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J Biol Chem, Vol. 273, Issue 23, 14392-14397, June 5, 1998
From the Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
A search of the yeast data base for a protein
homologous to Escherichia coli
UDP-N-acetylglucosamine pyrophosphorylase yielded UAP1 (UDP-N-acetylglucosamine
pyrophosphorylase), the Saccharomyces cerevisiae gene for UDP-N-acetylglucosamine
pyrophosphorylase. The Candida albicans and human homologs
were also cloned by screening a C. albicans genomic library
and a human testis cDNA library, respectively. Sequence analysis
revealed that the human UAP1 cDNA was identical to
previously reported AGX1. A null mutation of the S. cerevisiae UAP1 (ScUAP1) gene was lethal, and when
expressed under the control of ScUAP1 promoter, both
C. albicans and Homo sapiens UAP1
(CaUAP1 and HsUAP1) rescued the
ScUAP1-deficient S. cerevisiae cells. All the
recombinant ScUap1p, CaUap1p, and HsUap1p possessed
UDP-N-acetylglucosamine pyrophosphorylase activities in vitro. The yeast Uap1p utilized
N-acetylglucosamine-1-phosphate as the substrate, and
together with Agm1p, it produced UDP-N-acetylglucosamine from N-acetylglucosamine-6-phosphate. These results
demonstrate that the UAP1 genes indeed specify eukaryotic
UDP-GlcNAc pyrophosphorylase and that phosphomutase reaction precedes
uridyltransfer. Sequence comparison with other UDP-sugar
pyrophosphorylases revealed that amino acid residues,
Gly112, Gly114, Thr115,
Arg116, Pro122, and Lys123 of
ScUap1p are highly conserved in UDP-sugar pyrophosphorylases reported
to date. Among these amino acids, alanine substitution for
Gly112, Arg116, or Lys123 severely
diminished the activity, suggesting that Gly112,
Arg116, or Lys123 are possible catalytic
residues of the enzyme.
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.
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
The Eukaryotic UDP-N-Acetylglucosamine
Pyrophosphorylases
GENE CLONING, PROTEIN EXPRESSION, AND CATALYTIC MECHANISM*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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'.
-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).
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 |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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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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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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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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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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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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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB011272, AB011003, AB011004.
To whom correspondence should be addressed. Tel.: 81-467-47-2242;
Fax: 81-467-46-5320; E-mail: hisafumi.okabe{at}roche.com.
1 The abbreviations used are: UDP-GlcNAc, UDP-N-acetylglucosamine; ORF, open reading frame; GST, glutathione S-transferase; GlcN, glucosamine; Fru-6-P, fructose-6-phosphate; GlcN-6-P, glucosamine-6-phosphate; GlcN-1-P, glucosamine-1-phosphate; Man-6-P, mannose-6-phosphate; Man-1-P, mannose-1-phosphate; Gal-1-P, galactose-1-phosphate; Glc-1-P, glucose-1-phosphate; TLC, thin layer chromatography.
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REFERENCES |
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Discussion
References
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T. Kotake, D. Yamaguchi, H. Ohzono, S. Hojo, S. Kaneko, H.-k. Ishida, and Y. Tsumuraya UDP-sugar Pyrophosphorylase with Broad Substrate Specificity Toward Various Monosaccharide 1-Phosphates from Pea Sprouts J. Biol. Chem., October 29, 2004; 279(44): 45728 - 45736. [Abstract] [Full Text] [PDF]
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D. A. Bulik, P. van Ophem, J. M. Manning, Z. Shen, D. S. Newburg, and E. L. Jarroll UDP-N-acetylglucosamine Pyrophosphorylase, a Key Enzyme in Encysting Giardia, Is Allosterically Regulated J. Biol. Chem., May 5, 2000; 275(19): 14722 - 14728. [Abstract] [Full Text] [PDF]
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A. Wang-Gillam, I. Pastuszak, M. Stewart, R. R. Drake, and A. D. Elbein Identification and Modification of the Uridine-binding Site of the UDP-GalNAc (GlcNAc) Pyrophosphorylase J. Biol. Chem., January 14, 2000; 275(2): 1433 - 1438. [Abstract] [Full Text] [PDF]
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T. Mio, T. Yamada-Okabe, M. Arisawa, and H. Yamada-Okabe Saccharomyces cerevisiae GNA1, an Essential Gene Encoding a Novel Acetyltransferase Involved in UDP-N-acetylglucosamine Synthesis J. Biol. Chem., January 1, 1999; 274(1): 424 - 429. [Abstract] [Full Text] [PDF]
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A. Wang-Gillam, I. Pastuszak, and A. D. Elbein A 17-Amino Acid Insert Changes UDP-N-Acetylhexosamine Pyrophosphorylase Specificity from UDP-GalNAc to UDP-GlcNAc J. Biol. Chem., October 16, 1998; 273(42): 27055 - 27057. [Abstract] [Full Text] [PDF]
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F. Pompeo, Y. Bourne, J. van Heijenoort, F. Fassy, and D. Mengin-Lecreulx Dissection of the Bifunctional Escherichia coli N-Acetylglucosamine-1-phosphate Uridyltransferase Enzyme into Autonomously Functional Domains and Evidence That Trimerization Is Absolutely Required for Glucosamine-1-phosphate Acetyltransferase Activity and Cell Growth J. Biol. Chem., February 2, 2001; 276(6): 3833 - 3839. [Abstract] [Full Text] [PDF]
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) 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.
