Plant Physiology Preview. Published on March 28, 2011, as DOI:10.1104/pp.111.174706
1
UDP-sugar pyrophosphorylase – a new old mechanism for sugar
activation
*
Leszek A. Kleczkowski1 , Daniel Decker1 and Malgorzata Wilczynska2
1
Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, 90187 Umeå,
Sweden; 2Department of Medical Biochemistry, Umeå University, 90187 Umeå
*Corresponding author; e-mail: [email protected]
Abbreviations: aa, amino acid; AGPase, ADP-glc pyrophosphorylase; ara, arabinose; gal,
galactose; galA, galacturonic acid; GALT, gal-1-P uridyltransferase; glc, glucose; glcA,
glucuronic acid; man, mannose; PPi, pyrophosphate; SuSy, sucrose synthase; UAGPase,
UDP-N-acetylglucosamine pyrophosphorylase; UGE, UDP-glc epimerase; UGPase, UDP-glc
pyrophosphorylase; USPase, UDP-sugar pyrophosphorylase; xyl, xylose.
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Copyright 2011 by the American Society of Plant Biologists
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INTRODUCTION
Simple sugars (e.g. glc, gal) are the building blocks of disaccharides (e.g. sucrose) and
polysaccharides (e.g. cellulose, starch). To produce di- and polysaccharides, a given simple
sugar needs to be “activated”. This activation involves addition of a nucleoside group to a
sugar, resulting in the formation of a nucleoside 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
which can be glycosylated (Drickamer and Taylor 2006). Among nucleoside sugars, those with
uridyl group (UDP-sugars) are most prominent and they serve as precursors to primary
metabolites (e.g. sucrose 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-P, producing UDP-sugar 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 e.g. “UDP-ara/UDP-xyl pyrophosphorylase(s)” (Ginsburg
et al. 1956), “UDP-gal pyrophosphorylase” (Chaco et al. 1972, Lee et al. 1978, Smart and Pharr
1981, Studer-Feusi et al. 1999), “UDP-glcA pyrophosphorylase” (Hondo et al. 1983) or “UDPara 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 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 UTP-dependent
pyrophosphorylases, namely UDP-glc pyrophosphorylase (UGPase) (E.C. 2.7.7.9) which
exists as distinct UGPase-A and UGPase-B types, and UDP-N-acetyl-glucosamine
pyrophosphorylase (UAGPase) (E.C. 2.7.7.23), known in animals as AGX. These proteins
share low or very low identity at the amino acid (aa) level (below 20%) (Kleczkowski et al.
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2010). Phylogenetic analyses revealed that USPases, UGPases (either A- or B-type) and
UAGPases should be all categorized into distinct groups (Geisler et al. 2004, Litterer et al.
2006a,b, Okazaki et al. 2009, Kleczkowski et al. 2010).
In the present 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,b, Dai et al. 2006, Gronwald et al. 2008,
Damerow et al. 2010, Yang and Bar-Peled 2010), studies on transgenic plants with altered
USPase content (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 as to the possible roles of this multi-substrate-utilizing enzyme.
EVOLUTION OF USPASE AND GENE ORGANIZATION
In Fig. 1, we present a comprehensive phylogenetic tree for USPases, based on aa sequence
identity of the derived proteins. Most literature data on USPases concern proteins from plants
and protozoans (Leishmania and Trypanosoma), and there is usually about 30-35% identity
between USPases from these two groups. In most cases, genomes contain a single gene (USP)
for USPase. The only exception so far is genome of Physcomitrella, a moss, which has three
putative USP genes. One of those genes encodes a protein which has about 60% identity to
Arabidopsis 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 aa sequences. Surprisingly, based on aa 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 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 which evolved independently in some lineages (no aa identity at all to “common”
USPases), as is the case e.g. for bacterial UGPases which are not related by their aa sequence
to plant or animal UGPase, but carry out the same reaction (Kleczkowski et al. 2004). For
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instance, based on assays of extracts from skin fibroblasts, humans may contain separate
“UDP-gal pyrophosphorylase” and UGPase proteins (Chaco et al. 1972), the former possibly
analogous to USPase. However, using plant and Leishmania USPase aa sequences as
references, we found no USPase-like protein(s) coded by 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 reporter gene linked to USP promoter, the gene was
found to be expressed throughout at various tissues and organs, suggesting a house-keeping
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 upregulated 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 (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 sucrose 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 sucrose hydrolysis. The
involvement of the reverse USPase reaction, using UDP-sugar and PPi as substrates, would be
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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-1phosphates with UTP (forward reaction) and the corresponding UDP-sugars with PPi (reverse
reaction) as substrates. The sugar-1-P 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 UDPsugar 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 centre of mechanisms that provide
UDP-sugars for glycosylation reactions.
The USPase reaction is freely reversible, with slight preference for the
pyrophosphorolytic direction, with the Keq value of 0.2, as determined for purified pea 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, e.g. 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 aa level. The catalytic activity of USPase is initiated by binding of UTP or
UDP-sugar prior to the binding of sugar-1-P 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 Fig. 3, we present relative UTP- and sugar-1-P-dependent activities of purified
USPases from a variety of species. In those experiments, USPases were either purified from
plant extracts (pea), or overexpressed in E.coli and purified as recombinant proteins (for
Arabidopsis, soybean, 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-acetylglcA-1P, fucose-1-P, inositol-1-P and glc-6-P (Kotake et al. 2004, 2007, Litterer et al. 2006a,b,
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-P were in the
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range of 0.13-2.54 mM (Suppl. Table 1). 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-P than for pentose-1-P as a substrate. In the reverse reaction
(pyrophosphorolysis direction), the reported Km values were in the range of 0.03-0.72 mM and
0.13-1.01 mM for a given UDP-sugar and PPi, respectively (Suppl. Table 1).
Interestingly, a novel plant UGPase (so called UGPase-B), a chloroplastic enzyme
involved in sulfolipids 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, it is tempting to
speculate that UDP-gal required for the galactolipid synthesis is indeed produced by UGPaseB. 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-P 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,b, Schnurr
et al. 2006, Gronwald et al. 2008).
IS USPASE PRESENT IN ANIMALS?
It seems surprising that, based on aa 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) (see also Fig.
2). GALT is central to the Leloir pathway and carries out the reaction of: gal-1-P + UDP-glc
<----> glc-1-P + UDP-gal. In humans, genetic deficiency of the enzyme results in inability to
metabolize gal, and causes the disease galactosemia (Wang et al. 1998).
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In mice, knockouts lacking the GALT protein allowed to identify “UDP-gal
pyrophosphorylase” as an alternative route of conversion of gal-1-P to UDP-gal (Wehrli et al.
2007). This activity may belong to a yet unknown mice USPase or, more likely, to UGPase-A
that has a small residual activity with some UDP-sugars, in addition to its main activity with
UDP-glc. Purified human liver UGPase-A, 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 UDP-man 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). An
“UDP-gal pyrophosphorylase” activity reported for liver extracts (Abraham and Howell 1969)
likely corresponds to the nonspecific activity of human UGPase-A. Moreover, in yeast mutant
lacking GALT and unable to grow on gal in the media, overexpression with human UGPase-A
rescued the 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 own 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 can not be excluded.
Detailed analyses of USPase purified from Arabidopsis revealed 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 e.g. for some other enzymes involved in NDP-sugar formation: barley
AGPase isoforms targeted to different compartments upon alternative splicing (Kleczkowski
1996) or phosphorylated and non-phosphorylated isoforms of maize SuSy which are cytosolic
and plasmalemma-bound, respectively (Hardin et al. 2006, Kleczkowski et al. 2010)
There is also a body of evidence of UDP-glc dependent 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 UDPglc utilizing/ producing pyrophosphorylases, i.e. UGPases or UAGPase. In plants, synthesis
of both cellulose and callose occurs via the plasmalemma-bound 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
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efficient transfer of glc molecule to the cell wall components. Also, since synthesis of
hemicelluloses and pectins as well as protein N-glycosylation reactions are occurring in
endoplasmic reticulum (ER) and Golgi bodies (Gibeaut 2000), UDP-sugars used in these
processes must be produced there or transported from cytosol through the ER 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 it is composed of three distinct domains,
including large central domain and N- and C-terminal domains (Fig. 4). The central domain
contains a classic Rossmann fold (mixed β-sheet surrounded by α-helices) that is characteristic
of proteins that use nucleotides as substrates. The β-sheet of Rossmann fold is a structural basis
for active site of the enzyme. This general structural blueprint is shared by other UDP-glcproducing pyrophosphorylases (UGPase-A, UGPase-B and UAGPase) (Peneff et al. 2001,
Geisler et al. 2004, Roeben et al. 2006, McCoy et al. 2007, Steiner et al. 2007, Maruyama 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 β -sheet resembling that in C-terminal domain of human AGX (UAGPase), and a
left-handed β -helix that is characteristic of C-terminal domain of eukaryotic UGPase-A. The
distorted β-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 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 elongated cavity that is flanked from one side by nucleotide-binding loop (NB-loop)
and from the other side by sugar-binding loop (SB-loop) (Fig. 4). Upon substrates’ 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 accommodation of a variety of substrates. The USPase binding sites for C-5 and
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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 e.g. plant UGPase-A which is specific for
glc-1-P and UDP-glc, depending on direction of the reaction (Meng et al. 2008).
ON THE ROLE OF USPASE
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 sucrose and trehalose, the latter
metabolically related to trehalose-6-P which is an essential signalling 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 in plants, based on their
specific activities (Kleczkowski et al. 2010). However, the USPase reaction can be the major
mechanism for 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 glycosylation of proteins
and lipids (Karr 1972, Hayashi and Maclachlan 1984, Drickamer and Taylor 2006). In plants,
UDP-gal is also essential for synthesis of raffinose and stachyose, which are derivatives of
sucrose containing one and two gal molecules, respectively. Those carbohydrates are the main
carbon transporting compounds in the phloem of Cucurbitaceae family of plants (e.g. melon,
cucumber) (Dai et al. 2006).
The role of USPase has been most thoroughly studied in Arabidopsis, using both lossof-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 loss-of-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 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 to note 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 less 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.
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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 strategies 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 myo-inositol oxidation pathway, with UDP-glcA as an intermediate (Gronwald
et al. 2008) and in 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
synthesis of several glycoconjugates. This was indirectly, but elegantly, demonstrated (Lamerz
et al. 2010) by targeted deleting of 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 gal-containing glycoinositolphospholipids suggesting that 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 T. brucei, possibly because of
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 synthesis of UDPsugars (Fig. 2) and they may substitute for 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 USP-gene in an inducible construct (in the background of heterozygous
usp/USP mutant) is induced during the reproductive stage. This should facilitate the
formation of viable usp pollens and, subsequently, the production of homozygous mutants
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which would be crucial to study USPase role in vivo. Also, a recent study on male-sterile
UGPase-A knockout plants has suggested that male sterility can be circumvented by UDP-glc
supplementation to growth media (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 of UDP-sugar(s) produced by the
enzyme may overcome the reported male sterility of the usp/USP plants.
Currently, no USPase inhibitors are known. Since USPase is not present in animals, but
occurs in Leishmania or Trypanosoma which are parasites killing tens of thousands of people
every year (Yang and Bar-Peled 2010, Dickmanns et al. 2011), such an inhibitor could be used
as pharmacological means of neutralizing the parasite. Search for suitable inhibitors may
involve high throughput screening of chemical libraries (Kaiser et al. 2008) or the so called
structure-based inhibitor design strategy, based on architecture of 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 from those of UGPases and UAGPases in
crude extracts or partially purified preparations. The extent of 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 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, Suppl. Table
1). On the other hand, the structure of Leishmania protein can already provide a blueprint for
studies on other USPases, where the role of critical aa 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).
ACKNOWLEDGEMENTS
This work was supported (to L.A.K.) by The Swedish Research Council.
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12
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Legends to Figures
Fig. 1. Evolutionary tree of USPase proteins. Publicly available ClustalW2
(http://www.ebi.ac.uk/Tools/msa/clustalw2/) was used to analyze aa sequences acquired at
NCBI, and the resulting tree subsequently visualized using Treeview v.1.6.6 and Adobe
Photoshop CS3 Extended. The bar represents 0.1 substitutions per aa.
Fig. 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. GALT, gal-1P uridyltransferase; MUR4, UDP-xyl 4-epimerase; SuSy, sucrose synthase; UAGPase
(AGX), UDP-N-acetylglucosamine pyrophosphorylase; UDPG-DH, UDP-glc dehydrogenase;
UGDase, UDP-galacturonate decarboxylase; UGE, UDP-glc epimerase; UGPase, UDP-glc
pyrophosphorylase.
Fig. 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 major (Damerow et al. 2010)
and Trypanosoma cruzi (Yang and Bar-Peled 2010). Only activities with substrates showing
over 7% activity in comparison to that with the most active substrate are shown.
Fig. 4. Overall structure of USPase from Leshmania major. We show the structure as a
ribbon model, with the central, N- and C-terminal domains marked. Substrate (UDP-glc) is
shown in cyan, NB- and SB-loops that interact with nucleotide and sugar part of the substrate
(respectively) are shown in green and red. The loop (“I”) located at C-terminal domain
corresponds to the I-loop in human AGX 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 v1.8.7
software.
Fig. 5. The role of products of enzymatic reaction of USPase. Green boxes represent
products of USPase reaction.
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