al.,
2007; Meng et al., 2009a). Whether such a regulatory
mechanism exists for USPase is unknown at present.
The active site of USPase resembles that of other
pyrophosphorylases, and it is in the form of an elongated cavity that is flanked from one side by a nucleotide-binding loop (NB-loop) and from the other side
by a sugar-binding loop (SB-loop; Fig. 4). Upon substrate binding, the NB- and SB-loops move toward the
substrates (Dickmanns et al., 2011). This tightens the
active center and brings the substrates into a strained
conformation, which allows catalysis to occur. The
active site cavity of USPase is larger than that of
UGPase-A or UAGPase, apparently allowing for the
accommodation of a variety of substrates. The USPase
binding sites for C-5 and C- 6 of the sugar substrate
are flexible and are less shielded from the environment than the corresponding region of UGPase-A
(Dickmanns et al., 2011). This is consistent with
USPase using several alternative substrates, in contrast to plant UGPase-A, which is specific for Glc-1-P
and UDP-Glc, depending on the direction of the reaction (Meng et al., 2008).
ON THE ROLE OF USPASE
Figure 4. Overall structure of USPase from Leishmania. We show the
structure as a ribbon model, with the central, N-, and C-terminal
domains marked. Substrate (UDP-Glc) is shown in cyan, and NB- and
SB-loops that interact with the nucleotide and sugar part of the substrate
(respectively) are shown in green and red. The loop (“I”) located at the
C-terminal domain corresponds to the I-loop in human AGX (pdb code
IJV1) and is shown in pink. The model is based on x-ray structure with
pdb code 3OH4 (Dickmanns et al., 2011) and prepared using VMD
version 1.8.7 software.
UDP-sugars produced by USPase can be used in a
plethora of biochemical reactions carried out by hundreds of specific glycosyltransferases (Geisler-Lee
et al., 2006; Yonekura-Sakakibara, 2009; Fig. 5). For
instance, UDP-Glc can be used in the formation of
polysaccharides, such as cellulose and callose, but also
simple disaccharides, such as Suc and trehalose, the
latter metabolically related to trehalose-6-phosphate,
which is an essential signaling molecule (Schluepmann
et al., 2003). On the other hand, UDP-Glc is also
produced by both UGPase and SuSy, and those two
enzymes are believed to provide the bulk of UDP-Glc
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Kleczkowski et al.
Figure 5. The role of products of the enzymatic reaction of USPase.
Green boxes represent products of the USPase reaction.
in plants, based on their specific activities (Kleczkowski
et al., 2010). However, the USPase reaction can be the
major mechanism for the synthesis of several other
UDP-sugars (i.e. UDP-Gal, UDP-GlcA, UDP-Xyl, and
UDP-Ara). All of those activated sugars are used in the
formation of pectin and hemicellulose, two of the most
abundant biomolecules in nature, and are required for
the glycosylation of proteins and lipids (Karr, 1972;
Hayashi and Maclachlan, 1984; Drickamer and Taylor,
2006). In plants, UDP-Gal is also essential for the
synthesis of raffinose and stachyose, which are derivatives of Suc containing one and two Gal molecules,
respectively. Those carbohydrates are the main carbon-transporting compounds in the phloem of the
Cucurbitaceae family of plants (e.g. melon [Cucumis
melo], cucumber [Cucumis sativus]; Dai et al., 2006).
The role of USPase has been most thoroughly
studied in Arabidopsis using both loss-of-function
knockouts as well as “antisense” and overexpression
strategies. However, the exact physiological role of
plant USPase is still obscure, since no homozygous
mutants could be produced. This was because the lossof-function mutation in USP could not be transmitted
through the male gametophyte due to pollen sterility
(Schnurr et al., 2006; Kotake et al., 2007). The usp pollen
lacked the pectocellulosic inner layer in the cell wall
(Schnurr et al., 2006) and was shrunken and collapsed
in shape. It is unknown which particular UDP-sugar
deficiency leads to the usp pollen phenotype. In
this respect, it is interesting that plants deficient in
UGPase-A activity were also male sterile (Chen et al.,
2007; Mu et al., 2009; Park et al., 2010) or producing
fewer seeds (Meng et al., 2009b). Thus, both UGPase-A
and USPase are essential in reproductive processes.
Since UGPase-A carries out only UDP-Glc synthesis
(Meng et al., 2008), and assuming that UGPase-A is
active in the usp pollen, it appears that UDP-sugars in
general, not just UDP-Glc, are essential for proper
functioning of the pollen.
Antisense inhibition of USP expression in Arabidopsis led to a 75% decrease of USPase activity,
whereas overexpression of USP resulted in an up to
2.5-fold increase of USPase activity in transgenic
plants (Kotake et al., 2007). However, neither the
antisense nor overexpression strategy led to any
change in phenotype in transgenic plants, suggesting
that USPase is not rate limiting in plant growth/
development. In plants, USPase was proposed to be
involved in the myoinositol oxidation pathway, with
UDP-GlcA as an intermediate (Gronwald et al., 2008),
and in the recycling of monosaccharides released from
cell walls during rapid cell growth and cell division
(Kotake et al., 2004, 2007, 2010). On the other hand,
there are also other ways of making UDP-sugars,
catalyzed by distinct enzymes (Johansson et al., 2002;
Suzuki et al., 2003; Kotake et al., 2010; Fig. 2), and they
may compensate for USPase deficiency.
In Leishmania, USPase is a major mechanism to
produce UDP-Gal, which is essential for the synthesis
of several glycoconjugates. This was indirectly, but
elegantly, demonstrated (Lamerz et al., 2010) by targeted deleting of the UGPase-A gene, depriving the
parasite of UDP-Glc, which otherwise can be used to
produce UDP-Gal via UGE (Fig. 2). The ugp mutant
was not affected in the content of abundant Galcontaining glycoinositolphospholipids, suggesting
that the synthesis of UDP-Gal in Leishmania is independent of UDP-Glc supply. USPase was recently
found in Trypanosoma cruzi (Yang and Bar-Peled,
2010) but not in Trypanosoma brucei, possibly because
of the diverse nature of glycan structures and glycoconjugate composition in the trypanosomatid parasites (Damerow et al., 2010; Yang and Bar-Peled, 2010).
PERSPECTIVES
Despite USPase involvement in the production of
variety of UDP-sugars, not much is known on how
essential this process is. There are other enzymes contributing to the synthesis of UDP-sugars (Fig. 2), and
they may substitute for the USPase reaction. Also, the
exact role of plant USPase is difficult to study because
deletion of the USP gene results in male sterility, and it
has not been possible to produce homozygous mutants
(Litterer et al., 2006b; Kotake et al., 2007). This perhaps
can be overcome by using an inducible expression
system (Zuo and Chua, 2000), where the USP gene in an
inducible construct (in the background of the heterozygous usp/USP mutant) is induced during the reproductive stage. This should facilitate the formation of viable
usp pollen and, subsequently, the production of homozygous mutants, which would be crucial to study the
role of USPase in vivo. Also, a recent study on malesterile UGPase-A knockout plants has suggested that
male sterility can be circumvented by UDP-Glc supplementation to growth medium (Park et al., 2010). Assuming that uptake of other nucleotide sugars occurs in
vivo, this might prove to be an effective approach to
produce homozygous USPase mutants and might also
reveal which UDP-sugar(s) produced by the enzyme
may overcome the reported male sterility of the usp/
USP plants.
8
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Update on USPase
Currently, no USPase inhibitors are known. Since
USPase is not present in animals but occurs in Leishmania and Trypanosoma, which are parasites killing
tens of thousands of people every year (Yang and BarPeled, 2010; Dickmanns et al., 2011), such an inhibitor
could be used as a pharmacological means of neutralizing the parasite. The search for suitable inhibitors
may involve high-throughput screening of chemical
libraries (Kaiser et al., 2008) or the so-called structurebased inhibitor design strategy, based on the architecture of the active site of a protein (von Itzstein, 2008).
The latter approach may be most promising given that
the protozoan USPase structure has already been
solved (Dickmanns et al., 2011). Besides pharmacological applications, specific inhibitors of USPase could
be essential to distinguish, for instance, between
UDP-Glc-producing activity of USPase and those of
UGPases and UAGPases in crude extracts or partially
purified preparations. The extent of the sensitivity to a
given inhibitor may represent a distinctive feature of
USPase from a given species (Kleczkowski, 1994).
The crystallization of Leishmania USPase (Dickmanns
et al., 2011) has become a milestone in our understanding of the function/structure properties of this
protein. However, given that the protozoan USPase
has at most 35% identity to corresponding proteins
from other species, those USPases also need to be
crystallized in order to have detailed understanding
of their structures. This especially concerns details of
the active site, which, for different USPases, can accommodate distinct substrates with differing Km
values and differing specific activities (Fig. 3; Supplemental Table S1). On the other hand, the structure
of the Leishmania protein can already provide a blueprint for studies of other USPases, where the role of
critical amino acid groups can be experimentally
tested (e.g. by site-directed mutagenesis approaches).
Similar approaches with respect to barley UGPase-A
revealed regions crucial for (de)oligomerization and
those affecting substrate binding (Meng et al., 2009a).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Table S1. Km values of USPases from plants and protozoan
species.
Received February 18, 2011; accepted March 24, 2011; published March 28,
2011.
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