From the Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
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INTRODUCTION |
UDP-N-acetylglucosamine
(UDP-GlcNAc1) is a ubiquitous
and essential metabolite and plays important roles in several metabolic processes. In bacteria, it is known as a major cytoplasmic precursor of
cell wall peptide glycan and the disaccharide moiety of lipid A (1-3).
In eukaryotes, it serves as the substrate of chitin synthase, whose
product is shown to be essential for fungal cell wall (4). It is also
used in the GlcNAc moiety of N-linked glycosylation and
the GPI-anchor of cellular proteins (5).
Biosynthesis of UDP-GlcNAc has been extensively studied in bacteria,
and it requires the following enzymatic reactions: i) conversion of
fructose-6-phosphate (Fru-6-P) into glucosamine-6-phosphate (GlcN-6-P)
by glutamine:Fru-6-P amidotransferase; ii) conversion of GlcN-6-P into
glucosamine-1-phosphate (GlcN-1-P) by glucosamine (GlcN) phosphate
mutase; iii) acetylation of GlcN-1-P by GlcN-1-P acetyltransferase to
produce N-acetylglucosamine-1-phosphate (GlcNAc-1-P); and
iv) synthesis of UDP-GlcNAc from GlcNAc-1-P and UTP by GlcNAc-1-P uridyltransferase (also called UDP-GlcNAc pyrophosphorylase) (6-8). The Escherichia coli GlmS gene encodes glutamine:Fru-6-P
amidotransferase (9-11). E. coli GlmU specifies a
bifunctional protein with GlcN-1-P acetyltransferase and UDP-GlcNAc
pyrophosphorylase activities (12, 13).
In yeast Saccharomyces cerevisiae, Fru-6-P is converted
either into GlcN-6-P by glutamine:Fru-6-P amidotransferase or into mannose-6-phosphate by phosphomannose isomerase. GFA1 and
PMI have been shown to be the genes for glutamine:Fru-6-P
amidotransferase and phosphomannose isomerase, respectively (14, 15).
Then, GlcN-6-P is N-acetylated by an acetylase to become
GlcNAc-6-P, which is further converted into GlcNAc-1-P by GlcNAc
phosphate mutase (16). S. cerevisiae harbors four different
hexosephosphate mutase genes, PGM1 (17), PGM2
(18), SEC53 (19), and AGM1 (20). Among them,
AGM1 is responsible for the interconversion of GlcNAc-6-P
and GlcNAc-1-P (20). Interestingly, Agm1p has dual substrate
specificity; it also converts glucose-6-phosphate to
glucose-1-phosphate (Glc-1-P) (20). Finally, UDP-GlcNAc is produced
from GlcNAc-1-P by UDP-GlcNAc pyrophosphorylase. However, the
eukaryotic genes for GlcN-6-P acetylase and UDP-GlcNAc
pyrophosphorylase remain unidentified.
On the other hand, there are three UDP-sugar pyrophosphorylase genes in
S. cerevisiae reported to date. GAL7 (21) and
UGP1 (22) encode UDP-galactose (UDP-Gal)
pyrophosphorylase and UDP-glucose (UDP-Glc) pyrophosphorylase,
respectively. Recently, VIG9 was identified as the
GDP-mannose (GDP-Man) pyrophosphorylase gene by functional
complementation using the glycosylation defective vig9-1
mutant (23), and the possible amino acid sequence motif for the active
site of UDP-sugar pyrophosphorylase is proposed. Because all of these
enzymes preserve substrate specificity to a certain type of sugar,
there should be an enzyme specific to GlcNAc-1-P.
In an attempt to identify the gene for UDP-GlcNAc pyrophosphorylase, we
searched the S. cerevisiae genome data base and found that
the protein specified by YDL103C. The Candida
albicans and human homologs were also isolated and characterized.
From sequence comparison and mutation analysis, the probable catalytic
residues of UDP-sugar pyrophosphorylases are proposed.
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EXPERIMENTAL PROCEDURES |
Yeast Data Base Search and Screening of DNA Libraries--
An
amino acid sequence motif of
LXXGXGTXMXXXXPK where
X represents any amino acid was obtained by comparing the
amino acid sequences of E. coli GlmUp (EcGlm1p), S. cerevisiae Ugp1p (ScUgp1p), and Homo sapiens Ugp1p
(HsUgp1p), and was used to search the S. cerevisiae genome
data bases. The entire open reading frame of ScUAP1
(originally designated YDL103C) was amplified by polymerase chain reaction using the S. cerevisiae genomic DNA extracted
from strain A451 (MAT
can1, aro7, can1, leu2, trp1,
ura3) as a template, and cloned at the XbaI site of
pUC18 or pYEUra3 (Toyobo) generating pUC-ScUAP1 and pYEU-ScUAP1,
respectively. Primers used for polymerase chain reaction were
5'-AGATCTAGAATGACTGACACAAAACAGCT-3' and
5'-AGATCTAGATTATTTTTCTAATACTATAC-3'.
The C. albicans and human homologs of ScUAP1 were
cloned by screening a C. albicans genomic DNA library and a
human testis cDNA library using the 1.4-kilobase
EcoRI-EcoRI fragment of ScUAP1 as a
probe. Hybridization and washing of the filters were carried out under
stringent conditions (20 mM sodium phosphate (pH 7.2), 5×
SSC (1× SSC contains 150 mM NaCl and 15 mM
sodium citrate), 5× Denhardt's solution, 0.1% SDS, 25% formamide at
42 °C for hybridization; 0.1× SSC and 0.1% SDS at 50 °C
for washing). Bacterial cells and phages that were strongly hybridized
with the probe DNA were collected. After the third screening, DNA was
extracted from bacterial cells and phages, and the insert DNA was
cloned at the SmaI site of pUC19 for further plasmid
construction. Radiolabeling of the probe DNA was performed by the
random priming method using [
-32P]dCTP (24), and DNA
sequencing was carried out as described elsewhere (24). Construction of
the C. albicans genomic DNA library was already reported
(25). A human testis cDNA library was purchased from
CLONTECH (USA).
Expression and Purification of the Recombinant Proteins--
The
coding regions of ScUAP1, CaUAP1,
HsUAP1, and ScAGM1 were cloned at the
EcoRI (for ScUAP1 and ScAGM1) or
SmaI (for CaUAP1 and HsUAP1) site of
pGEX2T (26), and the resulting plasmids were transfected into E. coli JM109 to let them express recombinant yeast and human
proteins as a fusion product with glutathione S-transferase
(GST). Induction and expression of the recombinant Uap1 proteins was
carried out with isopropyl
-D-thio-galactopyranoside as
described (25, 26). At 4 h after the addition of
isopropyl-
-D-thio-galactopyranoside, the bacterial cells
were harvested, suspended in a buffer containing 20 mM
Tris-HCl (pH 7.5), 0.5 mM EDTA, 50 mM NaCl, 10 mM
-mercaptoethanol, 10%(v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, and lysed by sonication.
After cell debris were removed by centrifugation at 15,000 × g at 4 °C for 30 min, GST-Uap1 and GST-Agm1 fusion proteins were purified by glutathione Sepharose CL-4B column
chromatography, as described (26) and analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. The primers used for
amplifying the ScAGM1 open reading frame (ORF) were
5'-CGGGAATTCATAAGGTTGATTACGAGCAAT-3' and
5'-ATTGAATTCTCAAGCAGATGCCTTAACGTG-3'.
Assays for UDP-GlcNAc Pyrophosphorylase--
An assay for UDP-
GlcNAc pyrophosphorylase was performed in a 20 µl standard
reaction mixture containing 50 mM Tris-HCl (pH 8.3), 5 mM MgCl2, 20 µM GlcNAc-1-P, 10%
(v/v) glycerol, and 0.1 µM [
-32P]UTP
(specific activity 1 × 103 cpm/pmol) and
approximately 0.1 µg of the indicated recombinant proteins at
30 °C for 10 min. 2 µl of each reaction mixture were spotted onto
polyethyleneimine cellulose plates, and nucleotide sugars were
separated by thin layer chromatography (TLC) in a solution that was
prepared by mixing 6 g of
Na2B4O7/10H2O, 3 g of H3BO3, and 25 ml of ethylene glycol in 70 ml
of H2O (27). The radioactive spots were visualized by
autoradiography. An alternative high flux assay was carried out in 90 µl of reaction mixture containing 50 mM Tris-HCl (pH
8.3), 5 mM MgCl2, 25 µM UTP, 20 µM GlcNAc-1-P, 10% (v/v) glycerol, 1 mM
dithiothreitol, 0.4 units/ml pyrophosphatase (Sigma), and approximately
0.1 µg of the recombinant enzyme. After incubation at 30 °C for 10 min, 100 µl of the color reagent containing 0.03% (w/v) malachite
green, 0.2% (w/v) ammonium molybdate, and 0.05% (v/v) Triton X-100 in
0.7 N HCl was added to the reaction mixture, which was
followed by incubation at room temperature for 5 min. Inorganic
phosphate derived from the pyrophosphate and thereby representing the
enzyme activity was quantified by measuring optimal density at 655 nm.
Yeast Strains and Plasmids--
The entire ORF of
ScUAP1 was cloned at the XbaI site of pUC18 and
pYEUra3 (downstream of the GAL1 promoter), generating
pUC-ScUAP1 and pYEU-ScUAP1, respectively. Then the 539-base pair
StuI-BalI region of the ScUAP1 ORF in
pUC-ScUAP1 was excised and replaced by the LEU2 gene,
generating pUC-ScUAP1L. The haploid strain YPH499 (MATa
ura3, lys2, ade2, trp1,
his3, leu2) was transformed with pYEU-ScUAP1, and
ura+ transformants were further transfected with
pUC-ScUAP1L that had been digested with XbaI. The resulting
ura+ leu+ transformants, which grew in
galactose medium but not in glucose medium, were collected and used as
uap1
strain (MATa ura3, lys2, ade2, trp1, his3, leu2,
uap1
::LEU2 UAP1-URA3).
The S. cerevisiae agm1
null mutant strain was obtained by
a means similar to that for the ScUAP1 depletion. The entire
ORF of ScAGM1 was cloned at the XbaI site of
pUC18 and pYEUra3, generating pUC-ScAGM1 and pYEU-ScAGM1, respectively.
The 1.4-kilobase BalI-BglII region of the
ScAGM1 ORF in pUC-ScAGM1 was replaced by LEU2,
generating pUC-AGM1L. YPH499 cells were transformed with
pYEU-ScAGM1 and then with pUC-ScAGM1L that had been previously digested
with XbaI. The resulting ura+ leu+
transformants, which grew in galactose medium but died in glucose medium, were collected and used as agm1
strain
(MATa ura3, lys2, ade2,
trp1, his3, leu2, agm1
::LEU2
AGM1-URA3).
To test the ability of CaUAP1, HsUAP1, and the mutant
ScUAP1 to complement ScUAP1, the entire ORFs of
CaUAP1, HsUAP1, and the mutant ScUAP1 were cloned
in pRS414-1 where a 2.0-kilobase BglII-XbaI
fragment encompassing the ScUAP1 promoter was inserted at
the BamHI site of pRS414 (Stratagene). Thus, the
transcription of CaUAP1 and HsUAP1 from this
plasmid was under the control of the ScUAP1 promoter. The
resulting plasmids were transfected into uap1
cells.
After selection of trp+ cells in the presence of galactose,
they were transferred to plates containing glucose and further cultured
for 3 days.
Site-directed Mutagenesis--
A series of the ScUAP1
mutants harboring an alanine substitution for Gly111,
Gly112, Gly114, Thr115,
Arg116, Leu117, Pro122, or
Lys123 were generated by the oligonucleotide-directed dual
amber method as described (28) with Mutan-Express KmTM
(Takara). The entire ORF of the ScUAP1 gene was cloned at
the EcoRI site of pKF18k (Takara) using EcoRI
linker and hybridized with oligonucleotides containing the indicated
mutations. The resulting mutant ScUAP1 genes were excised
from the vector and ligated at the EcoRI site of pGEX-2T and
the BamHI site of pRS414-1. All the mutations were
confirmed by sequencing the DNA.
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RESULTS |
Cloning of the Yeast UDP-GlcNAc Pyrophosphorylase Gene--
Three
distinct UDP-sugar pyrophosphorylase activities are present in yeast.
In S. cerevisiae, the GAL7 (21), UGP1
(22), and VIG9 (23) genes have been shown to encode UDP-Gal
pyrophosphorylase, UDP-Glc pyrophosphorylase, and GDP-Man
pyrophosphorylase, respectively, but the gene for UDP-GlcNAc remains to
be established. Comparison of the amino acid sequences between E. coli UDP-GlcNAc pyrophosphorylase (GlmUp) and S. cerevisiae UDP-Glc pyrophosphorylase (Ugp1p) identified an amino
acid sequence motif,
L(X)2GXGTXM(X)4PK,
where X represents any amino acid. In an attempt to identify
the S. cerevisiae UDP-GlcNAc pyrophosphorylase gene, we
searched the yeast data base and found that PSA1 and
YDL103C could encode proteins with a sequence similar to the
above amino acid motif (Fig. 1).
PSA1 is identical to VIG9, which has been shown
to be the GDP-Man pyrophosphorylase gene. Accordingly, we asked whether
YDL103C specifies UDP-GlcNAc pyrophosphorylase. The Ydl103c
protein was expressed in E. coli as a fusion protein with
GST and purified by affinity column chromatography using glutathione-Sepharose CL-4B. The purified GST-Ydl103c fusion protein produced [32P]UDP-GlcNAc when incubated with GlcNAc-1-P
and [
-32P]UTP, whereas GST alone did not (Fig.
2). The above result demonstrates that
YDL103C is a gene for UDP-GlcNAc pyrophosphorylase, and, therefore, the gene was designated ScUAP1 (the S. cerevisiae UDP-GlcNAc pyrophosphorylase gene
1).

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Fig. 1.
Predicted amino acid sequences of the yeast
and human UDP-GlcNAc pyrophosphorylases. Amino acid sequences of
UDP-GlcNAc pyrophosphorylases of S. cerevisiae
(ScUap1p or Ydl103cp), C. albicans (CaUap1p), and
H. sapiens (HsUap1p) are aligned using the FASTA and BLAST
programs. Identical amino acids among the three proteins are
boxed. The amino acid sequence that matched
L(X)2GXGTXM(X)4PK
motif is underlined.
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Fig. 2.
Expression and enzyme activities of the yeast
and human UDP-GlcNAc pyrophosphorylases. The yeast and human
UDP-GlcNAc pyrophosphorylases were expressed in E. coli as a
fusion with GST and purified with glutathione-Sepharose beads.
A, approximately 1 µg of the purified recombinant proteins
were separated on a 10% SDS-polyacrylamide gel and stained with
Coomassie Brilliant Blue. Lane 1, GST; lane 2, GST-ScUap1p; lane 3, GST-CaUap1p; lane 4, GST-HsUap1p; lane 5, GST-ScAgm1p. The positions of the
protein size markers are indicated in kDa. B, approximately
0.1 µg of the purified recombinant proteins were incubated with
[ -32P]UTP and GlcNAc-1-P. The reaction products were
separated by polyethyleneimine cellulose TLC and visualized by
autoradiography. The positions of the UDP-GlcNAc, UDP-Glc, and UTP that
were visualized under UV light are indicated. Lane 1, no
protein; lane 2, GST; lane 3, GST-ScUap1p;
lane 4, GST-CaUap1p; lane 5, GST-HsUap1p.
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Because uridyltransfer to GlcNAc-1-P releases pyrophosphate from UTP,
we developed a conventional high-flux assay by adding pyrophosphatase
to the reaction mixture, which allows us to estimate the enzyme
activity from the amounts of inorganic phosphates produced after the
hydrolysis of pyrophosphates in the reaction mixture. By this assay, it
was demonstrated that the GST-ScUap1 fusion protein converted
GlcNAc-1-P to UDP-GlcNAc in a dose-dependent manner (see
below). Moreover, UTP was essential for the production of UDP-GlcNAc by
ScUap1p; none of ATP, GTP, and CTP were used as the substrate (not
shown).
Because UDP-GlcNAc is an essential metabolite serving as a precursor of
cell wall chitin, protein N-glycosylation, and GPI anchor in
yeast (4, 5), ScUAP1 may be an essential gene for viability
if it is the only UDP-GlcNAc pyrophosphorylase gene in S. cerevisiae. The S. cerevisiae uap1
null mutant
strain in which the endogenous UAP1 gene was disrupted, but
where episomal copies of UAP1 whose transcription was under
the control of GAL1 promoter were maintained, grew on
galactose plates but died on glucose plates. The cells of S. cerevisiae uap1
null mutant displayed an aberrant morphology;
most of the yeast cells fully swelled and some were lysed, which is a
phenotype quite similar to that caused by a null mutation of
AGM1, the gene for GlcNAc phosphate mutase (Fig.
3). This is suggestive that the
ScUAP1 is a sole UDP-GlcNAc pyrophosphorylase gene in
S. cerevisiae and that the most apparent defect resulting
from depletion of the functional UAP1 occurred in the cell
wall.

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Fig. 3.
Aberrant morphology caused by the depletion
of ScUAP. S. cerevisiae cells of the wild
type, uap1 harboring pYEU-ScUAP1, and agm1
harboring pYEU-ScAGM1 were spread on agar plates containing galactose
or glucose and incubated at 30 °C. Photographs were taken at 24 h.
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Identification of the UDP-GlcNAc Pyrophosphorylase Genes of Other
Organisms--
To gain more insight into the characteristics of
UDP-GlcNAc pyrophosphorylase, we intended to isolate the
ScUAP1 homologs from the pathogenic fungus C. albicans as well as from human. By screening a C. albicans genomic DNA library and a human testis cDNA library
with ScUAP1 DNA as a probe, CaUAP1, and
HsUAP1, C. albicans (Ca) and the human (Hs)
homologs of ScUAP1, were cloned and sequenced. The predicted
products of ScUAP1, CaUAP1, and HsUAP1 are highly related to each other (Fig. 1). Interestingly, the cloned
HsUAP1 cDNA was identical to the previously reported
AGX1 cDNA whose product is implicated as being an
antigen causing male infertility (29). Both of the recombinant CaUap1p
and HsUap1p, which were expressed in E. coli as a fusion
with GST, possessed UDP-GlcNAc pyrophosphorylase activities (Fig. 2),
confirming that CaUAP1 and HsUAP1 indeed specify
UDP-GlcNAc pyrophosphorylase. Furthermore, expression of
CaUAP1 or HsUAP1 under the control of the
ScUAP1 promoter supported the growth of the
UAP1-deficient S. cerevisiae cells even in the
presence of glucose. Thus, it appears that both the C. albicans and human UAP1 functionally complement
ScUAP1 (Fig. 4).

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Fig. 4.
Functional complementation of ScUAP1
by the C. albicans and human UAP1.
S. cerevisiae uap1 cells harboring pYEU-ScUAP1 were
further transformed with pRS414-1 bearing ScUAP1,
CaUAP1, or HsUAP1. Transfectants were spread on
agar plates containing galactose or glucose and incubated at 30 °C
for 3 days.
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Substrate Specificity of UDP-GlcNAc Pyrophosphorylase--
We
next examined the substrate specificity of UDP-GlcNAc pyrophosphorylase
using ScUap1p. ScUap1p reproducibly converted GlcNAc-1-P into
UDP-GlcNAc in the presence of UTP but did not utilize GlcNAc-6-P, galactose-1-phosphate (Gal-1-P) or mannose-1-phosphate (Man-1-P) as a
substrate (Fig. 5A).
Unexpectedly, the enzyme generated a spot whose mobility corresponded
to that of UDP-Glc from Glc-1-P, indicating the dual substrate utility
of Uap1p. However, Glc-1-P was much less efficient as shown in Fig.
5B. Consequently, ScUAP1 did not complement
ScUGP1 (data not shown).

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Fig. 5.
Substrate specificity of ScUap1p.
A, approximately 0.1 µg of the purified GST-ScUap1p was
incubated with [ -32P]UTP and 20 µM of
each sugar nucleotide. The reaction products were separated by
polyethyleneimine cellulose TLC and visualized by autoradiography. The
positions of the UDP-GlcNAc, UDP-Glc, and UTP that were visualized
under UV light are indicated. Lane 1, none; lane
2, GlcNAc-1-P; lane 3, GlcNAc-6-P; lane 4, GlcN-1-P; lane 5, Glc-1-P; lane 6, Gal-1-P;
lane 7, GalNAc-1-P; lane 8, Man-1-P.
B, the indicated amounts of the purified GST-ScUap1p were
incubated with 20 µM of the indicated nucleotide sugar,
UTP, and pyrophosphatase. The amounts of the released inorganic
phosphate that represent the enzyme activities were determined with
malachite green and ammonium molybdate.
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It is believed that the interconversion of GlcNAc-6-P and GlcNAc-1-P
precedes the uridyltransfer in vivo. This prompted us to add
the yeast GlcNAc phosphate mutase (ScAgm1p) to the reaction mixture. As
shown in Fig. 6A, together
with ScAgm1p, ScUap1p produced UDP-GlcNAc from GlcNAc-6-P, whereas
ScAgm1p alone did not. It is also demonstrated that hexosephosphate
mutases require Glc-1,6-P2 either as an activator or a
cofactor for the catalytic reaction (30, 31). Therefore, we examined
the effect of Glc-1,6-P2 on the synthesis of UDP-GlcNAc
from GlcNAc-6-P. The TLC analysis of the products indicated that
Glc-1,6-P2 was not essential for the interconversion of
GlcNAc-6-P and GlcNAc-1-P, because UDP-GlcNAc was produced from
GlcNAc-6-P by Agm1p and Uap1p even in the absence of
Glc-1,6-P2 (Fig. 6A). However, further
assessment of the importance of Glc-1,6-P2 for the reaction
by ScAgm1p revealed that the enhancement by Glc-1,6-P2 of
the mutase reaction was significant when the ScAgm1p concentration was
rather low (Fig. 6B).

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Fig. 6.
Production of UDP-GlcNAc from GlcNAc-6-P by
ScUap1p and ScAgm1p. A, approximately 0.1 µg of the
purified GST-ScUap1p (ScUap1p) was incubated with
[ -32P]UTP and 20 µM each of the
indicated sugar nucleotides in the presence or absence of approximately
0.1 µg of the purified GST-ScAgm1p. The reaction products were
separated by polyethyleneimine cellulose TLC and visualized by
autoradiography. The positions of the UDP-GlcNAc, UDP-Glc, and UTP that
were visualized under UV light are indicated. It should be noted that
UDP-Glc, which appeared in the presence of ScUap1p and
Glc-1,6-P2, was formed from a trace of Glc-1-P in the
Glc-1,6-P2. B, the indicated amounts of the
purified GST-ScAgm1p were incubated with approximately 0.1 µg of the
purified GST-ScUap1p, GlcNAc-6-P, and UTP in the presence ( ) or
absence ( ) of 20 mM Glc-1,6-P2. The amounts
of the released inorganic phosphate that represent the enzyme
activities were determined with malachite green and ammonium
molybdate.
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Possible Active Sites of ScUap1p--
Comparison of the amino acid
sequences among UDP-sugar pyrophosphorylases revealed that the region
between amino acid positions 111 and 123 of ScUap1p shares significant
sequence identity with other UDP-sugar pyrophosphorylases (Fig.
7). To verify the importance of this
region for the catalytic activity, the highly conserved amino acids in
this region, Gly111, Gly112,
Gly114, Thr115, Arg116,
Leu117, Pro122, and Lys123 were
replaced by alanine. As was done for the wild type ScUap1p, all the
mutant enzymes were expressed as a fusion with GST and purified by
affinity column chromatography (Fig.
8A). Although Gly114, Thr115, and Pro122 are also
highly conserved in known UDP-sugar pyrophosphorylases, replacement of
these amino acids by alanine only weakly impaired the enzyme activity.
In contrast, substitution of alanine for Gly112,
Arg116, or Lys123 severely diminished the
activity (Fig. 8B). Furthermore, G112A but not other mutants
displayed a higher Km value to GlcNAc-1-P (Table
I), and all of G112A, R116A, and K123A
failed to rescue the S. cerevisiae uap1
null mutant (data
not shown). None of the mutations significantly affected the
Km values in response to UTP (Table I). Taken
together, it was proposed that Gly112 serves as a binding
site for GlcNAc-1-P, and that Gly112, Arg116,
and Lys123 are possible catalytic residues.

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Fig. 7.
Comparison of the amino acid sequences of
UDP-sugar pyrophosphorylases. Amino acid sequences of ScUap1p are
compared with those of other UDP-sugar pyrophosphorylases using the
FASTA and BLAST programs. Identical amino acids among all the proteins
listed here are indicated by bold letters. Amino acids that
were replaced by alanine are marked by +. ScUap1p, S. cerevisiae UDP-GlcNAc pyrophosphorylase; CaUap1p,
C. albicans UDP-GlcNAc pyrophosphorylase;
HsUap1p, H. sapiens UDP-GlcNAc pyrophosphorylase;
ScUgp1p, S. cerevisiae UDP-Glc pyrophosphorylase
(22); EcGlmUp, E. coli UDP-GlcNAc
pyrophosphorylase (12, 13); HsUgp1p, H. sapiens
UDP-Glc pyrophosphorylase (33); EcUgp1p, E. coli
UDP-Glc pyrophosphorylase (34); ScVig9p, S. cerevisiae GDP-Man pyrophosphorylase (23).
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Fig. 8.
Effects on ScUap1p activity of alanine
substitution for the conserved amino acids. The ScUap1 mutant
proteins harboring an alanine substitution for each of the amino acids
that are highly conserved in UDP-sugar pyrophosphorylases were
expressed as a fusion with GST and purified with glutathione-Sepharose
beads. A, approximately 1 µg of the wild type and the
indicated mutant proteins were separated on a 10% SDS-polyacrylamide
gel (PAGE) and stained with Coomassie Brilliant Blue. The
position of GST-ScUap1p is indicated by the arrowhead.
B, approximately 0.1 µg of the purified GST and the
indicated mutant proteins were incubated with GlcNAc-1-P and UTP, and
the amounts of the released inorganic phosphate that represent the
enzyme activities were determined with malachite green and ammonium
molybdate.
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Table I
Characteristics of the mutant ScUap1p
The Km and kcat values of the
wild type and mutant enzymes to GlcNAc-1-P were determined from the
amounts of pyrophosphate released from UTP. The Km
values to UTP were also indicated in the right column.
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DISCUSSION |
In this paper, we have identified the eukaryotic UDP-GlcNAc
pyrophosphorylase genes. The expected amino acid sequences of the yeast
and human enzymes are well conserved, and both C. albicans and human enzymes functionally complement S. cerevisiae
UAP1. Although the yeast enzyme catalyzed uridyltransfer to
Glc-1-P, ScUap1p displayed a reasonable substrate specificity to
GlcNAc-1-P, because the enzyme utilized Glc-1-P much less efficiently
than GlcNAc-1-P. In fact, overexpression of ScUAP1 did not
overcome the lethal phenotype caused by a depletion of UGP1
in S. cerevisiae. Moreover, the enzyme did not recognize
GlcNAc-6-P, but together with ScAgm1p, it produced UDP-GlcNAc from
GlcNAc-6-P, demonstrating that the GlcNAc phosphate mutase reaction
precedes uridyltransfer in UDP-GlcNAc biosynthesis. However, we cannot
rule out the possibility that the results of the TLC assays and the
high flux assays may not be exactly the same, because in the high flux
assay the reverse reaction was eliminated by pyrophosphatase.
Both UDP-GlcNAc pyrophosphorylase and GlcN-1-P acetyltransferase
activities are authentic in E. coli GlmUp (9). It has also
been demonstrated that the N-terminal region is responsible for the
uridyltransfer, and acetylase activity resides in the C-terminal half
of GlmUp (13). Unlike bacterial UDP-GlcNAc pyrophosphorylase, the
eukaryotic enzymes seem not to be bifunctional, because ScUap1p did not
utilize GlcN-1-P as the substrate and the C-terminal portion of GlmUp
showed no significant sequence homology to any UDP-GlcNAc pyrophosphorylase. Thus, it is likely that in yeast, GlcN-6-P is first
acetylated by an as yet unidentified enzyme and then the mutase
reaction generates GlcNAc-1-P.
Phosphomannomutase and phosphoglucomutase require a sugar biphosphate
as a cofactor, which serves as a phosphate donor necessary to activate
the enzyme by phosphorylation (30, 31). In this study, ScAgm1p was able
to produce GlcNAc-1-P even in the absence of cofactor,
Glc1,6-P2, if a sufficient amount of ScAgm1p was present.
One possible explanation for this discrepancy is that a small portion
of the recombinant ScAgm1p was already phosphorylated and thereby
activated. However, this hypothesis is inconsistent with the recent
report by Oesterhelt et al. (31) that the plant and yeast
enzymes utilize a sugar diphosphate as a co-substrate.
Sequence comparisons of the UDP-sugar transferases revealed that there
is a region where the amino acid sequence is highly conserved among
most of the known UDP-sugar pyrophosphorylases. Alanine substitution
for Gly112, Arg116, or Lys123
severely diminished the enzyme activity and ability to complement the
wild type ScUAP1 gene, strongly suggesting that these amino acids are catalytic residues. Among these three amino acids,
Gly112 was shown to be a possible binding site to
GlcNAc-1-P, because G112A displayed an increased Km
value. In human UDP-Glc pyrophosphorylase, it was demonstrated that a
single mutation of Gly115 to Asp drastically impaired the
enzyme activity and caused cellular UDP-Glc deficiency (32). Sequence
comparison of the Uap and Ugp proteins reveals that Gly115
of the human Ugp1p corresponds to Gly112 of the yeast
Ugp1p. Thus, it is likely that Gly115 of the human Ugp1p
also serves as a Glc-1-P binding site. Curiously, Gal7p, which is known
as UDP-Gal pyrophosphorylase, shares no significant sequence homology
to known UDP-sugar pyrophosphorylases, and the conserved amino acids
essential for the catalytic activity of ScUap1p are not found in Gal7p
(21). This may imply that the catalytic mechanism of Gal7p differs from
those of other UDP-sugar pyrophosphorylases.
The human UDP-GlcNAc pyrophosphorylase cDNA turned to be identical
to the AGX1 cDNA. Although the physiological function of AGX1 remains to be established, it encodes an unknown
antigen expressed in infertile males and is implicated in
antibody-mediated human infertility (29). AGX1 is abundantly
expressed in testes, and only low levels of AGX1 mRNA
were detected in placenta, muscle, and liver (29). The reason why
testis expresses a higher level of AGX1 mRNA and how
UDP-GlcNAc pyrophosphorylase causes human male infertility await
further study. In addition, there is an additional AGX
cDNA, termed AGX2, which differs from AGX1 by
a 48-base pair insertion in the ORF. The level of AGX2
mRNA was not remarkably increased in testis; low but similar levels
of AGX2 mRNA were detected in testis, placenta, muscle,
and liver (29). Therefore, it may also be of interest to study how the internal 48-base pair insertion affects the UDP-GlcNAc
pyrophosphorylase activity.
We thank K. Saitoh for data base search and
sequence alignment, Y. Miyazaki and Y. Kitayama for assisting with the
experiments, and S. Miwa for reading the manuscript.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB011272, AB011003, AB011004.