Saccharomyces cerevisiae GNA1, an Essential Gene
Encoding a Novel Acetyltransferase Involved in
UDP-N-acetylglucosamine Synthesis*
Toshiyuki
Mio
,
Toshiko
Yamada-Okabe§,
Mikio
Arisawa
, and
Hisafumi
Yamada-Okabe
¶
From the
Department of Mycology, Nippon
Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530 and the § Department of Hygiene, School of Medicine,
Yokohama City University, 3-9 Fukuura, Kanazawa,
Yokohama 236-0004, Japan
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ABSTRACT |
The Saccharomyces cerevisiae gene,
YFL017C, for a putative acetyltransferase was
characterized. Disruption of YFL017C was lethal, leading to
a morphology similar to those caused by the depletion of
AGM1 or UAP1, the genes encoding
phospho-N-acetylglucosamine mutase and
UDP-N-acetylglucosamine pyrophosphorylase,
respectively. This implies the involvement of YFL017C in
UDP-N-acetylglucosamine synthesis. The recombinant protein
for YFL017C displayed phosphoglucosamine acetyltransferase
activities in vitro and utilized glucosamine 6-phosphate as
the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein
produced UDP-N-acetylglucosamine from glucosamine 6-phosphate. These results indicate that YFL017C specifies
glucosamine-6-phosphate acetyltransferase; therefore, the gene was
designated GNA1
(glucosamine-6-phosphate acetyltransferase). In addition, whereas bacterial
phosphoglucosamine acetyltransferase and
UDP-N-acetylglucosamine pyrophosphorylase activities are
intrinsic in a single polypeptide, they are encoded by distinct
essential genes in yeast. When the sequence of ScGna1p was compared
with those of other acetyltransferases, Ile97,
Glu98, Val102, Gly112,
Leu115, Ile116, Phe142,
Tyr143, and Gly147 were found to be highly
conserved. When alanine was substituted for these amino acids, the
enzyme activity for the substituted Phe142 or
Tyr143 enzymes was severely diminished. Although the
activity of Y143A was too low to perform kinetics, F142A displayed a
significantly increased Km value for acetyl-CoA,
suggesting that the Phe142 and Tyr143 residues
are essential for the catalysis.
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INTRODUCTION |
Acetyltransferases catalyze the acetylation of their specific
substrates using the cofactor acetyl-CoA (Ac-CoA). These enzymes play
important roles in a wide variety of biological processes, including
metabolism, chromatin structure, gene expression, and cell cycle
(1-3). At least one acetyltransferase is necessary for the
biosynthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), which
is an essential metabolite both in prokaryotes and eukaryotes. In
bacteria, UDP-GlcNAc is a major cytoplasmic precursor of the cell wall
peptide glycan, and it is also used as the disaccharide moiety of lipid
A (4-6). In eukaryotes, it serves as the substrate of chitin synthase
and is also utilized for the GlcNAc moiety of N-linked
glycosylation as well as the glycosylphosphatidylinositol-anchor of cellular proteins (7, 8).
In Escherichia coli, biosynthesis of UDP-GlcNAc starts
from the conversion of fructose 6-phosphate (Fru-6-P) into glucosamine 6-phosphate (GlcN-6-P) by glutamine:Fru-6-P amidotransferase. Then,
GlcN-6-P is converted into glucosamine 1-phosphate (GlcN-1-P) by
phosphoglucosamine (phospho-GlcN) mutase. Acetylation occurs on
GlcN-1-P, and the resulting N-acetylglucosamine 1-phosphate (GlcNAc-1-P) is uridylated by UDP-GlcNAc pyrophosphorylase (9-11). The
GlmS and GlmM genes of E. coli code
for glutamine:Fru-6-P amidotransferase (12-14) and phospho-GlcN mutase
(15), respectively. In addition, the product of the E. coli
GlmU gene is a bifunctional protein having both GlcN-1-P
acetyltransferase and UDP-GlcNAc pyrophosphorylase activities
(16, 17).
In Saccharomyces cerevisiae, GFA1 and
AGM1 are responsible for the synthesis of GlcN-6-P from
Fru-6-P and for the interconversion of N-acetylglucosamine
6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, respectively (18, 19). As
Agm1p utilizes phospho-N-acetylglucosamine (phospho-GlcNAc)
as the substrate, it has been assumed that acetyl transfer precedes the
mutase reaction, and, therefore, GlcN-6-P would serve as the recipient
of the acetyl moiety (19). In our previous paper, we reported that the
yeast UAP1 gene encodes UDP-GlcNAc pryophosphorylase and
demonstrated that, unlike the product of the E. coli GlmU
gene, phospho-GlcN acetyltransferase activity is not intrinsic in the
yeast Uap1 protein (20). The human UDP-GlcNAc pyrophosphorylase was
also identified and shown to be highly related to the yeast enzyme.
Curiously, the human enzyme turned out to be identical to the antigen
of male infertility patients (20-22). Furthermore, the human
enzyme recognizes both UDP-GlcNAc and UDP-GalNAc, but the deletion of
17 amino acids in the C-terminal region, which occurs physiologically
by alternative splicing, alters its substrate specificity (22).
In this paper, we report the S. cerevisiae gene
for phospho-GlcN acetyltransferase. The
enzyme utilizes GlcN-6-P as the substrate, and its deletion is lethal,
leading to a morphology similar to those observed in UAP1-
and AGM1-deficient cells. The important residues for the
catalysis are proposed on the basis of mutation analysis.
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EXPERIMENTAL PROCEDURES |
Yeast Data Base Search and Screening of DNA Libraries--
The
entire open reading frame
(ORF)1 of
ScGNA1 (YFL107C) was amplified by polymerase
chain reaction with the S. cerevisiae genomic DNA extracted
from strain A451 (MAT
can1 aro7 can1 leu2 trp1 ura3) as a
template. It was then cloned at the XbaI site of pUC18 or
pYEUra3 (Toyobo), generating pUC-ScGNA1 and pYEU-ScGNA1, respectively.
Primers used for polymerase chain reaction were 5'-AAGGATCCAGCTTACCCGATGGATTTTATATA-3' and
5'-AAGAATTCCTATTTTCTAATTTGCATTTCCAC-3'. The Candida albicans
homolog of ScGNA1 was cloned by screening a C. albicans genomic DNA library as a probe using the 0.2-kilobase fragment of the C. albicans genomic DNA, whose sequence was
available in the C. albicans data base and was found to be
related to YFL017C. The filters were hybridized and washed
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). After the third screening,
DNA was extracted from bacterial cells that were strongly hybridized
with the probe DNA, and the insert DNA was cloned between the
BamHI and SalI sites of pUC19 for further plasmid
construction. Radiolabeling of the probe DNA was performed by the
random priming method using [
-32P]dCTP (23), and DNA
sequencing was carried out as described elsewhere (23). Construction of
the C. albicans genomic DNA library was already reported
(24).
Expression and Purification of the Recombinant Proteins--
The
coding regions of ScGNA1 and CaGNA1 were cloned
between the BamHI and EcoRI sites of pGEX-2T
(25), and the resulting plasmids were transfected into E. coli JM109 to express the recombinant yeast proteins as a fusion
with glutathione S-transferase (GST). Induction and
expression of the recombinant Gna1 proteins was carried out with
isopropyl-
-D-thiogalactopyranoside as described (24,
25). At 4 h after the addition of
isopropyl-
-D-thiogalactopyranoside, the bacterial cells
were harvested, suspended in a buffer containing 20 mM
Tris-HCl (pH 7.5), 0.5 mM EDTA, 10%(v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, and lysed by sonication.
After cell debris was removed by centrifugation at 15,000 × g at 4 °C for 30 min, GST-Gna1 fusion proteins were
purified by glutathione-Sepharose CL-4B column chromatography, as
described (25), and analyzed by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis. The primers used for
amplifying the CaGNA1 ORF were
5'-AAGGATCCTTACCACAAGGTTATACATTCAGA-3' and
5'-AAGAATTCCTAGAATCTACATACCATTTCAAC-3'.
Assays for Phospho-GlcN Acetyltransferase--
Because CoA
reacts with 2-nitrobenzoic acid and releases 4-nitrothiophenolate (26,
27), an assay for phospho-GlcN acetyltransferase was performed in 50 µl of a reaction mixture containing 50 mM Tris-HCl (pH
7.5), 5 mM MgCl2, 200 µM
GlcN-6-P, 200 µM Ac-CoA, 10% (v/v) glycerol, and
approximately 0.1 µg of the recombinant GST-Gna1p fusion protein.
After incubation at 30 °C for the indicated time, the reaction was
terminated by adding 50 µl of a solution containing 50 mM
Tris-HCl (pH 7.5) and 6.4 M guanidine hydrochloride and
then 50 µl of a solution containing 50 mM Tris-HCl (pH
7.5), 1 mM EDTA, and 20 µM 2-nitrobenzoic
acid. The amounts of CoA produced by the Gna1 protein were estimated
from that of 4-nitrothiophenolate by measuring the optimal density at
412 nm. The UDP-GlcNAc synthesis assay was carried out in a 20 µl
standard reaction mixture containing 50 mM Tris-HCl (pH
8.3), 5 mM MgCl2, 200 µM
GlcN-6-P, 200 µM Ac-CoA, 10% (v/v) glycerol, 0.1 µM [
-32P]UTP (specific activity, 1 × 103 cpm/pmol) and approximately 0.1 µg each of Gna1,
Agm1, and Uap1 proteins at 30 °C for 10 min. Two microliters of the
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 (28). The radioactive spots were visualized
by autoradiography.
Yeast Strains and Plasmids--
The entire ORF of
ScGNA1 was cloned between the BamHI and
SalI sites of pUC18 and pYEUra3 (downstream of the GAL1
promoter), generating pUC-ScGNA1 and pYEU-ScGNA1, respectively. Then,
the 133-base pair EcoT14I-EcoT14I region of the
ScGNA1 ORF in pUC-ScGNA1 was excised and replaced by the
LEU2 gene, generating pUC-ScGNA1L. The haploid strain YPH499
(MATa ura3, lys2, ade2,
trp1, his3, leu2) was transformed with
pYEU-ScGNA1, and ura+ transformants were further
transfected with pUC-ScGNA1L that had been digested with
SalI and SmaI. The resulting ura+
leu+ transformants, which grew in galactose medium but not
in glucose medium, were collected and used as gna1
strain
(MATa ura3, lys2, ade2,
trp1, his3, leu2 gna1
::LEU2
GNA1-URA3). The S. cerevisiae uap1
null mutant
(MATa ura3, lys2, ade2,
trp1, his3, leu2 uap1
::LEU2 UAP1-URA3) and agm1
null mutant (MATa
ura3, lys2, ade2, trp1, his3, leu2 agm1
::LEU2 AGM1-URA3)
strains were already described in the previous paper (20).
To test the ability of CaGNA to complement
ScGNA1, the entire ORF of CaGNA1 was cloned at
the BamHI site (downstream of the ADH1 promoter)
of pGBT9-T (24). Thus, the transcription of CaGNA1 from this
plasmid was under the control of the ADH1 promoter. The
resulting plasmid was transfected into gna1
cells. After the trp+ cells were selected 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 ScGNA1
mutants harboring an alanine substitution for Ile97,
Glu98, Val102, Gln107,
Gly112, Leu115, Ile116,
Leu119, Phe142, Tyr143, or
Gly147 were generated by the oligonucleotide-directed dual
amber method as described (29) with Mutan-Express KmTM
(Takara). The entire ORF of the ScGNA1 gene was cloned
between the BamHI and EcoRI sites of pKF18k
(Takara) using EcoRI linker and hybridized with
oligonucleotides containing the indicated mutations. The resulting
mutant ScGNA1 genes were excised from the vector and ligated
with pGEX-2T, which had been digested with BamHI and
EcoRI. All the mutations were confirmed by sequencing the DNA.
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RESULTS |
Identification of the Yeast GlcN-6-P Acetyltransferase
Gene--
Previously, we demonstrated that unlike the bacterial
enzyme, the eukaryotic UDP-GlcNAc pyrophosphorylase is a monofunctional protein with no phospho-GlcN acetyltransferase activity (20). When
searching the S. cerevisiae data base, we found
YFL017C on chromosome VI encodes a protein with homology to
known acetyltransferases. To examine the physiological function of
YFL017C, we created an S. cerevisiae strain in
which the endogenous YFL017C gene was disrupted, but where
episomal copies of YFL017C, whose transcription was under
the control of the GAL1 promoter, were maintained. The resulting strain grew on galactose plates, but died on glucose plates,
confirming that YFL017C is an essential gene. When cultured on glucose plates, most of the YFL017C-deficient cells
swelled and often then lysed. This morphology was quite similar to that caused by the depletion of AGM1 or UAP1 (Fig.
1), suggesting that YFL017C is
involved in the synthesis of UDP-GlcNAc. Although the expected product
of YFL017C showed only a limited sequence similarity to the
C-terminal phospho-GlcN acetyltransferase domain of the bacterial
GlmU gene product (15% identity, see below),
YFL017C was called GNA1
(glucosamine synthesis
acetyltransferase 1) in this study.

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Fig. 1.
Aberrant morphology caused by the depletion
of YFL017C. S. cerevisiae cells of the wild
type, gna1 harboring pYEU-ScGNA1, uap1
harboring pYEU-ScUAP1, and agm1 harboring pYEU-ScAGM1
were spread on agar plates containing glucose and incubated at
30 °C. Photographs were taken at 24 h.
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If GNA1 is really a gene required for an essential metabolic
pathway, such as UDP-GlcNAc synthesis, it should be preserved in other
organisms. A search of the C. albicans data base revealed a
partial nucleotide sequence that shared sequence similarity with
S. cerevisiae GNA1 (ScGNA1). By screening the
C. albicans genomic DNA library with the DNA fragment of the
above nucleotide sequence as a probe, we were able to clone and
sequence the C. albicans homolog of GNA1
(CaGNA1). The predicted products of ScGNA1 and
CaGNA1 are closely related to each other (Fig.
2) (44% sequence identity over the
entire protein). In addition, when expressed under the control of the
ADH1 promoter, CaGNA1 rescued an S. cerevisiae gna1
null mutant (data not shown), demonstrating
that the CaGNA1 functionally complements ScGNA1.
Furthermore, a search of the Schizosaccharomyces pombe and
Caenorhabditis elegans data bases revealed that S. pombe SPAC16E8 and C. elegans B0024-12 encode proteins
which share a high degree of sequence identity with ScGna1p over the
entire protein (25.2% identity between ScGna1p and the SPAC16E8
product and 28.9% identity between ScGna1p and the B0024-12 product)
(Fig. 2). Therefore, we referred to these genes as S. pombe
GNA1 (SpGNA1) and C. elegans GNA1
(CeGNA1), respectively. Although the functionality of
SpGNA1 and CeGNA1 remains to be established, all
the above results strongly support the idea that GNA1
encodes ubiquitous acetyltransferase necessary for UDP-GlcNAc synthesis.

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Fig. 2.
Predicted amino acid sequences of the product
of YFL017C and its homologs in other organisms. The
amino acid sequence of the product of S. cerevisiae YFL017C
(ScGna1p) was compared with those of the C. albicans (CaGna1p), C. elegans
(CeGna1p), and S. pombe (SpGna1p)
homologs using the FASTA and BLAST programs. Amino acids that are
identical among the four proteins are boxed.
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In UDP-GlcNAc synthesis, there is a process involving acetyltransferase
that is the acetylation of phospho-GlcN. Accordingly, we asked whether
GNA1 specifies phospho-GlcN acetyltransferase. Both ScGna1
and CaGna1 proteins were expressed in E. coli as a fusion
with GST and then purified by affinity column chromatography (Fig.
3A). When incubated with
GlcN-6-P and Ac-CoA, GST-ScGna1p and GST-CaGna1p produced CoA, whereas
GST alone did not (Fig. 3B). The amounts of CoA released
from Ac-CoA reached a plateau within 5 min (Fig. 3B), and
GlcN-6-P was preferably utilized as the substrate (Fig. 3C).
Although small amounts of CoA were produced when higher concentrations
of GlcN-1-P were present (at concentrations higher than 100 µM), the Km value for GlcN-1-P was
about 25 times higher than that for GlcN-6-P (3.0 mM for
GlcN-1-P versus 124 µM for GlcN-6-P) (Fig.
3C, Table I). No CoA
production was observed when galactosamine 1-phosphate (GalN-1-P) was
used as the substrate (Fig. 3C).

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Fig. 3.
Expression and enzyme activities of the yeast
phospho-GlcN acetyltransferase. The S. cerevisiae and
C. albicans Gna1 proteins were expressed in E. coli as a fusion with GST and purified with glutathione-Sepharose
beads. A, approximately 1-µg amounts of the purified
recombinant proteins were separated on a 12.5% SDS-polyacrylamide gel
and stained with Coomassie Brilliant Blue. Lane 1; GST,
lane 2; GST-ScGna1p, lane 3; GST-CaGna1p. The
positions of the protein size markers are indicated in kilodaltons.
B, approximately 0.1 µg of the purified recombinant
GST-ScGna1p and GST-CaGna1p were incubated with 200 µM
each of GlcN-6-P and Ac-CoA. At the indicated times, the reaction was
terminated and the amounts of the released CoA, which represent the
enzyme activities, were determined by measuring the optical densities
at 412 nm. , GST-ScGna1p; , GST-CaGna1p; , GST. C,
approximately 0.1 µg of the purified recombinant GST-ScGna1p was
incubated with 200 µM Ac-CoA and the various
concentrations of the indicated phosphoamino sugars. The enzyme
activities were determined after incubation at 30 °C for 10 min as
in B. , GlcN-6-P; , GlcN-1-P; , GalN-1-P.
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Table I
Characteristics of the mutant ScGna1p
The Km and kcat values of the
wild type and mutant enzymes for Ac-CoA were determined from the
amounts of CoA produced from Ac-CoA. The Km values
for GlcN-6-P are also indicated in the right column. ND, not
determined.
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Synthesis of UDP-GlcNAc from GlcN-6-P in Vitro--
Previously, we
demonstrated that together with Agm1p, Uap1p produced UDP-GlcNAc from
GlcNAc-6-P (20). If Gna1p really catalyzes the acetylation of GlcN-6-P
and produces GlcNAc-6-P, UDP-GlcNAc would be synthesized from GlcN-6-P
in the presence of Gna1p, Agm1p, and Uap1p. As expected, GST-ScGna1p
and GST-CaGna1p, but not GST alone, produced
[32P]UDP-GlcNAc from GlcN-6-P, when the yeast Agm1p,
Uap1p, and [
-32P]UTP were added to the reaction
mixture (Fig. 4). A minor spot corresponding to UDP was also observed in each lane. This is presumably due to a minor contamination of [
-32P]UDP in the
[
-32P]UTP preparation, because it appeared even in the
absence of the enzyme (Fig. 4). Synthesis of UDP-GlcNAc from GlcN-6-P
required acetyl-CoA, GlcN-6-P, and Gna1p, confirming that Gna1p
generates GlcNAc-6-P using Ac-CoA (Fig.
5). Although Gna1p did not efficiently utilize GlcN-1-P as the substrate in the acetyltransferase assay (Fig.
3C), [32P]UDP-GlcNAc was efficiently
synthesized from GlcN-1-P when Agm1p, Uap1p, and [32P]UTP
were present (Fig. 5). Moreover, phospho-GlcNAc mutase remained an
essential factor even for the synthesis of UDP-GlcNAc from GlcN-1-P; no
[32P]UDP-GlcNAc was detected in the absence of Agm1p
(Fig. 5). This result suggests that Agm1p recognizes both phospho-GlcN
and phospho-GlcNAc as the substrates; Agm1p might first convert
GlcN-1-P into GlcN-6-P, providing the substrate for Gna1p, and then
might attack GlcNAc-6-P which was generated from GlcN-6-P by Gna1p.
Taken together, we concluded that GNA1 encodes GlcN-6-P
acetyltransferase, and defined GNA1 as
glucosamine-6-phosphate
acetyltransferase 1.

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Fig. 4.
Production of UDP-GlcNAc from GlcN-6-P by
ScUap1p, ScAgm1p, and ScGna1p. Approximately 0.1 µg of the
purified GST-ScGna1p was incubated with 0.1 µg of GST-ScUap1p, 0.1 µg of GST-ScAgm1p, 0.1 µM [ -32P]UTP,
200 µM GlcN-6-P, and 200 µM Ac-CoA. After
incubation at 30 °C for 10 min, the reaction products were separated
by polyethyleneimine-cellulose TLC and visualized by autoradiography.
The positions of UDP-GlcNAc, UDP, and UTP were visualized under UV
light. Lane 1, none; lane 2, GST; lane
3, GST-ScGna1p; lane 4, GST-CaGna1p.
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Fig. 5.
Factors required for the production of
UDP-GlcNAc from GlcN-1-P. Approximately 0.1 µg of the purified
GST-ScGna1p was incubated with 0.1 µg of GST-ScUap1p and 0.1 µM [ -32P]UTP in the presence or absence
of 0.1 µg of GST-ScAgm1p, 200 µM GlcN-1-P, and 200 µM Ac-CoA. After incubation at 30 °C for 10 min, the
reaction products were separated by polyethyleneimine-cellulose TLC and
visualized by autoradiography. The positions of UDP-GlcNAc, UDP, and
UTP visualized under UV light are indicated.
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Possible Active Sites of ScGna1p--
A number of
acetyltransferases have been identified in various organisms. When the
amino acid sequence of ScGna1p was compared with known and putative
acetyltransferases, we found that although the bacterial GlmU protein
is not highly related to ScGna1p, some of the amino acids in the two
short regions of ScGna1p are highly conserved among several
acetyltransferases (Fig. 6). These
regions, designated domain I and domain II, encompass the amino acid
positions from 97 to 117 and from 142 to 147, respectively. To examine
the importance of these domains for catalysis, we mutated the highly conserved amino acids in these regions Ile97,
Glu98, Val102, Gly112,
Leu115, Ile116, Phe142,
Tyr143, and Gly147 to alanine. As a control, a
nonconserved Leu119 was also converted to alanine. After
confirming the nucleotide sequences, all the mutant enzymes were
expressed as a fusion with GST and purified by affinity column
chromatography (Fig. 7A). As
shown in Fig. 7B, alanine substitutions for
Val102, Gly112, Leu115,
Ile116, Leu119, and Gly147 only
weakly impaired the activity. In contrast, those for Phe142
and Tyr143 severely diminished the activity. Furthermore,
although the activity of Y143A was too low to perform kinetics, alanine
substitution for the neighboring amino acid Phe142
increased the Km values for Ac-CoA and GlcN-6-P by
about 10- and 5-fold, respectively (Table I). The activities of I97A and E98A were also affected to a lesser extent (Fig.
7B). Interestingly, these two mutants displayed different
features of the binding affinities to the cofactor and the substrate.
Mutation of Ile97 increased the Km value
for GlcN-6-P by about 3-fold, whereas that of Glu98 raised
the Km value for Ac-CoA by about 2.5-fold (Table I).

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Fig. 6.
Comparison of the amino acid sequences of
several acetyltransferases. The amino acid sequence of ScGna1p was
compared with those of other acetyltransferases using the FASTA and
BLAST programs. Amino acids that were replaced by alanine are marked by
+. A nonconserved amino acid, Leu119, was mutated as a
control. ScGNA1, S. cerevisiae GlcN-6-P
acetyltransferase; CaGNA1, C. albicans GlcN-6-P
acetyltransferase; SpGNA1, S. pombe GlcN-6-P
acetyltransferase; CeGNA1, C. elegans GlcN-6-P
acetyltransferase; HsSSAT (GenBankTM accession
number U40369), Homo sapiens spermidine/spermine
N1-acetyltransferase (36).
CcSTAT (GenBankTM accession number U01945),
Camphylobacter coli streptothricin acetyltransferase (37);
SlSTAT (GenBankTM accession number M16183),
Streptomyces lavendulae streptothricin acetyltransferase
(38); CfAAC (GenBankTM accession number Z54241);
Citrobacter freundii aminoglycoside
6'-N-acetyltransferase (39); HsAA-NAT
(GenBankTM accession number X14672), H. sapiens
arylamine N-acetyltransferase (40); RatAA-NAT
(GenBankTM accession number U40803), Rattus
norvegicus N-acetyltransferase (41); ScNAT2
(GenBankTM accession number L25608), S. cerevisiae methionine N-acetyltransferase (42);
EcGlmU (GenBankTM accession number AE000450),
E. coli GlmU protein (17).
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Fig. 7.
Effects on ScGna1p activity of alanine
substitution for the conserved amino acids. The wild type and the
mutant ScGna1 proteins bearing an alanine substitution for each of the
amino acids that are highly conserved in several acetyltransferases
listed in Fig. 6 were expressed as a fusion with GST and purified with
glutathione-Sepharose beads. A, approximately 1-µg amounts
of the wild type (WT) and the indicated mutant proteins were
separated on a 12.5% SDS-polyacrylamide gel and stained with Coomassie
Brilliant Blue. The positions of the protein size markers and
GST-ScUap1p are indicated in kilodaltons and by the
arrowhead, respectively. B, approximately 0.1 µg of the purified wild type (WT) and the indicated mutant
proteins were incubated with 200 µM each of GlcN-6-P and
Ac-CoA at 30 °C for 10 min, and the amounts of the released CoA that
represent the enzyme activities were determined as described in Fig. 3.
The activities of the mutant enzymes are indicated as a percent of that
of the wild type.
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In human spermine/spermidine
N1-acetyltransferase (HsSSAT), it was
demonstrated that the amino acid sequence motif RGFGIGS beginning at
the position of 101 is required for the Ac-CoA binding and that
Arg101 and the proximal glycine loops in this motif are
essential for the enzyme activity (30). Moreover, the double mutant
R101A/E152K acts as a dominant negative (31). In our sequence
alignment, Arg101 and Gly106 of HsSSAT
correspond to Gln107 and Gly112 of ScGna1p,
respectively (Fig. 6). As indicated above, G112A restored the
significant activity, and other amino acids in the RGFGIGS motif are
not highly conserved in Gna1p-related acetyltransferases listed in
Table I and Fig. 6. Accordingly, we mutated Gln107 of
ScGna1p to alanine. Though the effect of the mutation of
Gln107 was not as severe as those of Phe142 and
Tyr143, the activity and the kcat values of
Q107A were significantly lower than that of the wild type, and the
Km value for Ac-CoA was about five times higher than
that of the wild type enzyme (Fig. 7B, Table I). From these
results, it was speculated that Phe142 and
Tyr143 of ScGna1p are essential residues for the catalysis,
and that Ile97, Glu98, and Gln107
also locate in the active pocket and support the binding of the cofactor and the substrate.
 |
DISCUSSION |
In this paper, we have identified the yeast GNA1 as the
gene for phospho-GlcN acetyltransferase, and demonstrated that unlike the case in bacteria, yeast UDP-GlcNAc pyrophosphorylase and
phospho-GlcN acetyltransferase are encoded by distinct genes. Because
UAP1, AGM1, GNA1, and GFA1
are all essential genes (18-20), every step of the UDP-GlcNAc
synthesis is essential for the viability of yeast cells. In addition,
yeast has only one type of acetylated amino sugar, that is GlcNAc;
neither GalNAc nor N-acetylmannosamine is present in yeast
cells. Thus, Gna1p is expected to be the sole amino sugar
acetyltransferase present in yeast cells.
ScGna1p showed a relatively higher Km value
for Ac-CoA (228 µM) compared with those of other
acetyltransferases (e.g. 3 µM for HsSSAT and
3.8 µM for choline acetyltransferase) (30-32). This is
also true for the E. coli phospho-GlcN acetyltransferase (320 µM) (26). Probably, low affinity to Ac-CoA is unique
feature of phospho-GlcN acetyltransferases. The Km
value of ScGna1p for GlcN-6-P was also comparatively high (124 µM). The enzyme displayed a reasonable substrate
specificity to GlcN-6-P, however, because the Km
value for GlcN-1-P was about 25 times greater than that for
GlcN-6-P. Nevertheless, ScGna1p produced UDP-GlcNAc from GlcN-1-P in
the presence of Agm1p, Uap1p, and UTP. Because Uap1p does not utilize
GlcNAc-6-P as the substrate (20), the results obtained in this study
indicate that Agm1p has a broad substrate specificity and recognizes
phospho-GlcN as well. In fact, not only phospho-GlcNAc but also
phosphoglucose can be the substrate of Agm1p (19).
Although PAT1, which denotes putative
acetyltransferase, is not listed in the yeast
data base, a search of the literature revealed that the amino acid
sequence of ScGna1p is identical to that of previously reported Pat1p
(33). PAT1 was isolated as the gene that suppressed the
histone H1-dependent growth of the S. cerevisiae
YRL4 cells (33). Depletion of Pat1p function results in multiple
phenotypical changes. The PAT1-deficient cells displayed
enlarged cell volume, increased sensitivity to the microtuble inhibitor, benomyl, aberrant spindle structure, and incompletion of
cytokinesis. Furthermore, by fluorescence-activated cell sorter analysis, it was demonstrated that PAT1 is required for
multiple steps in the cell cycle, such as exit from G0,
progression of DNA synthesis, and mitosis (33). All these facts suggest
a physiological link between UDP-GlcNAc synthesis and cell cycle
progression. As UDP-GlcNAc is mainly used as the cell wall precursor in
yeast, we wonder whether Gna1p has additional substrates, such as core histones, whose acetylation is essential for gene expression and cell
cycle progression; however, no histone acetyltransferase activity was
detected in the recombinant ScGna1p.
Lin et al. (33) cloned the PAT1/GNA1 homolog from
S. pombe by the functional complementation of an S. cerevisiae pat1
null mutation. Although the amino acid sequence
of the C-terminal region of S. pombe Pat1p (SpPat1p)
is identical to that of SpGna1p, the previously identified SpPat1p has
245 additional amino acids at the N terminus (33). Because we could not
find the DNA sequence corresponding to SpPat1p in the S. pombe data base, the presence of the extra N-terminal amino acids
in SpPat1p may be unique to a certain S. pombe strain.
Sequence comparisons of the various acetyltransferases revealed that
ScGna1p contains two short domains, where several amino acids are
highly conserved among several known and putative acetyltransferases. By the mutation study, we found that Phe142 and
Tyr143 of ScGna1p are crucial amino acids for the catalytic
reaction and that Ile97, Glu98, and
Gln107 also play important roles in the binding of the
cofactor and the substrate. As mentioned before, arginine at the
position of 101 and the proximal glycine loops of HsSSAT have been
implicated to be the active site of the enzyme (30, 31). Although
Arg101 of HsSSAT is converted to glutamine in the yeast
Gna1 proteins, our results indicate that Gln107 of ScGna1p
is also involved in the Ac-CoA binding. In contrast, alanine
substitution for the highly conserved Gly112, which
corresponds to the third glycine in the RGFGIGS motif, had little
effect on the activity of ScGna1p, and other glycines in the RGFGIGS
motif are not highly conserved in ScGna1p and its homologs. Recently,
the three-dimensional structures of the yeast histone acetyltransferase
(Hat1p) and Serratia marcescens aminoglycoside 3-N-acetyltransferase, both of which belong to the
GCN5-related superfamily, were solved, and the
R/Q-X-X-G-X-G motif, where
X denotes some variations, was demonstrated to form an
important part of the pyrophosphate-binding loop (34, 35).
Nevertheless, our results suggest that Phe142 and
Tyr143 in domain II of Gna1p play more important roles in
the Ac-CoA binding. Because neither Phe142 nor
Tyr143 is conserved in most of GCN5-related
N-acetyltransferases, it is still difficult to predict the
exact roles of these two amino acids in catalysis. Possibly,
Phe142 and Tyr143 in domain II are involved in
the interaction with the adenine base of Ac-CoA.
 |
ACKNOWLEDGEMENTS |
We thank S. Fukuchi for data base search and
sequence alignment, Y. Miyazaki for assisting with the experiments,
and S. B. Miwa for reading the manuscript.
 |
FOOTNOTES |
*
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) AB017626, AB017627, AB017628, and AB017629 (for
ScGNA1, CaGNA1, SpGNA1, and
CeGNA1, respectively).
¶
To whom all correspondence should be addressed: Dept. of
Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura,
Kanagawa, 247-8530, Japan. Tel.: 81-467-47-2242; Fax: 81-467-46-5320;
E-mail: hisafumi.okabe{at}roche.com.
 |
ABBREVIATIONS |
The abbreviations used are:
ORF, open reading
frame;
GST, glutathione S-transferase..
 |
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