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INTRODUCTION |
L-Glutamine:D-fructose-6-phosphate
amidotransferase (hexose-isomerizing) EC 2.6.1.16, known under a
trivial name of glucosamine-6-phosphate (GlcN-6-P)1 synthase,
catalyzes the complex reaction involving ammonia transfer and sugar
phosphate isomerization: L-glutamine + D-fructose 6-phosphate
D-glucosamine
6-phosphate + L-glutamate. This reaction is the first
committed step of the cytoplasmic biosynthetic pathway leading to the
formation of uridine 5'-diphospho-N-acetylglucosamine
(UDP-GlcNAc). The final product of this pathway is an activated
precursor of numerous macromolecules containing amino sugars, including
chitin and mannoproteins in fungi, peptidoglycan and
lipopolysaccharides in bacteria, and glycoproteins in mammals. GlcN-6-P
synthase belongs to the class II amidotransferase family but is unique
among other amidotransferases due to its apparent inability to use
exogenous ammonia as a nitrogen donor (1). The enzyme is widely
distributed in nature, and its activity has been detected in almost
every organism and tissue; several genes coding for GlcN-6-P synthase have been cloned and sequenced (for review see Ref. 1). However, only
prokaryotic GlcN-6-P synthases have been purified to apparent homogeneity from Escherichia coli and from the thermophilic
bacteria Thermus thermophilus (3). The E. coli
enzyme has been crystallized (4), and a structure of the
glutamine-binding domain has been elucidated (5). Availability of the
pure protein has facilitated extensive studies on its structure and
molecular mechanism of the enzymatic reaction (6-8).
On the other hand, there is very little known of the molecular
structure of eukaryotic GlcN-6-P synthases, and all the studies performed so far have been done on partially purified preparations. Several lines of evidence indicate that the eukaryotic enzyme could be
different from its prokaryotic counterpart. Comparison of the available
gene sequences has revealed a relatively large region (about 200 base
pairs) that is lacking in the prokaryotic proteins (9). Eukaryotic but
not prokaryotic GlcN-6-P synthases are the subject of feedback
inhibition by UDP-GlcNAc (10). Sensitivity to this inhibitor in
Blastocladiella emersonii (11) and probably in
Aspergillus nidulans (12) enzymes is modulated by reversible phosphorylation/dephosphorylation mediated by protein kinase(s) and phosphatase(s).
Fungal GlcN-6-P synthase is a subject of interest as a potential target
in antifungal chemotherapy (13). Rationally designed oligopeptides
containing an inhibitor of this enzyme showed promising chemotherapeutic effect in the murine model of disseminated candidiasis (14). Moreover, the fungal enzyme is a probable point of regulation of
chitin biosynthesis. In the opportunistically pathogenic fungus Candida albicans, activity of this enzyme increases
4-5-fold during yeast-to-mycelia (Y
M) morphological
transformation (15), correlating with a similar change in a chitin
content in the cell wall (16). This transformation is considered to be
a virulence factor during pathogenesis of human tissues (17).
The GFA1 gene encoding C. albicans GlcN-6-P
synthase has been recently cloned and sequenced (9). In the present
communication we describe the results of our further studies concerning
overexpression of the GFA1 gene in yeast, purification and
characterization of properties of the gene product, aimed especially at
regulation of its activity during morphological transformation of
C. albicans cells.
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Culture Conditions
Saccharomyces cerevisiae BJ1991 (MAT
pep4-3
prb1 ura3 leu2 trp1) was provided by I. Purvis (Glaxo Group
Research, Greenwood, UK). C. albicans ATCC 10261 was a gift
from M. Payton (GIMB, Geneva, Switzerland). C. albicans and
yeast cells were grown in YPD medium (2% glucose, 2% bacterial
peptone, 1% yeast extract) at 28 °C with shaking at 200 rpm.
Morphological Transformation
C. albicans cells grown overnight in YPD were
harvested, washed with saline, and starved overnight in saline at
4 °C. Starved cells were used to inoculate either the YCB/BSA medium
containing 1.17% yeast carbon base, 1% glucose, and 0.2% bovine
serum albumin or the Lee's medium (18). Yeast form cells grew
efficiently in both media at pH 4.5, 28 °C, and Y
M
transformation was performed at pH 6.5, 37 °C. Efficiency of the
morphological transformation was assessed by cell counting in a Burker chamber.
Bacterial Strains and Plasmids
E. coli DH5
F' (Life Technologies, Inc.) was used
for plasmid selection and amplification. Plasmid YEpGW42 (8.7 kb)
carrying the S. cerevisiae GFA1 gene on a 3.5-kb
EcoRI fragment inserted into YEp352 (19) was a gift from W. Tanner (Regensburg, Germany). YEpMA91 was a yeast shuttle vector
carrying the LEU2 marker and the promoter and terminator from
PKG1 separated by a BglII site (20).
DNA Isolation and Manipulations
Standard procedures were used for the isolation and subcloning
of plasmid DNA fragments (21). Methods for Southern and Northern analyses were the same as cited previously (9). PCR amplification was
for 30 cycles (1 min at 94 °C, 2 min at 50 °C, and 3 min at 72 °C) followed by 8 min at 72 °C then cooling to 40 °C. The
reaction mix used standard concentrations recommended by Perkin-Elmer. The primers were designed to incorporate BamHI sites at
either end of the structural gene, while maintaining an optimum
environment around the start codon (22): 5'-oligo, 5'-GAG AAA AAT ggA
Tcc TAT TAA Aaa ATG TGT GG-3'; 3'-oligo, 5'-CAG ACA ggA TcC
ATT TTC ATT ACT CAA CAG-3'. The start codon and stop
anti-codon are underlined. Mismatches are in
lowercase.
Yeast Transformation
S. cerevisiae deletion strains YRSu3-21 and YRSu3-31
were propagated in YPD containing D-glucosamine, 5 mg
ml
1. The cells were transformed by the lithium acetate
method (23). Selection for transformants was for LEU+ on
YNB minimal agar plates. YRS-C65 and YRS-C53 transformants were
propagated in defined YNB media containing 1% glucose, 0.65% YNB, and
appropriate supplements at 50 µg/ml and then transferred to YPD medium.
Purification of the Enzyme
Preparation of Crude Extract--
YRSC-65 cells (10 g wet
weight) from the overnight culture on YPD were harvested by
centrifugation (5,000 × g, 4 °C, 10 min) and washed
with cold buffer A (25 mM potassium phosphate buffer, pH
6.8, 1 mM EDTA). Cell paste was mixed with 20 g of
alumina and frozen. The mixture was carefully thawed and cells were
disrupted by grinding in a mortar. Buffer B (25 mM
potassium phosphate buffer, pH 6.8, 1 mM EDTA, 1 mM DTT) was added in small portions until the cell paste
became sticky, and grinding was continued. Cell debris and alumina were
spun down (10,000 × g, 4 °C, 5 min). Supernatant was saved, and the solid residue was extracted again with buffer B,
followed by centrifugation. Both supernatants were combined and
centrifuged (35,000 × g, 4 °C, 45 min). Precipitate
was discarded, and supernatant was saved as a crude extract.
Protamine Treatment--
Solution containing 1% protamine
sulfate in buffer B was added dropwise to the crude extract (1 ml per
70 mg of protein present in the crude extract), stirred moderately at
4 °C. The precipitated solid was harvested. Supernatant was
discarded, and precipitate was washed with buffer B and then combined
with 6 ml of buffer C (0.1 M pyrophosphate buffer, pH 6.8, 1 mM EDTA, 1 mM DTT, 10 mM
Fru-6-P). This suspension was stirred for 30 min at 4 °C and centrifuged (10,000 × g, 4 °C, 10 min). Precipitate
was discarded, and supernatant was saved as a pyrophosphate extract.
FPLC on Mono Q--
Pyrophosphate extract containing GlcN-6-P
synthase activity was filtered through the 0.22-µm Millipore membrane
filter, diluted 1:2 with buffer D (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT), and loaded on Mono Q HR
5/5 FPLC column equilibrated with buffer D. The column was washed with
5 ml of buffer D, and elution was performed with a linear 0-0.5
M NaCl gradient in buffer D at 1.0 ml min
1.
Active fractions were pooled and concentrated by ultrafiltration with
Centricon 10 device.
FPLC on Superdex 200--
The concentrated active fraction from
Mono Q was loaded on Superdex 200 HR 10/30 column equilibrated with
buffer B containing 0.15 M NaCl. Proteins were eluted with
the same buffer at the flow rate of 0.5 ml min
1. Active
fractions were pooled.
Determination of GlcN-6-P Synthase Activity
Colorimetric Method--
A standard incubation mixture consisted
of 10 mM Fru-6-P, 10 mM
L-glutamine, 1 mM EDTA, 1 mM DTT,
50 mM potassium phosphate buffer, pH 6.8, and appropriately
diluted enzyme preparation and inhibitors when necessary. Final
concentration of the pure GlcN-6-P synthase was 0.5-1.0 µg
ml
1. The reaction was started by adding the enzyme,
incubated at 37 °C for 30 min, and terminated by heating at
100 °C for 1 min. The concentration of GlcN-6-P formed by the
enzyme, determined by the modified Elson-Morgan procedure (24),
increased linearly for at least 60 min. One unit of specific activity
was defined as an amount of enzyme that catalyzed the formation of 1 µmol of GlcN-6-P min
1 mg protein
1.
Spectrophotometric Assay--
L-Glutamate formed by
GlcN-6-P synthase was determined by coupling with glutamate
dehydrogenase, essentially as described by Badet et al. (2).
This method was used to confirm the results obtained from kinetic
experiments performed at low concentrations of the substrates.
Determination of GlcN-6-P Synthase Activity in
Situ--
C. albicans cells grown in Lee's medium were
harvested and suspended in 8.5-ml portions of 0.1 M
imidazole/HCl buffer, pH 7.0, containing 0.2 M KCl and 0.1 M MgCl2, at cell density 1.2-1.6 × 109 cells ml
1. Aliquots (1.5 ml) composed of
toluene/ethanol/Triton X-100, 5:20:2, were added to the cell
suspensions, and the mixtures were vortexed for 5 min at room
temperature. The cells were washed three times with 50 mM
potassium phosphate buffer, pH 6.8, containing 1 mM EDTA
and 1 mM DDT and suspended in the same buffer at
108 cells ml
1. L-Glutamine, 10 mM, and Fru-6-P, 10 mM, and inhibitors when necessary were added, and the suspensions were incubated for 30 min at
37 °C. Cells were removed by centrifugation, and GlcN-6-P concentration was assayed in the supernatant, as described above.
Inactivation of GlcN-6-P Synthase with Glutamine Analogs
Incubation mixtures containing 5 µg of GlcN-6-P synthase, 50 mM potassium phosphate buffer, pH 6.8, 1 mM
EDTA, 15 mM Fru-6-P, and inactivators at various
concentrations in a total volume of 1 ml were incubated at 25 °C. To
follow an inactivation of the enzyme, 200-µl aliquots were withdrawn
from the mixtures, applied at the tops of 1-ml columns packed with
Sephadex G-25 (equilibrated previously with 50 mM potassium
phosphate buffer, pH 6.8), and centrifuged (500 × g, 1 min, 4 °C). Under these conditions the unbound inhibitor was
separated from the enzyme, and protein was recovered in clean test
tubes. Appropriate effluent aliquots were used for the determination of
the residual enzyme activity.
Chemical Modification of the Enzyme
GlcN-6-P synthase, 5 µg, was incubated with group-specific
reagents under following conditions: (a) with NTCB, IAA,
phenylmethylsulfonyl fluoride, and NAI in 50 mM potassium
phosphate buffer, pH 6.8, containing 1 mM EDTA;
(b) with DEP in 50 mM potassium phosphate buffer, pH 6.0, containing 1 mM EDTA; (c) with
BD in 50 mM bicarbonate buffer, pH 8.5; (d) with
CMC in 50 mM MES buffer, pH 6.0; (e) with PLP in
50 mM HEPES buffer, pH 6.5, in the dark, followed by 1-h
reduction with 20 mM NaBH3CN. All the
incubations were run in a total volume of 1 ml, at 25 °C, except
that with DEP was run at 4 °C. Aliquots (200 µl) withdrawn from
the incubation mixtures at appropriate time intervals were further
processed as described above.
Determination of an Isoelectric Point
Chromatofocusing was performed on a Mono P HR 5/5 column. The
purified GlcN-6-P synthase (200 µg) was dissolved in 25 mM Bis-Tris-HCl, pH 6.3, as a starting buffer, and a pH 6 to 4 gradient was generated during the elution with 20 ml of Polybuffer
74 (diluted 1:10 in water) solution, pH 4. Samples (0.5 ml) were
collected, and pH values and the GlcN-6-P synthase activity were measured.
Phosphorylation of GlcN-6-P Synthase in Vitro
GlcN-6-P synthase, 100 µg ml
1, was preincubated
for 0-120 min at 25 °C with 10 µM cAMP, 1 mM ATP, cAMP-dependent protein kinase from beef
heart (30 units ml
1), and/or protein kinase inhibitor
from rabbit muscle (10 µg ml
1), as indicated. Each
sample contained 10 mM EDTA and 40 mM NaF. Samples were assayed for GlcN-6-P synthase activity and sensitivity to
0.5 mM UDP-GlcNAc.
Determination of Stoichiometry of Phosphorylation
GlcN-6-P synthase (10 µg ml
1, 30 pmol) was
phosphorylated by incubating with cAMP-dependent protein
kinase (5 µg ml
1, 62 pmol), [
-32P]ATP
(0.5 mM, 4 µCi nmol
1), 10 µM
cAMP, 10 mM EDTA in a total volume of 800 µl of 50 mM Tris-HCl, pH 7.4, at 25 °C for various times. At time
intervals, 2× 50 µl samples of the incubation mixture were
collected. One of them was treated with protein kinase inhibitor, 1 µg ml
1, and after appropriate dilution assayed for
GlcN-6-P synthase activity. In the second sample, the phosphorylation
was stopped by adding Laemmli sample buffer, and the mixture was boiled
for 3 min and then subjected to SDS-PAGE. Gels were stained with
Coomassie Brilliant Blue, and bands corresponding to GlcN-6-P synthase
were cut from the gel and counted for radioactivity in Beckman LS 3801 scintillation counter. Blanks obtained from control mixtures containing all components except protein kinase, processed as described above, were subtracted from the particular values.
Molecular Weight Determination
Gel filtration was performed on Superdex 200 HR 10/30, eluted at
0.5 ml min
1 with 25 mM potassium phosphate
buffer, pH 6.8, containing 0.15 M NaCl, 1 mM
DTT, and 1 mM EDTA. Protein elution was followed at 280 nm,
and GlcN-6-P synthase activity was measured colorimetrically in 0.5-ml
samples. Native PAGE was run at 4-9% acrylamide concentration, and
the data were treated as described (25).
Cross-linking
Native GlcN-6-P synthase, 0.5 mg ml
1 in 50 mM triethanolamine buffer, pH 8.0, containing 0.15 M KCl, was treated at 25 °C with 0.01% glutaraldehyde
for 8 h. SDS was added to 0.6%, the samples were incubated at
37 °C for 1 h and then submitted to SDS-PAGE analysis run in
3% gel with cross-linked rabbit phosphorylase b oligomers
as molecular mass markers.
Other Methods
Small quantities of yeast or C. albicans cells were
broken by the small scale glass beads procedure (26). Phosphoglucose isomerase activity was assayed according to the procedure of Stein (27)
and glutaminase activity by the method of Holcenberg (28). Glucose
6-phosphate concentration was assayed as described by Lowry and
Passoneau (29). Discontinuous SDS-PAGE was performed by the method of
Laemmli (30) with 5% stacking gel and 7.5% separating gel. Native
PAGE was performed as described previously (31). Protein was assayed by
the Bradford procedure (32) with bovine serum albumin as a standard.
Materials
Glutamine analogs were synthesized by Dr. R. Andruszkiewicz,
Technical University of Gda
sk. [
-32P]ATP (4 µCi nmol
1) was from Amersham, UK. Other reagents were
from Sigma.
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RESULTS |
Construction of Deletion Strains, Overexpression Plasmid, and Yeast
Transformation--
EcoRI fragment of the YEpGW42 plasmid
containing the S. cerevisiae GFA1 gene was ligated into the
EcoRI site of vector pBR325 (6 kb, Life Technologies, Inc.).
The backbone of the deletion casette was obtained by band purification
of an 8.5-kb fragment after digestion with XhoI and
BglII. A 1.1-kb HindIII fragment encoding a
functional URA3 gene was obtained by digestion of pSPUR1, band-purified, and ligated into the polylinker of pGEM-7Zf(+) (3,000 base pairs, Promega). Transformants that gave white colonies on
isopropyl
-D-thiogalactoside, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside plates were isolated. The 1.1-kb
fragment was then excised by digestion with XhoI and
BamHI, thus generating URA3 fragment containing ends compatible with the deletion cassette backbone. Ligation yielded
plasmid pRS where residues
31 to 971 of GFA1 had been replaced with the URA3 gene. Yeast strain BJ1991 was
transformed with the mixture of the restriction fragments excised with
SspI, and URA3 colonies were selected. Four were picked and
shown to require glucosamine for growth on YPD plates. YRSu3-21 and
YRSu-31 were isogenic deletion strains except for the orientation of
insertion of the URA3 gene. Northern analysis confirmed that
these strains did not produce any mRNA hybridizing to the
GFA1 gene (Fig. 1), and no
GlcN-6-P synthase activity could be detected.

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Fig. 1.
Northern analysis of GFA1
gene expression. Total mRNA isolated from deletion
strains YRSu3-21, YRSu3-31 and transformants YRSC-65, YRSC-53 was
subjected to quantitative Northern analysis. Samples were probed with
the BamHI fragment (0.8 kb) of the YEpGW42 plasmid,
containing the S. cerevisiae GFA1 gene.
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The C. albicans GFA1 gene (9) was amplified by PCR using
primers that incorporated BamHI sites 5' and 3' to the
coding sequence. The PCR 2.1-kb product was digested with
BamHI, band-purified, and ligated into the BglII
site of pMA91. Restriction mapping was used to determine that obtained
plasmids YEpRSC-65 and YEpRSC-53 contained the GFA1 gene in
the correct orientation relative to the PGK1 promoter. These
plasmids were transformed into strains YRSu3-21 and YRSu3-31. They
complemented the glucosamine auxotrophy, restored GlcN-6-P synthase
activity, and a GFA1 transcript of the correct size was
detected by Northern analysis (Fig. 1).
Several isolated YRSC-65 and YRSC-53 transformants when grown in YPD
medium reproducibly produced C. albicans GlcN-6-P synthase constituting 5-7% of total cytoplasmic proteins, as revealed by densitometric SDS-PAGE analysis.
Enzyme Purification and Kinetic Properties--
The C. albicans GlcN-6-P synthase, overproduced by YRSC-65 cells, was
purified to at least 97% homogeneity with 52% yield using a four-step
procedure involving protamine sulfate precipitation, ion-exchange
chromatography, and gel filtration, as summarized in Table
I. Two enzymes that could affect the
kinetic measurements, namely phosphoglucose isomerase and glutaminase,
were not precipitated with protamine sulfate and therefore absent from
the pyrophosphate extract and more purified preparations. Such a
purification could be completed in 2 days. Isolation from 10 g of
wet weight cells afforded 4.4 mg of the electrophoretically homogenous
protein. The enzyme was relatively stable in crude extract, when stored at
20 °C; repeated thawing and freezing did not affect its
activity for at least 1 month. More purified preparations were stable
at
20 °C for weeks when stored in 50% glycerol. The presence of 1 mM DTT and 10 mM Fru-6-P was essential for the
enzyme stability. The pure enzyme exhibited pH optimum 6.8 ± 0.05 in Tris, Bis-Tris, HEPES, Mops, and phosphate buffers.
Km for L-Gln was 1.56 mM,
Km for Fru-6-P was 1.41 mM, and
kcat was 1150 min
1. The
Km values were also determined for the enzyme
present in crude extracts prepared from C. albicans ATCC
10261 Y and M cells. The former exhibited Km for
L-Gln was 1.52 mM and Km for
Fru-6-P was 1.45 mM, whereas the latter had the same
Km for Fru-6-P, but the Km for
L-Gln was 0.82 mM, and an inhibition of the
enzyme activity by an excess of this substrate was observed (details
not shown). Substrate inhibition was not detected for the pure enzyme
and for the enzyme present in the crude extract from C. albicans Y cells.
Isoelectric Point--
Pure GlcN-6-P synthase was chromatofocused
using a 6 to 4 pH gradient on MonoP HR 5/5 FPLC column. The enzyme was
partially denatured during column development, but an activity profile
(not shown) enabled an estimation of an isoelectric point of 4.6 ± 0.05.
Studies on the Enzyme Structure--
Molecular weight of the
C. albicans GlcN-6-P synthase subunit was determined by
SDS-PAGE. From the plot of lg(Mr)
versus migration distance, Mr = 79,500 was obtained, which is in a good agreement with the value
79,482, deduced from the gene sequence. The molecular weight of the
native protein was determined by gel filtration and native PAGE run
under variety of acrylamide concentrations. The first method gave
reproducibly Mr = 340,000 and the latter Mr = 330,000 (Fig.
2). In both methods only single bands
were detected.

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Fig. 2.
Determination of the molecular weight of
native GlcN-6-P by native PAGE run under variety of polyacrylamide
concentrations. GlcN-6-P synthase and the following marker
proteins: 1, bovine serum albumin (66 kDa); 2, alcohol dehydrogenase (150 kDa); 3, -amylase (200 kDa);
4, xanthine oxidase (280 kDa); 5, apoferritin
(443 kDa); urease (483 kDa) were applied to 4, 5, 6, 7.5, and 9%
polyacrylamide gels and Coomassie Brilliant Blue-stained. For each
protein a graph of a logarithmic function of the relative mobility
versus acrylamide concentration was constructed
(inset shown the graph obtained for GlcN-6-P synthase). The
slopes of the graphs were plotted against molecular masses. Position of
GlcN-6-P synthase is indicated by an arrow.
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The native enzyme was treated with glutaraldehyde for 8 h. SDS
treatment of such a preparation and subsequent separation of its
components by SDS-PAGE led to the appearance of bands corresponding to
Mr = 80,000, 178,000, and 325,000 (not shown).
It may be assumed that the bands corresponded to the monomeric,
dimeric, and tetrameric forms of the enzyme, respectively. No band
corresponding to the possible trimeric form was found.
Pure native C. albicans GlcN-6-P synthase was incubated with
several chemical reagents under conditions ensuring selective modification of particular amino acid residues. Samples drawn from the
reaction mixtures at time intervals were subjected to enforced gel
filtration, and activity of GlcN-6-P synthase was determined in
effluent aliquots. Incubation with Cys-directed NTCB and IAA,
Arg-directed BD, His-directed DEP, Lys-directed PLP, Asp/Glu-directed
CMC, and Tyr-directed NAI led to a time- and
concentration-dependent irreversible modification of the
enzyme, whereas Ser-directed phenylmethylsulfonyl fluoride had no
effect. In each case the inactivation was complete at the appropriate reagent concentration. The pattern of the plots of apparent
inactivation velocity constants (kapp)
versus inactivator concentration derived from these
experiments were the straight lines originating at zero point (not
shown), thus indicating single-step reactions. Kinetic analysis of the
results afforded the apparent second-order rate constants
k1, summarized in Table
II. These values were rather low, except
that found for DEP. Reaction orders determined from the plots of
lg(kapp) = n lg([I]) were found to
be in the 0.87-1.18 range. The residues modified by the group-specific
reagents were not unequivocally identified, but the protective effect
of enzyme substrates was studied. Presence of L-Gln in
incubation mixtures protected the enzyme against inactivation caused by
NTCB and IAA, whereas Fru-6-P prevented inactivation caused by DEP, BD,
PLP, and NAI. Fru-6-P afforded only partial protection against CMC.
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Table II
Chemical modification of C. albicans GlcN-6-P synthase
Pure GlcN-6-P synthase was incubated with group-specific reagents, an
excess of inactivating agent was removed by enforced gel filtration,
and a residual enzyme activity was determined. Inactivation rate
constants (k1) were determined from the plots of
apparent rate constants kapp versus
inactivator concentration. In protective experiments, either 10 mM L-Gln or 10 mM D-Fru-6-P was included in the
incubation mixture containing inactivator at concentrations causing
complete inactivation in the absence of the substrate, and
kapp' was determined.
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Inhibition of the Enzyme Activity--
The pure GlcN-6-P synthase
was inhibited by a number of glutamine analogs, competitively in
respect to L-Gln and uncompetitively in respect to Fru-6-P.
Inhibitory constants for these compounds are summarized in Table
III.
N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropanoic
acid and
N3-D-trans-2,3-epoxysuccinamoyl-L-2,3-diaminopropanoic
acid inactivated the enzyme in a time- and
concentration-dependent manner. The plots of half-times of
inactivation t1/2 versus inactivator
concentration were hyperbolic in both cases (not shown), thus
indicating that inactivation was a two-step process with formation of a
reversible complex preceding an irreversible modification. Kinetic
analysis of the inactivation data, according to the method of Kitz and
Wilson (33), provided the kinetic constants
Kinact and k2, also shown
in Table III.
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Table III
Inhibition and inactivation of GlcN-6-P synthase by glutamine
analogs
For inhibition, residual activity of pure GlcN-6-P synthase was assayed
as described under "Experimental Procedures" in the presence of
different concentrations of glutamine analogs, except the
L-Gln concentrations were variable. Data were analyzed
using Lineweaver-Burk plots. Inhibitory constants Ki were
determined from the secondary plots of kapp
versus inhibitor concentration, derived from the
Lineweaver-Burk plots. For inactivation, pure GlcN-6-P synthase was
incubated with FMDP or EADP in the presence of 10 mM
Fru-6-P. An excess of inactivating agent was removed by enforced gel
filtration, and a residual enzyme activity was determined. Limiting
inactivation rate constants k2 and inactivation
constants kinact were determined from the plots of
inactivation half-times t1/2 versus
reciprocal of inactivator concentration.
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Pure recombinant GlcN-6-P synthase and the enzyme present in the crude
extract prepared from the mycelial forms of C. albicans ATCC
10261 were very poorly inhibited by UDP-GlcNAc. The maximal level of
inhibition noted for 5 mM and higher concentrations of UDP-GlcNAc was as low as 32%, whereas the enzymes present in a crude
extracts obtained from the YRSC-65 and C. albicans ATCC 10261 yeast-like cells were quite sensitive to this inhibitor with
IC50 = 0.67 and 0.69 mM, respectively (Fig.
3). The sensitivity to the inhibitor was
actually lost after the second step of purification, i.e.
precipitation with protamine sulfate and subsequent elution with
pyrophosphate buffer. Such preparations obtained from crude extracts
prepared either from YRSC-65 or ATCC 10261 cells demonstrated poor
sensitivity, comparable to that of the pure protein.

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Fig. 3.
Inhibition of GlcN-6-P synthase by
UDP-GlcNAc. Residual activity of pure and crude GlcN-6-P synthase
was assayed as described under "Experimental Procedures." Crude
extract from YRSC-65 ( ), crude extract from C. albicans
ATCC 10261, Y forms ( ), crude extract from C. albicans, M
forms ( ), pure GlcN-6-P synthase ( ).
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Since the previous literature data suggested a possible role of sugar
phosphates in modulation of GlcN-6-P synthase sensitivity to
UDP-GlcNAc, we compared the effects of different structurally related
sugar phosphates on enzyme inhibition by this sugar nucleotide. The
data presented in Fig. 4 clearly show
that among the compounds tested, only glucose 6-phosphate enhanced the
enzyme sensitivity to UDP-GlcNAc. This effect was Glc-6-P
concentration-dependent and a relatively high level of this
sugar phosphate, >10 mM, was necessary to reach the full
enzyme sensitivity to UDP-GlcNAc. All the tested sugar phosphates
alone, at concentrations <20 mM, had no direct effect on
GlcN-6-P synthase activity.

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Fig. 4.
Effect of sugar phosphates on inhibition of
pure C. albicans GlcN-6-P synthase by UDP-GlcNAc.
Residual activity of GlcN-6-P synthase (GS) treated by
UDP-GlcNAc, 0.8 mM, was assayed as described under
"Experimental Procedures" in the presence or absence of sugar
phosphates, 10 mM. Each bar represents the mean
of three determinations ± S.D. Inset, influence of
Glc-6-P on inhibition of pure GlcN-6-P synthase by 0.8 mM
UDP-GlcNAc. Each point represents the mean of three determinations ± S.D.
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Inhibition of pure GlcN-6-P synthase by UDP-GlcNAc in the presence of
Glc-6-P was non-competitive, both in respect to Fru-6-P (Fig.
5) and to L-Gln (not shown).
The Hill coefficient and the inhibitory constant, determined from the
log (v/vi
1) = log
1/Ki + nH log [I] plot
(Fig. 5, inset), were nH = 0.91 and
Ki = 0.52 mM, respectively. The
non-competitive mode of inhibition was not changed in the absence of
Glc-6-P, but the nH value dropped to 0.54, whereas the Ki increased to 3.7 mM.

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Fig. 5.
Kinetic analysis of GlcN-6-P synthase
inhibition by UDP-GlcNAc. Non-competitive inhibition in respect to
Fru-6-P. Activity of pure GlcN-6-P synthase was assayed as described
under "Experimental Procedures," except the Fru-6-P concentrations
were variable in the presence of 0.5 mM UDP-GlcNAc and 10 mM Glc-6-P ( ); 0.5 mM UDP-GlcNAc ( ); 10 mM Glc-6-P or no additives ( ). Each point is the
mean of three determinations. Inset, Hill plots derived for
inhibition of pure GlcN-6-P synthase by UDP-GlcNAc measured under
standard conditions in the absence ( ) or presence ( ) of 10 mM Glc-6-P.
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Phosphorylation of the C. albicans GlcN-6-P Synthase in
Vitro--
The pure enzyme was tested as a possible substrate for
commercially available cAMP-dependent protein kinase from
beef heart. The GlcN-6-P synthase was subjected to the kinase action
under conditions optimal for protein phosphorylation. GlcN-6-P synthase activity and sensitivity to UDP-GlcNAc were monitored. The 30-min incubation with protein kinase in the presence of ATP and cAMP resulted
in a more than 100% increase of GlcN-6-P synthase-specific activity,
whereas the sensitivity to the physiological feedback inhibitor was
only slightly affected (Fig. 6). cAMP
alone and a combination of cAMP and ATP had no effect on GlcN-6-P
synthase, whereas the presence of the kinase inhibitor from rabbit
muscle abolished the kinase effect, thus demonstrating that the
observed enhancement of GlcN-6-P synthase activity is a consequence of protein phosphorylation. The enhancement of GlcN-6-P synthase activity
upon the action of cAMP-dependent protein kinase was time-dependent and showed saturation kinetics with the
maximum value observed after 90 min (Fig. 6, inset). Results
of another experiment, shown in Fig. 7,
in which GlcN-6-P synthase was phosphorylated with
[
-32P]ATP as a substrate, demonstrated correlation
between GlcN-6-P synthase activity and an extent of its
phosphorylation. The stoichiometry of the phosphorylation determined
under optimal conditions was 1.21 ± 0.08 mol of P/mol of GlcN-6-P
synthase.

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Fig. 6.
Changes in activity of C. albicans
GlcN-6-P synthase and its sensitivity to UDP-GlcNAc after
treatment with cAMP-dependent protein kinase. Pure
GlcN-6-P synthase, 100 µg ml 1, was preincubated for 30 min at 25 °C with 10 µM cAMP, 1 mM ATP,
cAMP-dependent protein kinase from beef heart (30 units/ml), and/or protein kinase inhibitor from rabbit muscle (10 µg
ml 1), as indicated. Each sample contained 10 mM EDTA and 40 mM NaF. Samples were assayed for
GlcN-6-P synthase activity in the absence (black bars) and
presence (gray bars) of 0.8 mM UDP-GlcNAc. Each
bar represents the mean of three determinations ± S.D.
Inset, time course of changes in GlcN-6-P synthase activity
subjected to the action of protein kinase. Samples containing both
enzymes, cAMP, ATP, EDTA. and NaF at concentrations indicated above
were incubated at 25 °C for 120 min and assayed for GlcN-6-P
synthase activity at times indicated.
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Fig. 7.
Dependence of GlcN-6-P synthase activity on
extent of phosphorylation of this enzyme. 8 µg of GlcN-6-P
synthase was incubated with 4 µg of cAMP-dependent
protein kinase, [ -32P]ATP (0.5 mM, 4 µCi
nmol 1), 10 µM cAMP, 10 mM EDTA
in a total volume of 800 µl of 50 mM Tris-HCl, pH 7.4, at
25 °C for various times. At intervals samples of the incubation
mixture were collected, assayed for GlcN-6-P synthase activity, and
subjected to SDS-PAGE. Bands corresponding to GlcN-6-P synthase were
cut from the gel and counted for radioactivity. Results are the means
of three independent determinations ± S.D.
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Changes in the GlcN-6-P Synthase Properties during Y
M
Transformation of C. albicans Cells--
GlcN-6-P synthase activity
and sensitivity to UDP-GlcNAc were measured in situ in
C. albicans cells stimulated in defined media to Y
M
transformation by temperature and pH shift. Data summarized in Table
IV show about 5-fold increase in the
enzyme activity measured in cells incubated in the Lee's amino acid
medium, pH 6.5, at 37 °C and this enhancement was correlated with
the progress of morphological transition. On the other hand, the enzyme activity was practically constant when the cells were incubated in the
Lee's medium, pH 4.5, at 30 °C, i.e. under conditions
ensuring the yeast-like morphology (detailed data not shown). Induction of Y
M transformation resulted also in an immediate desensitization of GlcN-6-P synthase to UDP-GlcNAc. Similar changes in the GlcN-6-P synthase activity and sensitivity to inhibition caused by UDP-GlcNAc were observed when morphological transformation was induced in YCB/BSA
medium (not shown).
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Table IV
Changes in the GlcN-6-P synthase activity and its sensitivity to
UDP-GlcNAc measured in situ during Y M transformation of C. albicans
Y M morphological transformation of C. albicans ATCC
10261 was induced in Lee's medium, pH 6.5. Cells were incubated at
37 °C; samples of the cell suspension were collected at intervals,
and GlcN-6-P synthase activity was assayed in situ, as
described under "Experimental procedures," in the presence or
absence of 0.5 mM UDP-GlcNAc. Progress of morphological
transformation was assessed by cell counting. Data are the means of
three determinations.
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The Glc-6-P concentration in cytosol of C. albicans cells,
measured after overnight starvation, fell to 0.85 ± 0.05 mol mg dry weight
1 from the value of 2.9 ± 0.15 nmol mg
dry weight
1 found in Y cells harvested from the rich YPD
medium. Incubation of the starved cells in Lee's or YCB/BSA medium, pH
4.5, at 30 °C, i.e. under conditions ensuring the yeast
morphology, caused an increase in Glc-6-P concentration to 3.2 ± 0.2 nmol mg dry weight
1 after 1 h, followed by
constant, slow decrease. On the other hand, the maximal level of
Glc-6-P detected in the cells induced to Y
M transformation in
Lee's or YCB/BSA, pH 4.5, at 37 °C, was as low as 1.3 ± 0.3 mg dry weight
1. The low level of Glc-6-P was practically
constant during transformation.
 |
DISCUSSION |
The C. albicans GFA1 gene was overexpressed in S. cerevisiae, thus allowing the purification of its product,
i.e. C. albicans GlcN-6-P synthase. It should be
mentioned that the GFA1 gene of the yeast host was knocked
out, thus allowing an efficient purification of C. albicans
GlcN-6-P synthase, not contaminated by its yeast-derived counterpart.
From the relationship between the specific activity of the pure protein
and that present in the crude extract prepared from the C. albicans cells, it can be estimated that GlcN-6-P synthase is a
very minor component of the C. albicans cytosolic protein
pool (<0.025%). This content was increased to 5-7% in YRSC-65
recombinant cells. Such a level of overexpression, albeit not very
high, is comparable to that achieved previously for the E. coli GlcN-6-P synthase (34) and quite sufficient for quick and
effective purification of the enzyme.
A relatively simple four-step procedure was elaborated which allowed
isolation of the electrophoretically pure enzyme from recombinant
cells. The overproduced C. albicans GlcN-6-P synthase could
be almost quantitatively precipitated from crude extract with protamine
sulfate and recovered with good yield by elution from the precipitate
with a pyrophosphate buffer. This step allowed separation of the enzyme
from bulk amount of accompanying proteins. The pyrophosphate extract,
containing about 25% pure GlcN-6-P synthase but not phosphoglucose
isomerase and glutaminase, could be used for most kinetic measurements,
since the results were not different from those obtained for the pure
protein. The Mono Q chromatography, albeit carried out as quickly as
possible, always caused a substantial loss of the enzyme activity.
However, a relative instability of GlcN-6-P synthases from other
sources during an ion exchange chromatography was previously reported
(35). The described method can be easily scaled up if a Mono Q column
of higher capacity is used.
The properties of the pure C. albicans GlcN-6-P synthase
could be compared with those reported for the pure bacterial enzyme or
partially purified preparations of eukaryotic GlcN-6-P synthase. The
Km for L-glutamine is 2-3 times higher
than most of the values determined so far, but Km
values for Fru-6-P and the pH optima are similar to those reported
previously. We could confirm a previous observation of Chiew et
al. (15) on a substantial increase of the enzyme affinity to
L-glutamine occurring during Y
M transformation of
C. albicans cells. An isoelectric point of the enzyme is 0.3 pH units lower than that found previously for its bacterial counterpart
(2) but similar to the value of 4.5 found for the rat hepatoma enzyme
(36). The acidic character of the protein explains its high affinity to protamine.
The bacterial GlcN-6-P synthase was previously unequivocally found to
be a dimer of 70-kDa subunits (2). Our results clearly demonstrate that
the fungal enzyme is a homotetramer of 79.5-kDa subunits. Such a
quaternary structure seems to be a general feature of eukaryotic
GlcN-6-P synthase, since molecular masses ranging from 300 to 380 kDa
were previously reported for partially purified enzymes from eukaryotic
sources (10, 36-38).
Chemical modification studies provided preliminary information on amino
acid residues essential for the enzyme activity. Two such residues,
Cys-1, the only catalytic residue located at the glutamine-binding
domain, and Lys-608, participating in sugar phosphate isomerization at
the Fru-6-P-binding domain, were previously unequivocally identified in
E. coli GlcN-6-P synthase (2, 39). Since the C. albicans enzyme was effectively protected by glutamine against
inactivation caused by cysteine-directed reagents, NTCB and IAA, and by
Fru-6-P against lysine-directed PLP and, on the other hand, respective
Cys-1 and Lys-708 residues are present in the highly conserved regions
of the fungal protein (9), there is little doubt that these residues
were actually modified in our experiments and play the same role as in
the bacterial enzyme.
The C. albicans GlcN-6-P synthase was effectively
inactivated by a histidine-directed reagent DEP under conditions
preventing interaction with the reactive cysteinyl residue. A
protective effect of Fru-6-P suggests a location of this essential
residue at the Fru-6-P-binding domain, where it could probably act as a
general acid-base catalyst in isomerization of fructose imine phosphate. The previous investigation of the bacterial enzyme, irreversibly modified by DEP, provided evidence for the presence of an
essential histidine at the glutamine-binding domain (40). The authors
were not able to identify this residue, due to the instability of the
DEP-derivatized histidyl residues. However, the later x-ray studies of
the glutamine-binding domain of E. coli GlcN-6-P synthase
did not reveal any histidyl residue participating in substrate binding
or catalysis at this domain (5), thus denying a previously formulated
conception of Cys-His-Asp catalytic triad (41). In our opinion the
"essential" histidyl residue modified upon the action of DEP on the
bacterial enzyme could be a moiety situated next to any really
essential site. The most likely candidate seems to be His-97, since
Asn-98 and Gly-99 were reported to be involved in stabilization of a
tetrahedral intermediate (5). The respective His-123 residue of the
C. albicans enzyme, adjacent to Asn-124 and Gly-125, does
not seem to be accessible for chemical modification, since we did not
observe any protective effect of L-glutamine against
chemical modification of this protein caused by DEP.
In contrast to the glutamine-binding domain, amino acids participating
in catalysis and substrate binding at the Fru-6-P-binding domain are
not known, except the lysyl residue mentioned above. Our present
studies provided preliminary evidence suggesting the presence of
essential histidyl, arginyl, and tyrosyl residues at this site. The
essential arginyl residue could be involved in phosphate binding. Such
a role was previously suggested for the essential arginyl residue
present at the active site of phosphoglucose isomerase, an enzyme
catalyzing a reaction closely resembling sugar phosphate isomerization
taking place at the Fru-6-P-binding site of GlcN-6-P synthase (42). On
the other hand, an essential tyrosyl residue should be most likely
involved in formation of hydrogen bonds stabilizing transition state
intermediates. A precise location of a putative essential Glu/Asp
moiety is not clear. The identity and actual roles of putative
essential histidyl, arginyl, Glu/Asp, and tyrosyl residues remain to be
revealed, especially by site-directed mutagenesis. This work is in
progress in our laboratory.
In our previous work we reported that C. albicans GlcN-6-P
synthase, like its other eukaryotic counterparts, is inhibited by
UDP-GlcNAc (9). However, the IC50 value is in the
millimolar range, much higher than that found previously for the rat
liver (10), Neurospora crassa (43), human liver (44), or
jack bean (45) enzymes but is comparable to the yeast protein (19). The
non-competitive character of inhibition in respect to both substrates
indicates that the UDP-GlcNAc-binding site is different from the active
site. A similar situation was found previously for the N. crassa enzyme (43). However, it should be noted that in the case
of other eukaryotic GlcN-6-P synthases, strikingly different modes of
inhibition were reported, especially in respect to Fru-6-P. It was
competitive for rat liver and plant enzymes (10, 45) or uncompetitive
in A. nidulans protein (12). It is not clear if these
results reflect the real differences in enzyme structure and properties
or were a consequence of working with complex protein mixtures instead
of the pure enzyme.
We found that the sensitivity of C. albicans GlcN-6-P
synthase to UDP-GlcNAc was dependent on the presence of another
specific effector, Glc-6-P. The previous reports suggesting that this
compound could affect the properties of eukaryotic GlcN-6-P synthases
were rather confusing (46). Having the pure fungal enzyme we were able
to demonstrate unequivocally that Glc-6-P actually modulates the
sensitivity of C. albicans GlcN-6-P synthase in a
concentration-dependent manner. In the absence of Glc-6-P
an enzyme affinity to UDP-GlcNAc is low, and the binding of this
inhibitor is regulated by the strong negative cooperativity. This
effect allows only partial, less than 50%, inhibition even at very
high concentrations of UDP-GlcNAc. The presence of Glc-6-P enhances the
enzyme affinity to the inhibitor and prevents the allosteric interactions.
Frisa and Sonnenborn (47) and Etchebehere et al. (48)
provided evidence for the involvement of cAMP-dependent
protein kinase and protein phosphatases 2A and 2C in reversible
sensitization and desensitization of Blastocladiella
emersonii GlcN-6-P synthase to inhibition caused by UDP-GlcNAc
(47, 48). Our results show that phosphorylation and dephosphorylation
are involved in the regulation of the C. albicans enzyme
activity but not sensitivity to the feedback inhibitor. The latter
seems to be entirely dependent on the intracellular Glc-6-P level.
However, it is known that the biosynthesis of cAMP, being a part of one
of the signal transduction systems, is often conjugated with changes in
the intracellular concentration of Glc-6-P (49). Several authors showed
that different signal transduction systems involving
cAMP-dependent and Ca2+-dependent
protein kinases operate during Y
M transformation (50, 51). A
diffusable cAMP analog, dibutyryl cAMP, effectively triggers germ tube
formation (52), whereas specific inhibitors of
cAMP-dependent kinase inhibit germination under in
vivo conditions (53). On the other hand, although several
different environmental factors were reported to promote the
morphological shift, it is believed that the low glucose level in the
growth medium, neutral pH resulting in a slow glucose uptake and/or
serum proteins, protein amino acids or
N-acetyl-D-glucosamine as the main carbon
source, alternative to glucose, are the most important (54, 55). In this respect it is noteworthy that the cAMP cascade in C. albicans can be activated by glucagon (56), a human hormone
produced by pancreas in response to low glucose level in the blood.
It was previously demonstrated that the GFA1 mRNA level
substantially increased at the beginning of yeast-to-mycelia shift but
then fell down, despite further progress in morphological transformation (9). On the other hand, enhancement of GlcN-6-P synthase
activity is correlated with extent of germination of C. albicans cells (Ref. 15 and this work), and immediate
desensitization of the enzyme to UDP-GlcNAc is observed at the onset of
Y
M transition (this work). These observations rather exclude a
possibility of transcriptional regulation of GlcN-6-P synthase
production in response to factors stimulating germination. Therefore,
it seems possible to put forward a working hypothesis on the mechanism of post-transcriptional regulation of GlcN-6-P synthase activity in
C. albicans. In Y cells containing less than 1% of chitin
in their cell walls (16), grown in a rich medium, this enzyme remains in the semi-inhibited state, since the the intracellular concentration of its feedback inhibitor, UDP-GlcNAc, reported to be about 1 mM (57), exceeds the IC50 value of the enzyme.
Induction of germ tube formation lowers the intracellular level of
Glc-6-P and thus desensitizes the GlcN-6-P synthase to UDP-GlcNAc and, on the other hand, triggers cAMP formation. cAMP-activated protein kinase phosphorylates GlcN-6-P synthase thus enhancing its activity. The modified molecules of GlcN-6-P synthase can satisfy a requirement for the amino sugar, substantially enhanced in mycelial cells, containing 4-5% chitin in their walls (15). We are fully aware of the
fact that the actual situation is probably much more complex, and our
model may be valid only under specific conditions of germination used
by us.
Further studies, especially aimed at identification of endogenous
cAMP-dependent kinase involved in phosphorylation of
GlcN-6-P synthase and its phosphorylation site, are necessary to
confirm the above hypothesis and understand the complexity of
regulation of this enzyme in C. albicans. A recent
discovery, purification and characterization of the sole
cAMP-dependent protein kinase from C. albicans
(53), should obviously facilitate this study.