Leishmania UDP-sugar pyrophosphorylase: The missing link in galactose salvage?

Article abstract of DOI:10.1074/jbc.M109.067223

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.

Full text of DOI:10.1074/jbc.M109.067223

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 2, pp. 878–887, January 8, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Leishmania UDP-sugar Pyrophosphorylase
□
S
THE MISSING LINK IN GALACTOSE SALVAGE?
Received for publication, September 17, 2009, and in revised form, November 9, 2009 Published, JBC Papers in Press, November 11, 2009, DOI 10.1074/jbc.M109.067223
Sebastian Damerow‡, Anne-Christin Lamerz‡1, Thomas Haselhorst§, Jana Fu¨hring‡, Patricia Zarnovican‡,
Mark von Itzstein§2, and Franc¸oise H. Routier‡3
From the ‡Department of Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany and
the §Institute for Glycomics, Griffith University (Gold Coast Campus), Parklands Drive, Southport, Queensland 4222, Australia
The Leishmania parasite glycocalyx is rich in galactose-
containing glycoconjugates that are synthesized by specific
glycosyltransferases that use UDP-galactose as a glycosyl
donor. UDP-galactose biosynthesis is thought to be predom-
inantly a de novo process involving epimerization of the
abundant nucleotide sugar UDP-glucose by the UDP-glucose
4-epimerase, although galactose salvage from the environ-
ment has been demonstrated for Leishmania major. Here, we
present the characterization of an L. major UDP-sugar pyro-
ent manifestations ranging from self-healing cutaneous lesions
to fatal visceral forms.
Leishmania parasites are coated by a dense glycocalyx
composed of glycosylphosphatidylinositol-like structures,
which is essential for parasite survival in the sandfly vector
and, at least for some species, for promastigote infectivity in the
mammalian host (1). This glycocalyx is particularly rich in
galactose occurring either in the pyranosic form (Gal) or the
more unusual furanosic form (Galf).4 Its biosynthesis thus
phosphorylase able to reversibly activate galactose 1-phos- depends on the availability of the nucleotide-activated sugar
phate into UDP-galactose thus proving the existence of the UDP-galactopyranose (UDP-Gal), which can be interconverted
Isselbacher salvage pathway in this parasite. The ordered into UDP-galactofuranose (UDP-Galf) by the specific enzyme
UDP-galactopyranose mutase (2, 3). Consequently, mutants
deficient in the formation of UDP-Galf or in the transport of
UDP-Gal into the secretory pathway organelles present an
altered glycocalyx associated with parasite attenuation (4–7). A
route to UDP-Gal formation is via epimerization of the abun-
dant nucleotide sugar UDP-glucose (UDP-Glc) by the UDP-Glc
4-epimerase (8). The biosynthesis of UDP-Gal is thus inti-
mately linked to glucose metabolism (Fig. 1). Because the
trypanosomatid parasites Trypanosoma brucei and Trypano-
soma cruzi are unable to take up galactose from the environ-
ment (9, 10), the UDP-Glc 4-epimerase is indispensable for bio-
synthesis of UDP-Gal and derived glycoconjugates in these
organisms and is essential for their survival (11–14). In con-
trast, a salvage pathway for UDP-Gal synthesis is known to
occur in Leishmania because radiolabeled Gal is taken up by
promastigotes and incorporated into surface molecules (15).
Gal most likely enters cells by a family of hexose transporters
(16) before being converted into galactose 1-phosphate (Gal-
1-P) by the putative galactokinase present in the genome
(LmjF35.2740). Deletion of three of these hexose transporters
in Leishmania mexicana revealed their importance in the
growth, infectivity, and survival of the parasite underlining the
importance of monosaccharide salvage in both promastigotes
and amastigotes (16, 17).
bisubstrate mechanism and high affinity of the enzyme for UTP
seem to favor the synthesis of nucleotide sugar rather than their
pyrophosphorolysis. Although L. major UDP-sugar pyrophos-
phorylase preferentially activates galactose 1-phosphate and
glucose 1-phosphate, the enzyme is able to act on a variety of
hexose 1-phosphates as well as pentose 1-phosphates but not
hexosamine 1-phosphates and hence presents a broad in vitro
specificity. The newly identified enzyme exhibits a low but sig-
nificant homology with UDP-glucose pyrophosphorylases and
conserved in particular is the pyrophosphorylase consensus
sequence and residues involved in nucleotide and phosphate
binding. Saturation transfer difference NMR spectroscopy
experiments confirm the importance of these moieties for sub-
strate binding. The described leishmanial enzyme is closely
related to plant UDP-sugar pyrophosphorylases and presents a
similar substrate specificity suggesting their common origin.
Trypanosomatid parasites of the genus Leishmania, the
causal agent of the human disease leishmaniasis, are character-
ized by a digenetic life cycle with a promastigote stage in the
sandfly vector and an amastigote stage in mammalian macro-
phages. According to World Health Organization reports,
more than 20 million people are infected worldwide and pres-
Gal-1-P is usually activated into UDP-Gal by a UDP-glucose:
␣-D-galactose-1-phosphate uridylyltransferase enzyme (EC
2.7.7.12 encoded by the gene GALT) via the Leloir pathway. A
clear homologue of this activating enzyme is, however, not
found in the Leishmania major genome. Alternatively, incor-
poration of Gal-1-P into uridine nucleotide by a pyrophospho-
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S5.
The nucleotide sequence(s) reported in this paper has been submitted to the
GenBankTM/EBI Data Bank with accession number(s) EU249268.
1 Present address: Roche Diagnostics GmbH, Sandhofer Strasse 116, 68305
Mannheim, Germany.
2 Recipient of support from the Australian Research Council and the National 4 The abbreviations used are: Galf, galactofuranose; GalA, galacturonuic acid;
Health and Medical Research Council.
GALT, UDP-glucose:␣-D-galactose-1-phosphate uridylyltransferase; STD,
saturation transfer difference; UGP, UDP-glucose pyrophosphorylase; USP,
UDP-sugar pyrophosphorylase.
3 To whom correspondence should be addressed. Tel.: 49-511-532-9807; Fax:
49-511-532-3947; E-mail: Routier.Francoise@mh-hannover.de.
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L. major UDP-sugar Pyrophosphorylase
mM isopropyl 1-thio-␤-D-galactopyranoside. After 20 h, the
cells were harvested by centrifugation (6000 ϫ g, 15 min, 4 °C)
and washed with phosphate-buffered saline.
A bacterial pellet obtained from 500 ml of Power Broth solu-
tion was resuspended in 15 ml of Ni2ϩ-chelating buffer ANi (50
mM Tris/HCl, pH 7.8, 300 mM NaCl), including protease inhib-
itors (40 ␮g/ml bestatin (Sigma), 4 ␮g/ml pepstatin (Sigma), 0.5
␮g/ml leupeptin (Serva), and 1 mM phenylmethylsulfonyl fluo-
ride (Roche Applied Science)). Cells were lysed by sonication
with a microtip (Branson Sonifier, 50% duty cycle, output con-
trol 5, eight 30-s pulses for 8 min), and cell debris was removed
by centrifugation (20,000 ϫ g, 15 min, 4 °C). The soluble frac-
tion was loaded onto a 1-ml HisTrap HP Ni2ϩ-chelating col-
umn (GE Healthcare). After a 20-ml wash with buffer ANi (50
mM Tris/HCl, pH 8, 300 mM NaCl), the column was eluted with
20 ml of buffer ANi containing 40 mM imidazole followed by a
final elution step of 5 ml of buffer ANi containing 300 mM imid-
FIGURE 1. UDP-galactose synthesis in L. major. The abbreviations used are
as follows: GK, galactokinase (EC 2.7.1.6); USP, UDP-sugar pyrophosphorylase azole. The fractions containing L. major USP were pooled and
(EC 2.7.7.64); GALT, UDP-glucose:␣-D-galactose-1-phosphate uridylyltrans-
passed over a HiPrep 26/10 desalting column (GE Healthcare)
ferase;GALE, UDP-galactose4-epimerase(EC5.1.3.2);UGP, UDP-glucosepyro-
phosphorylase (EC 2.7.7.9); PGM, phosphoglucomutase (EC 5.4.2.2); and HK,
hexokinase (EC 2.7.1.1). The GALT gene is absent from L. major genome sug-
gesting the absence of the Leloir pathway.
to exchange buffer ANi to buffer AQ (50 mM Tris/HCl, pH
8.0). The sample was then loaded on a 1-ml Q-Sepharose FF
anion-exchange column (GE Healthcare) that was successively
washed and eluted with 20 ml of buffer AQ, 20 ml of buffer AQ
rolytic reaction has been reported in mammals and constitutes
containing 100 mM NaCl, and a final volume of 5 ml of buffer
the Isselbacher pathway (Fig. 1) (18), although a UTP:␣-D-ga-
AQ containing 300 mM NaCl. Again, the fractions containing
lactose-1-phosphate uridylyltransferase (EC 2.7.7.10) has never
the recombinant L. major USP were pooled and exchanged to
been identified. In stark contrast, plants exhibit an enzyme with
standard buffer (Tris/HCl, pH 7.8, 10 mM MgCl2) via HiPrep
broad specificity called UDP-sugar pyrophosphorylase (USP;
26/10 column. Purified samples were snap-frozen in liquid
EC 2.7.7.64) that has been recently involved in this alternative
nitrogen and stored in standard buffer at Ϫ80 °C.
pathway for Gal activation (19–21).
Complementation of E. coli DEV6 galU Mutant—Comple-
Analysis of an L. major UDP-glucose pyrophosphorylase
mentation of the E. coli DEV6 galU mutant strain was per-
formed as described previously by Lamerz et al. (22).
(UGP) deletion mutant5 revealed the presence of Gal-contain-
ing molecules underlining the existence of a UDP-Gal biosyn-
thetic pathway independent of UDP-Glc biosynthesis. Here, we
report the identification, cloning, and characterization of an L.
major UDP-sugar pyrophosphorylase (USP) (EC 2.7.7.64) with
broad substrate specificity, including Gal-1-P and glucose
1-phosphate (Glc-1-P). The enzyme identified by homology
with its plant orthologues (19, 21) suggests the presence of the
Isselbacher pathway in Leishmania.
Size-exclusion Chromatography—Size-exclusion chroma-
tography on a Superdex 200 10/300 GL column (10 ϫ 300 mm)
(GE Healthcare) was used to determine the quaternary organi-
zation of the recombinant L. major USP. The column was equil-
ibrated with 50 ml of standard buffer (50 mM Tris/HCl, pH 7.8,
10 mM MgCl2 loaded with 100 ␮l of one of the following stan-
dard proteins: bovine carbonic anhydrase (3 mg/ml), bovine
serum albumin (10 mg/ml), yeast alcohol dehydrogenase (5
mg/ml), potato ␤-amylase (4 mg/ml), and thyroglobulin (3
mg/ml) (protein standard kit; Sigma) or with purified recombi-
nant His6-tagged L. major USP (4 mg/ml) and eluted at a flow
rate of 1 ml/min. The apparent molecular weight was deter-
mined by standard curve.
EXPERIMENTAL PROCEDURES
Cloning, Expression, and Purification of His6-tagged L. major
USP—The entire open reading frame of L. major UDP-sugar
pyrophosphorylase (LmjF17.1160) was amplified with the
primer set ACL115 (CTG ACT CCA TAT GAC GAA CCC
GTC CAA CTC C) and ACL116 (CTT AGC GGC CGC ATC
AAC TTT GCC GGG TCA GCC G), containing integrated
restriction sites for NdeI and NotI, respectively, and inserted
into a pET22b expression vector (Novagen), containing a C-ter-
minal His6 tag. For recombinant expression, the vector was
transformed into Ca2ϩ-competent Escherichia coli BL21(DE3)
via heat shock. Cells were grown in Power Broth (AthenaES) at
37 °C to an absorbance of 1.0 and transferred to 15 °C. The
expression was induced at an absorbance of 1.2 by addition of 1
In Vitro Enzyme Assays—The formation of pyrophosphate in
the forward reaction was detected with the EnzChekꢀ pyro-
phosphate assay kit (Molecular Probes). The assay medium
contained 50 mM Tris/HCl, pH 7.8, 10 mM MgCl2, 1 mM dithio-
threitol, 0.2 mM 2-amino-6-mercapto-7-methylpurine ribo-
nucleoside, 0.03 units of inorganic pyrophosphatase, 2.0 units
of purine nucleoside phosphorylase, and varying amounts of
sugar 1-phosphate and UTP ranging from 0.5 to 3 mM. Enzyme
reactions were performed at 25 °C in a total volume of 100 ␮l
and started by the addition of USP. A control without USP was
used for normalization.
5 A.-C. Lamerz, S. Damerow, B. Kleczka, M. Wiese, G. vanZanbergen, A. Wenzel,
J. Lamerz, F. F. Hsu, J. Turk, S. M. Beverley, and F. H. Routier, unpublished
work.
UTP produced in the reverse reaction was converted into 1
eq of inorganic phosphate by E. coli CTP-synthase in presence
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L. major UDP-sugar Pyrophosphorylase
of ATP, L-Gln, and the cofactor GTP. Inorganic phosphate was signal intensities of a reference spectrum (I0). The STD signal
then quantified using the EnzChekꢀ pyrophosphate assay kit with the highest intensity was set to 100%, and other STD sig-
(Molecular Probes) but omitting the first coupling enzyme. For nals were calculated accordingly (23).
these experiments, the CTP-synthase gene was recombinantly
RESULTS
cloned from E. coli XL1-blue in a pET22b expression vector
with a primer set, including NdeI and NotI restriction sites
Identification of a Putative UDP-sugar Pyrophosphorylase in
(SD13, CTT ACA TAT GCA TCA TCA TCA TCA TCA CGC Leishmania Genome—Although enzymatic epimerization of
TAG CGG ATC CAT GAC AAC GAA CTA TAT TTT TGT UDP-glucose is clearly not the sole source of UDP-galactose in
GAC C; SD14, CTT AGC GGC CGC TTA CTT CGC CTG Leishmania major (15),5 the genome of this parasite lacks
ACG TTT CTG G). The N-terminal His-tagged CTP-synthase an obvious UDP-glucose:␣-D-galactose-1-phosphate uridylyl-
was expressed and purified as described above for the USP but transferase enzyme (EC 2.7.7.12). BLAST searches of the L.
without anion-exchange chromatography. The assay mixture major genome revealed, however, the existence of a gene
for the reverse reaction contained 50 mM Tris/HCl, pH 7.8, 10 (LmjF17.1160) displaying ϳ32% identity with pea sprout USP
mM MgCl2, 1 mM dithiothreitol, 0.2 mM 2-amino-6-mercapto- (19) and 15% identity with L. major UGP (22, 24). This gene is
7-methylpurine ribonucleoside, 1 mM ATP, 1 mM L-Gln, 0.25 extremely conserved among Leishmania species and is also
mM GTP, 3 ␮g of CTP-synthase, 2.0 units of PNP, and 2 mM found in T. cruzi but not in T. brucei. In L. major, the encoded
UDP-sugar and pyrophosphate in a final volume of 100 ␮l. The protein referred to here as L. major USP or LmjUSP contains
reaction was initiated by addition of USP and normalized to 630 amino acids and has a theoretical molecular mass of 69 kDa.
buffer control.
An alignment of L. major USP and UGP is presented in Fig. 2A
Measurements were performed in 96-well half-area flat-bot- and highlights the conservation of residues essential for cata-
tom microplates (Greiner Bio-One) with the Power-WaveTM lytic activity of pyrophosphorylase and for nucleotide sugar
340 KC4 system (Bio-Tek). To exclude cross-reactions, all sub- binding (25, 26). The LmjUSP basic residues Lys-134, His-224,
strates and cofactors of coupling enzymes were tested against and Lys-434 (corresponding to LmjUGP Lys-95, His-191, and
USP inhibition or competition and vice versa (data not shown). Lys-380) are strictly conserved and predicted to be involved in
The determinations of Km and Vmax values were performed phosphate binding (26). Similarly, with the exception of Val-
using varying substrate concentrations up to 12 triplicates, 120, which is a lysine residue in UGPs, the amino acids involved
whereas the second substrate was set to a constant saturating in uridine binding are preserved. In contrast, only two of the
concentration. The initial linear rates (y) were plotted against residues of UGPs interacting with the glucose moiety (Gly-256
the substrate concentrations (x), and the Michaelis-Menten and Asn-308) are conserved in USPs (Fig. 2A and supplemental
kinetics was analyzed in PRISM using nonlinear regression Fig. S1).
(y ϭ Vmaxꢁx/(Km ϩ x)).
The phylogenetic tree presented in Fig. 2B demonstrates that
SDS-PAGE Analysis and Immunoblotting—SDS-PAGE was LmjUSP is clearly distinct from the UDP-Glc-synthesizing
performed according to Laemmli. Protein samples were sep- enzyme UGP as well as the UDP-N-acetylglucosamine pyro-
arated on SDS-polyacrylamide gels composed of a 5% stack- phosphorylase and UDP-glucose:␣-D-galactose-1-phosphate
ing gel and a 10% separating gel. Protein bands were visual- uridylyltransferase (GALT). Trypanosomatid USPs clusters
ized by Coomassie Brilliant Blue staining. For Western blot with the plant and algal USPs but constitute a separate branch.
analysis, proteins were transferred to nitrocellulose mem-
L. major USP Is Involved in Biosynthesis of UDP-glucose
branes (Schleicher & Schu¨ll). His6-tagged proteins were and/or UDP-galactose—To demonstrate the role of L. major
detected using the penta-His antibody (Qiagen) at a concentra- putative USP in the metabolism of galactose, we first investi-
tion of 1 ␮g/ml and a goat anti-mouse Ig alkaline phosphatase gated its ability to complement the growth defect of E. coli galU
conjugate (Jackson ImmunoResearch).
mutant strain DEV6. This specific bacterial strain is unable to
STD NMR—All STD NMR experiments were performed on a grow on agar-containing galactose as the only carbohydrate
Bruker Avance DRX 600 MHz spectrometer equipped with a source due to a mutation in UGP, which prevents synthesis of
triple axis cryoprobe at 298 K in 50 mM deuterated Tris buffer, UDP-Glc and subsequent depletion of the cytotoxic Gal-1-P
pH 7.8, and 10 mM MgCl2. The protein was saturated with a by the UDP-glucose:␣-D-galactose-1-phosphate uridylyltrans-
cascade of 40 selective Gaussian-shaped pulses of 50 ms dura- ferase enzyme. Upon transformation with LmjUSP cDNA, the
tion with a 100-␮s delay between each pulse resulting in a total ability of these bacteria to grow on galactose-containing media
saturation time of ϳ2 s. The on- and off-resonance frequency was restored (supplemental Fig. S2) indicating the involvement
was set to 0.7 and 40 ppm, respectively. In a typical STD NMR of L. major USP in the activation of Gal-1-P either dependently
experiment, 0.5 ␮M recombinant USP was used, and all inves- (via the Leloir pathway) and/or independently of UDP-Glc bio-
tigated ligands were added at a molecular ratio (protein/ligand) synthesis (via the Isselbacher pathway) (Fig. 1).
of 1:100. A total of 1024 scans per STD NMR experiment were
L. major USP Is a Monomer Exhibiting Broad Substrate
acquired, and a WATERGATE sequence was used to suppress Specificity—Oligomerization has been shown to regulate the
the residual HDO signal. A spin lock filter with strength of 5 activity of barley UGP (27) but not that of L. major UGP (22).
kHz and duration of 10 ms was applied to suppress protein We have thus investigated the oligomerization state of L. major
background. Relative STD effects were calculated according to C-terminally His-tagged USP (LmjUSP-His6) expressed in
the equation ASTD ϭ (I0 Ϫ Isat)/I0 ϭ ISTD/I0 by comparing the E. coli BL21 (DE3) cells and purified to homogeneity via nickel
intensity of the signals in the STD-NMR spectrum (ISTD) with affinity, anion-exchange, and size-exclusion chromatographies
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L. major UDP-sugar Pyrophosphorylase
FIGURE2. RelationofL. majorUDP-sugarpyrophosphorylasewithUDP-glucosepyrophosphorylases, UDP-GalNAcpyrophosphorylases, andUDP-glucose:
␣-D-galactose1-phosphateuridylyltransferases. A, aminoacidsequencealignmentofL. majorUSPandUGP. ResiduesinvolvedinUDP-glucosebindingofUGPare
marked below for contact with the nucleotide (square), glucose (diamond), or phosphate (circle); the pyrophosphorylase consensus sequence is boxed. B, unrooted
phylogenetic tree of selected sequences (EBI-ClustalW2 multiple alignment). The abbreviations used are as follows: USP, UDP-sugar pyrophosphorylase (EC 2.7.7.64);
UGP, UDP-glucose pyrophosphorylase (EC 2.7.7.9); GALT, UDP-glucose:␣-D-galactose 1-phosphate uridylyltransferase; UAP, UDP-GalNAc pyrophosphorylase (EC
2.7.7.23); At, A. thaliana; Cm, Cucumis melo; Hs, Homo sapiens; Lb, Leishmania braziliensis; Lm, L. major; Mm, Mus musculus; Ot, O. tauri; Pf, Plasmodium falciparum; Sc,
Saccharomyces cerevisiae; Tb, T. brucei; Tc, T. cruzi. Noncharacterized putative proteins are marked with an asterisk.
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L. major UDP-sugar Pyrophosphorylase
FIGURE 3. Purification and oligomerization status of recombinant L. major UDP-sugar pyrophosphorylase. Purification of E. coli-expressed L. major
USP-His6 was followed by SDS-PAGE with Coomassie staining (A) and anti-His6 Western blotting (B). Lane 1, bacterial lysate; lane 2, flow-through of the nickel
column; lane 3, desalted eluate of the nickel column; lane 4, flow-through of anion-exchange column; lane 5, desalted eluate of anion-exchange column.
Determination of quaternary organization was performed by size-exclusion chromatography (C). Protein standards are indicated by arrows (a–e: 669, 200, 150,
66, and 29 kDa), and the apparent molecular mass of LmjUSP-His6 was determined by standard curve (inset). LmjUSP-His6 elutes at a retention volume of 14.65
ml (solid line), corresponding to a protein of 69.7 kDa. The activity pattern (dashed line) confirms that the monomer is the active form. Elution was traced by
SDS-PAGE Coomassie staining (D) and anti-His6 Western blotting (E) of fractions.
(Fig. 3). The recombinant L. major USP eluted as a single peak different NADH-based assay system (28) (data not shown).
at 14.65 ml corresponding to an apparent molecular mass of Alternatively, the formation of UTP was followed to analyze the
69.7 kDa calculated from the log molecular weight versus reten- synthesis of sugar 1-phosphate from nucleotide sugar and pyro-
tion volume plot (Fig. 3C, inset). The theoretical mass (70.4 phosphate (reverse reaction). In this assay, UTP was utilized
kDa) to an apparent molecular mass ratio of 1.01 clearly indi- by E. coli CTP-synthase (29) generating free inorganic phos-
cates that the recombinant His6-tagged LmjUSP is a monomer. phate that could be detected using the same principle. The
This result was confirmed using untagged LmjUSP expressed in substrate specificity of the enzyme was determined by one of
E. coli and partially purified by anion-exchange and size-exclu- the employed in vitro assays (Table 1). Gal-1-P and Glc-1-P
sion chromatography (data not shown). Moreover, we demon- appeared to be the main substrates of LmjUSP, in agreement
strated that the monomeric form (identified by PAGE and with the ability of the enzyme to functionally complement
Western blotting in Fig. 3, D and E, respectively) is the active E. coli DEV6 galU mutants. Like UGP, LmjUSP acts reversibly
form of the enzyme by assaying each fraction for their ability in vitro and could efficiently use the substrates UDP-Gal and
to synthesize UDP-glucose with the assay described below UDP-Glc to produce Gal-1-P and Glc-1-P, respectively. Impor-
(Fig. 3C).
tantly, significant activity, albeit at lower level, was also
Because of its homology with plant USPs, LmjUSP was detected when D-xylopyranose 1-phosphate (Xyl-1-P), UDP-␤-
anticipated to have broad substrate specificity; versatile L-arabinopyranose (UDP-L-Ara), or UDP-␣-D-galacturonate
enzymatic assays that allow testing of the forward or reverse (UDP-GalA) was used as substrate, whereas GlcNAc-1-phos-
reaction with various substrates were thus established. The phate and UDP-GalNAc were not converted. Similarly, ␣-D-
synthesis of UDP-Glc, UDP-Gal, or other UDP-sugar from mannose 1-phosphate (Man-1-P) and ␣-L-fucose 1-phosphate
their respective sugar 1-phosphate and UTP (forward reac- (Fuc-1-P) were not activated by LmjUSP in the presence of
tion) generates pyrophosphate as a by-product that can be GTP. Our results clearly establish that the leishmanial enzyme
monitored using the Enz-Chek pyrophosphate kit (Invitrogen). exclusively utilizes UTP for activation of Gal-1-P in accordance
This system is based on hydrolysis of pyrophosphate and sub- with expectations. Together, these data establish the pyrophos-
sequent enzymatic reaction of inorganic phosphate with 2-ami- phorylase activity of the candidate protein and demonstrate its
no-6-mercapto-7-methylpurine ribonucleoside to produce broad substrate specificity that includes Gal-1-P and Glc-1-P.
ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine
L. major USP Follows Simple Michaelis-Menten Kinetics—
absorbing at 360 nm. In pilot experiments, the possibility of The kinetic parameters of the purified enzyme were deter-
ribose 1-phosphate cross-reactions with LmjUSP was therefore mined for the main substrates of the forward (Gal-1-P, Glc-1-P,
excluded by competitive testing with the substrate Glc-1-P in a and UTP) and reverse (UDP-Gal, UDP-Glc, and pyrophos-
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L. major UDP-sugar Pyrophosphorylase
phate) reaction. Additionally, UDP-L-Ara and UDP-GalA, UTP. In contrast, the affinity and maximum velocity of the
which represent analogues of UDP-Gal in which the C6 group is
either absent or substituted, were analyzed. L. major USP fol-
lowed Michaelis-Menten kinetics with all substrates tested
enzyme with UDP-Gal and UDP-Glc are comparable (Km
ϭ
148 or 174 ␮M and Vmax ϭ 134 or 157 ␮mol/min/mg, respec-
tively) resulting in an identical efficiency of the enzyme with
these two substrates of 51% when compared with the efficiency
obtained with UTP. Assuming that the sugar moiety of hexose
1-phosphate and UDP-hexose binds to the same site, the higher
affinity of LmjUSP for the UDP-sugar suggests that the nucle-
otide moiety plays a major role in substrate binding. This
hypothesis is consistent with the high Km value for UTP. As
expected, the affinity and turnover for UDP-L-Ara and UDP-
GalA are low in agreement with the observation that these
nucleotide sugars are poorer substrates (Tables 1 and 2) (19, 20,
32). Thus, the absence of the C6 hydroxyl group and particu-
larly its substitution leads to a drastic reduction of the enzyme
efficiency.
(supplemental Fig. S3). The calculated kinetic parameters Vmax
,
Km, kcat, and derived catalytic efficiency (kcat/Km) are summa-
rized in Table 2. Interestingly, Gal-1-P seems to be preferred
over Glc-1-P (Km ϭ 860 and 1706 ␮M, respectively) suggesting
a slight influence of the C4 hydroxyl group stereochemistry in
substrate binding. Nevertheless, the affinity of L. major USP for
these two hexose 1-phosphates is rather low and remarkably
lower than the affinity of the enzyme for UTP (Km ϭ 860 and
1706 ␮M versus 98 or 116 ␮M), the latter being comparable with
the affinity of various UGPs for UTP (22, 30, 31). This striking
difference is reflected by the low efficiency of the enzyme with
hexose 1-phosphates when compared with the co-substrate
Because of its high affinity for LmjUSP, UTP might bind first
to the enzyme followed by Gal-1-P or Glc-1-P thus favoring the
forward reaction despite the low affinity of the enzyme for Gal-
1-P and Glc-1-P. In line with this assumption, the turnover
rates measured for the hexose 1-phosphates were similar to the
turnover rate measured for UTP (ϳ200 sϪ1) and higher than
the one of UDP-Gal, UDP-Glc, or pyrophosphate (ϳ170 sϪ1).
Altogether, the kinetic data and their comparison with data
obtained with L. major UGP (24) suggest an ordered bi-bi sub-
strate mechanism.
TABLE 1
L. major UDP-sugar pyrophosphorylase substrate specificity
Relative enzymatic activities of purified LmjUSP were determined in the presence of
different acceptor substrates. All acceptor substrates were used in rate nonlimiting
concentrations (3 mM sugar 1-phosphates, 0.8 mM UTP, 2 mM UDP-sugars, and
pyrophosphate). The highest activity obtained was set to 100%. Errors are given as
standard deviations from triplicate measurements.
Relative activity
%
A. Forward reaction (؉UTP)
Sugar 1-phosphate
Gal-1-P
100 Ϯ 2.6
88 Ϯ 6.8
Glc-1-P
L. major USP Follows an Ordered Bi Bi Mechanism—To gain
insight into substrate binding to L. major USP and substantiate
a sequential binding mode of substrates to the enzyme, protein-
ligand interactions were investigated by STD NMR. This pow-
erful method (33, 34) is capable of identifying ligand-binding
epitopes of a ligand when bound to a target protein. Ligand
protons that are in close contact with the enzyme receive a
higher degree of saturation via the protein resulting in the
observation of stronger STD NMR signals compared with
ligand protons that do not interact with the protein surface and
Xyl-1-P
12 Ϯ 1.0
GalA-1-P
GlcNAc-1-P
Man-1-P
1.6 Ϯ 0.1
0.6 Ϯ 0.03
0.007 Ϯ 0.004
B. Reverse reaction (؉PPi)
UDP-sugar
UDP-Gal
83 Ϯ 1.2
100 Ϯ 4.1
UDP-Glc
UDP-L-Arap
UDP-GalA
33 Ϯ 0.3
7.1 Ϯ 0.1
UDP-GalNAc
0.013 Ϯ 0.0015
C. Forward reaction (؉galactose L-phosphate)
NTP
UTP
CTP
ATP
GTP
1
100 Ϯ 13
0.2 Ϯ 0.2
0.7 Ϯ 0.2
0.3 Ϯ 0.6
are solvent-exposed. H NMR spectra were first recorded to
assign signals and ensure that the enzyme was active in the
forward and reverse reactions under the applied assay condi-
TABLE 2
L. major UDP-sugar pyrophosphorylase kinetic parameters
Indicated substrate parameters were determined in presence of rate nonlimiting and constant co-substrate concentrations (0.8 mM UTP, 2 mM Gal-1-P, 3 mM Glc-1-P, 2
mM pyrophosphate, and 2 mM UDP-sugars), in order to generate a pseudo first-order reaction type. The highest efficiency obtained was UTP set to 100% for both directions
(A and B). Errors are given as standard deviations from triplicate measurements.
Efficiency
Substrate
Km
Vmax
kcat
k
cat/Km
%
Ϫ1
Ϫ1
␮M
␮mol/min/mg
s
M
Ϫ1s
A. Forward reaction
Gal-1-P
860 Ϯ 34
1706 Ϯ 144
98 Ϯ 10
190 Ϯ 3
166 Ϯ 6
175 Ϯ 7
181 Ϯ 11
219 Ϯ 3
191 Ϯ 6
201 Ϯ 8
209 Ϯ 13
2.59 ϫ 105 Ϯ 1.42 ϫ 104
1.14 ϫ 105 Ϯ 1.34 ϫ 104
2.09 ϫ 106 Ϯ 2.80 ϫ 105
1.83 ϫ 106 Ϯ 3.90 ϫ 105
12 Ϯ 0.7
5 Ϯ 0.6
Glc-1-P
UTP (Gal-1-P)
100 Ϯ 13.4
88 Ϯ 18.7
UTP (Glc-1-P)
116 Ϯ 18
B. Reverse reaction
UDP-Gal
148 Ϯ 8
174 Ϯ 9
134 Ϯ 2
157 Ϯ 3
63 Ϯ 3
11 Ϯ 1
152 Ϯ 3
161 Ϯ 2
70 Ϯ 5
13 Ϯ 1
154 Ϯ 2
180 Ϯ 3
72 Ϯ 4
13 Ϯ 1
174 Ϯ 4
185 Ϯ 2
81 Ϯ 5
15 Ϯ 1
1.07 ϫ 106 Ϯ 7.18 ϫ 104
1.06 ϫ 106 Ϯ 7.18 ϫ 104
1.98 ϫ 105 Ϯ 3.60 ϫ 104
1.67 ϫ 104 Ϯ 5.61 ϫ 103
5.80 ϫ 105 Ϯ 4.51 ϫ 104
4.92 ϫ 105 Ϯ 1.91 ϫ 104
8.12 ϫ 104 Ϯ 1.40 ϫ 104
2.08 ϫ 104 Ϯ 4.88 ϫ 103
51 Ϯ 3.4
51 Ϯ 3.4
9 Ϯ 1.7
1 Ϯ 0.3
28 Ϯ 2.2
24 Ϯ 0.9
4 Ϯ 0.7
1 Ϯ 0.2
UDP-Glc
UDP-L-Arap
373 Ϯ 48
790 Ϯ 193
307 Ϯ 18
383 Ϯ 11
1018 Ϯ 112
728 Ϯ 113
UDP-GalA
PPi (UDP-Gal)
PPi (UDP-Glc)
PPi (UDP-L-Arap)
PPi (UDP-GalA)
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L. major UDP-sugar Pyrophosphorylase
tions. STD NMR experiments revealed strong STD NMR sig-
nals for UTP, whereas the related nucleotides ATP, GTP, and
CTP result in low, if any, STD NMR signal intensity (supple-
mental Fig. S4). This result strongly suggests that ATP, GTP,
and CTP have low affinity for USP. The clear signals arising
from the ribosyl proton H4rib, uridyl protons H5uri and H6uri
,
and in particular from the ribosyl proton H1rib of UDP-Gal
indicate their close vicinity to the protein surface. Interestingly,
UDP and UMP show little affinity for LmjUSP emphasizing the
importance of the UTP ␥-phosphate in binding (supplemental
Fig. S5). In contrast, the STD NMR spectra of UDP-Gal, UDP-
Glc, UDP-GalA, and UDP-L-Ara display strong STD NMR
effects, indicating high affinity (Fig. 4) for LmjUSP. The STD
NMR spectrum of UTP revealed close proximity of the H1rib
,
H4rib, H5uri, and H6uri protons when bound to LmjUSP,
whereas the H2rib, H3rib, and H5/H5Јrib protons seem less
important in the binding event. This interaction is strength-
ened by the sugar moiety notably in the case of UDP-Gal and
UDP-Glc. Remarkably, most of the Gal protons are in close
vicinity of the protein surface with the H4gal, H2gal, H3gal, and
H6gal protons making the most significant contributions to the
binding event. In comparison, the glucose moiety of UDP-Glc
receives less saturation suggesting a lesser involvement in the
binding event to the enzyme. The relative STD NMR effects of
the H1glc (25%), H2glc (17%), and H3glc (30%) protons clearly
demonstrate a less intimate contact with the protein and there-
fore a likely poorer affinity of UDP-Glc for USP. Despite their
different stereochemistry, the H4gal and H4glc protons show no
drastic difference in relative STD NMR effects, in good agree-
ment with the specificity of the enzyme. Finally, the STD NMR
spectra of UDP-Ara and UDP-GalA emphasize the importance
of the H6gal/glc and H6Јgal/glc proton contacts with the protein
surface. UDP-Ara seems to bind to the enzyme in a similar
manner as UDP-Gal, but loss of H6 and H6Ј results in a reduced
affinity. In contrast, the steric hindrance and/or negative charge
of the carboxylic group of GalA appears to strongly influence
binding of the sugar as only weak STD NMR effects in complex
with the enzyme are observed.
FIGURE 5. Ordered bi-substrate mechanism of L. major UDP-sugar pyro-
phosphorylase. STD NMR spectrum for Gal-1-P does not present any signal
demonstrating absence of binding to L. major USP (d). In the presence of UTP,
Gal-1-P binds to the enzyme, and UDP-Gal is formed as shown by relative STD
NMR signals for these compounds (e). 1H NMR spectra of Gal-1-P (a); UDP-Gal
(b) and a mixture of Gal-1-P and UTP showing conversion into UDP-Gal (c)
were acquired for reference.
sequential binding mode with the nucleotide sugar binding
prior to pyrophosphate supported by the observation that the
affinity of the enzyme for pyrophosphate seems to be influ-
enced by the nucleotide sugar (Table 2).
DISCUSSION
Interestingly, no binding of Gal-1-P, Glc-1-P, or Xyl-1-P to
LmjUSP was observed by STD NMR spectroscopy. However,
Although Leishmania promastigotes are known to incorpo-
addition of UTP to the sugar 1-phosphate/LmjUSP mixture rate Gal taken from the environment into surface molecules
resulted in strong and specific STD NMR signals for H4 and H2 (15), enzymes involved in the salvage pathway for UDP-Gal
protons of Gal-1-P (Fig. 5). This phenomenon, suggestive of an synthesis had not yet been reported. The activation of this
ordered bi-substrate mechanism with UTP binding preceding monosaccharide was lately shown to be independent from
the hexose 1-phosphate entry, was previously observed with the UDP-Glc synthesis because an L. major UGP deletion mutant
LmjUGP, and a conformational change was proposed (22). The still expresses Gal-containing molecules.5 Herein, we report the
subsequently determined x-ray crystal structure clearly re- identification and characterization of an L. major UDP-sugar
vealed a conformational change upon complexion with UTP pyrophosphorylase able to reversibly activate Gal-1-P into
(26). It is therefore not unreasonable to assume that USP binds UDP-Gal constituting the Isselbacher pathway for UDP-Gal
UTP in a similar mode. Interestingly, STD NMR signals could synthesis.
be clearly observed for various UDP-sugars even in the absence
L. major USP presents a clear homology with plant USPs and
of pyrophosphate. In the reverse reaction, the data suggest a a modest but significant homology with UGPs and UDP-N-
FIGURE 4. STD NMR spectra of UDP-sugars complexed with L. major UDP-sugar pyrophosphorylase. STD NMR spectra are shown for UDP-glucose (A),
UDP-galactose (B), UDP-galacturonate (C), and UDP-L-arabinopyranose (D). All spectra were recorded at 298 K, 600 MHz using deuterated Tris buffer (50 mM, pH
7.8) and MgCl2 (10 mM) in D2O. The on resonance frequency was set to 0.7 ppm and the off resonance to 40 ppm. The residual water signal was removed by
applyingaWATERGATEsequence. EpitopemapswereconstructedbycalculatingrelativeSTDNMReffectsaccordingtotheformulaASTD ϭ(I0 ϫIsat)/I0 ϭISTD/I0
(see supplemental material) using the H1rib proton to 100% (bar chart). Each of the analyzed UDP-sugars shows strong STD NMR signals.
JANUARY 8, 2010•VOLUME 285•NUMBER 2
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L. major UDP-sugar Pyrophosphorylase
acetylglucosamine pyrophosphorylases over the entire se- ization of UDP-Glc for synthesis of UDP-Gal, because the hex-
quence. In particular, the pyrophosphorylase glycine-rich con- ose transporters of these parasites are unable to transport Gal
sensus motif (25, 26) essential for catalysis is highly conserved (9, 10). Nevertheless, the genome of T. cruzi contains homo-
and additional residues involved in uridine and phosphate logues of galactokinase and USP, which might be involved in
binding. As highlighted by STD NMR spectroscopic studies, recycling galactose originating from degradation of glycocon-
interactions of the uridine moiety of nucleotide sugars or UTP jugates in the endo-lysosomal compartment or plays a role in
with LmjUSP are similar to those observed with LmjUGP and salvage pathways of other sugars. T. cruzi is the only one of the
play a significant role in substrate binding. This leading role of three trypanosomatids that synthesizes UDP-Rha, UDP-Xyl,
the nucleotide moiety is observed in many enzymes involved in and its precursor UDP-GlcA (8). Like the plant enzyme, T. cruzi
glycosylation, for example UDP-galactopyranose mutase (35), USP might be involved in the synthesis of these nucleotide sug-
sialyltransferases (36), and pyrophosphorylases from E. coli ars. In Arabidopsis, USP is particularly important in pollination
(37), and might even hold true for nucleotide sugar transporters and possibly converts Gal-1-P, Ara-1-P, and Rha-1-P secreted
(38).
by the pistil (21). In T. cruzi, however, the precise function of
Intriguingly, residues interacting with the glucose moiety in the Xyl and Rha metabolism is still unclear.
UGPs are not conserved in USPs, which probably accounts for
The trypanosomatid USPs are closely related to plant
the broader specificity toward monosaccharide 1-phosphates USPs and hypothetical proteins of the diatoms Phaedacty-
and UDP-sugars of the latter. Like plant USPs, LmjUSP is able lum tricornutum and Thalassiosira pseudonana and green
to convert reversibly and efficiently both Glc-1-P and Gal-1-P algae Micromonas pusilla, Ostreococcus tauri, Ostreococcus
with a slight preference for Gal-1-P. Pentose 1-phosphates such lucimarinus, and Chlamydomonas reinhardtii, which suggest
as Xyl-1-P and Ara-1-P can also be activated in vitro by Leish- the common origin of these genes. Moreover, USP homo-
mania or plant USPs, albeit with a reduced efficiency reflecting logues are found in ciliate protozoa (Paramecium tetraurelia
their lower affinity for the enzyme and underlining the contri- and Tetrahymena thermophila) and Apicomplexa (Toxo-
bution of the hexoses H6 and H6Ј protons to binding. In con- plasma gondi, Cryptosporidium sp., and Plasmodium sp.) but
trast, GalA-1-P is a poor substrate of LmjUSP. It is reasonable to seem absent from Percolozoa, Loukozoa, and Metamonada.
assume that the carboxylic acid group of GalA creates either The discovery of a plant-like enzyme common to several patho-
steric hindrance or more likely an unfavored electrostatic gens opens new perspectives for the development of a pesti-
potential leading to weak interactions of the uronic acid with cide-like drug as already proposed for the apicomplexan para-
the leishmanial enzyme. Different from the plant enzymes, all sites (40). Like in mammals (41) and yeast (42), accumulation of
USPs identified in Leishmania species present an 18-amino Gal-1-P might reveal toxic for the parasite.
acid insertion near the uridine-binding site that contains the
Acknowledgments—We thank Nikolay Nifant’ev (Zelinsky Institute of
Organic Chemistry, Russian Academy of Sciences) for gift of xylose
1-phosphate. UDP-galacturonic acid and UDP-L-arabinose were
purchased by CarboSource, supported in part by National Science
Foundation RCN Grant 0090281.
conserved residues G223H224. Although not yet proven, this
insertion might be responsible for the substrate differences
observed for plant enzymes. The role of these additional amino
acids awaits a crystal structure of LmjUSP.
Despite its lower affinity for Ara-1-P, Arabidopsis thaliana
USP seems to play a central role in the salvage of this pentose in
vivo (21). In Leishmania, however, where D-Ara is present, the
monosaccharide is exclusively activated by GDP, and a putative
GDP-Ara pyrophosphorylase has been identified in the genome
(8). In addition to GDP-␣-D-Ara, L. major promastigote synthe-
sizes UDP-Glc, UDP-Gal (in the pyranosic and furanosic form),
UDP-GlcNAc, GDP-Man, and low amounts of GDP-Fuc, but
neither UDP-Xyl nor its precursor UDP-GlcA are produced (8).
Considering the specificity of LmjUSP for UDP-activated sug-
ars, its inability to act on hexosamine 1-phosphate, and the
characterization or presence in the genome of specific pyro-
phosphorylases for the activation of GDP-activated sugars and
UDP-GlcNAc (8, 39), Leishmania USPs most likely play a role
in the salvage of galactose and glucose exclusively. Moreover,
and despite the fact that USP is able to act reversibly, the
ordered Bi Bi mechanism of the enzyme and its high affinity for
UTP, a naturally abundant metabolite, presumably ensures the
synthesis of nucleotide sugar rather than their pyrophospho-
rolysis. Remarkably, LmjUSP seems to have evolved a slight
preference for Gal-1-P over Glc-1-P in good agreement with
the presence of Gal in many of their surface glycoconjugates.
In contrast to Leishmania parasites, the trypanosomatids T.
brucei and T. cruzi are thought to rely exclusively upon epimer-
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JANUARY 8, 2010•VOLUME 285•NUMBER 2
JOURNAL OF BIOLOGICAL CHEMISTRY 887

Glycobiology and Extracellular Matrices:
Leishmania UDP-sugar Pyrophosphorylase:
THE MISSING LINK IN GALACTOSE
SALVAGE?
Sebastian Damerow, Anne-Christin Lamerz,
Thomas Haselhorst, Jana Führing, Patricia
Zarnovican, Mark von Itzstein and Françoise
H. Routier
J. Biol. Chem. 2010, 285:878-887.
doi: 10.1074/jbc.M109.067223 originally published online November 11, 2009
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