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INTRODUCTION |
UDP-N-acetylglucosamine (UDP-GlcNAc), the
nucleotide-activated form of N-acetylglucosamine, plays a
very important role in the biochemistry of all living organisms. In
bacteria, it is required for the biosynthesis of essential
cell-envelope components, namely peptidoglycan (1), lipopolysaccharides
(2, 3), and teichoic acids (4), and for the formation of the
enterobacterial common antigen (5).
Conditional-lethal mutants of Escherichia coli altered in
the biosynthesis of this essential precursor are characterized by a
cell-lysis phenotype (6-10). The four-step formation of UDP-GlcNAc from fructose-6-P has been now completely elucidated in this bacterial species (6, 7, 9, 11-13). It involves the successive actions of
GlcN-6-P synthase, phosphoglucosamine mutase, GlcN-1-P
acetyltransferase, and GlcNAc-1-P uridyltransferase
(GlmU1; also named UDP-GlcNAc
pyrophosphorylase). We showed earlier that the two latter activities
were carried by a single 456-amino acid protein, the product of a gene
we named glmU located just upstream from the GlcN-6-P
synthase glmS gene at 84 min on the E. coli
chromosome (7, 14). The glmU gene has been identified in
some other bacterial species, in particular Neisseria
gonorrhoeae (15) and Bacillus subtilis (16).
The bifunctional E. coli GlmU enzyme has been purified to
homogeneity, and its kinetic parameters were determined (7, 13, 17). A
complete loss of acetyltransferase activity was observed following
incubation of the enzyme in the absence of reducing agent or treatment
with thiol-specific reagents. Site-directed mutagenesis experiments
further demonstrated the important role of two of the four cysteines of
GlmU, namely residues Cys307 and Cys324, for
acetyltransferase activity (17). The GlmU protein has been shown to
exhibit a number of characteristics, which suggested that the
acetyltransferase and uridyltransferase activities may reside in
separate catalytic domains: (i) the substrates, products, and effectors
of the acetyltransferase reaction did not inhibit the uridyltransferase
activity and vice versa (13, 18); (ii) the
intermediate GlcNAc-1-P was clearly released from the acetyltransferase domain prior to transformation by the uridyltransferase domain (18);
(iii) portions of the GlmU amino acid sequence showing similarities
with that of other previously characterized XDP-sugar pyrophosphorylase
and acetyltransferase activities were located in the
N-terminal portion and the second third of the protein, respectively (13, 15, 19); (iv) the acetyltransferase but not the
uridyltransferase was shown to be inactivated by thiol-specific reagents, and the two cysteine residues whose alteration resulted in
dramatic decreases of acetyltransferase activity were identified in the
second moiety of the protein sequence (17); (v) mutagenesis of residues
that are important for uridyltransferase activity did not affect
acetyltransferase at all (Ref. 20 and data not shown); (vi) the fusion
of a His6 tag at the C terminus of the protein resulted in
a 20-fold decrease of acetyltransferase activity, without change in its
uridyltransferase activity (17).
The crystal structure of a truncated form of the GlmU enzyme,
GlmU-Tr331, was recently resolved at 2.25- and 2.3-Å resolution in the
absence or presence of UDP-GlcNAc, respectively (20). The crystal
structure is composed of two distinct domains connected by a long
-helical arm: a N-terminal domain resembling the
dinucleotide-binding Rossmann fold and a C-terminal domain adopting a
left-handed parallel
-helix structure (L
H) that is also found in
homologous bacterial acyl- and acetyltransferases (21-24).
We here report the construction of truncated forms of this enzyme,
which confirms that the bifunctional enzyme is composed of two
autonomously folding and functional domains of roughly equivalent
sizes, the N-terminal one exhibiting uridyltransferase activity and the
C-terminal one acetyltransferase activity. It is also shown that trimer
organization is essential for expression of the acetyltransferase
activity and that the catalytic site of the latter should be formed by
complementary regions from adjacent monomers.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Growth Conditions--
E. coli
strains JM83 (ara
[lac-proAB]
rpsL thi
80 dlacZ
M15) (25) and DH5
(supE44
lacU169 hsdR17 recA1 endA1 gyrA96 thi-1
relA1
80 dlacZ
M15) (Life Technologies, Inc.)
were used as hosts for plasmids and for the overproduction of wild-type and mutant GlmU enzymes. Strain UGS83 (JM83
glmU::kan [pGMU]), which carries an
inactivated copy of the glmU gene on the chromosome and a
wild-type copy of glmU on a plasmid whose replication is thermosensitive, was used in complementation experiments (7). The
plasmid vector pTrcHis30 for expression of proteins under a
N-terminal histidine-tagged form has been described previously (17).
2YT (26) was used as culture medium, and growth was monitored at 600 nm
with a Shimadzu UV-1601 spectrophotometer. For strains carrying drug
resistance genes, antibiotics were used at the concentrations of 100 (ampicillin), 35 (kanamycin), and 25 (chloramphenicol)
µg·ml
1.
Construction of Expression Plasmids--
Standard procedures for
molecular cloning (27) and E. coli cell transformation (28)
were used. The pFP1 and pFP3 plasmids, allowing expression of the
wild-type and N-terminal His6-tagged forms of GlmU,
respectively (under control of the strong trc promoter), have been described previously (17). DNA fragments encoding C-terminally truncated enzymes (i.e. GlmU-Tr227, -Tr250, and
-Tr331) were generated by PCR using the following oligonucleotides as primers: 5'-GGACGGGATCCTTGAATAATGCTATGAGCGTAGTGA-3'
as a sense primer, 5'-GCAGCTGCAGTCACACGCCTTCTACTTCGCT-3' for Tr227, 5'-GCATCTGCAGTCATAACAGCAGTTTTTCAGCCTG-3' for Tr250, and 5'-CTCAGCTGCAGGACGCAATCAGGCAAACGG-3' for Tr331 as
antisense primers, respectively. The resulting PCR products were
purified, digested with BamHI and PstI (in bold),
and inserted into the pTrcHis30 vector. DNA fragments
encoding N-terminally truncated enzymes (i.e. GlmU-del78,
-del130, -del227, -del233, and -del250) were similarly generated by PCR
using the following primers:
5'-AGCCCTGCAGAATCACTTTTTCTTTACCGG-3' as an antisense primer,
and 5'-GTGCTTGGATCCGAGCAGCTGGGTACGGGT-3' for del78,
5'-ATTGGTGGATCCACGGTGAAACTGGATGATCCG-3' for del130,
5'-GTAGAAGGATCCAATAACCGCCTGCAACTC-3' for del227, 5'-CGCCTGGGATCCTCCCGTCTGGAGCGTGTT-3' for del233, and
5'-AAACTGGGATCCGCAGGCGTTATGCTGCGC-3' for del250 as
sense primers, respectively. PCR products were purified, digested with
BamHI and PstI (in bold), and inserted into the pTrcHis30 vector. The pFP1kan plasmid was
constructed by inserting the 1.28-kilobase pair HincII
kanamycin resistance cartridge from pUC4K (Amersham Pharmacia Biotech)
at the ScaI site lying within the ampicillin resistance gene
from pFP1.
Preparation of Crude Extracts and Enzyme
Purification--
E. coli cells carrying plasmids described
in this work were grown at 37 °C in 2YT-ampicillin medium (0.5-liter
cultures). When the optical density (OD) of the culture reached 0.4, IPTG was added at a final concentration of 1 mM and growth
was continued for 3 h. Cells were harvested and washed with 40 ml
of cold 20 mM potassium phosphate buffer, pH 7.4, containing 0.5 mM MgCl2 and 0.1%
-mercaptoethanol. The cell pellet was suspended in 5 ml of the same
buffer supplemented with a mixture of protease inhibitors: 1 mM leupeptin, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 20 µg·ml
1 trypsin inhibitor. Cells were
disrupted by sonication in the cold and the resulting suspension was
centrifuged at 4 °C for 30 min at 200,000 × g. The
supernatant was dialyzed against 100 volumes of the same buffer.
SDS-PAGE analysis of proteins were performed as described previously
(29). The different His6-tagged enzymes were purified as
reported recently (17), basically following the steps in the
manufacturer's (Qiagen) recommendations: binding of
His6-GlmU on Ni2+-nitrilotriacetate-agarose
(Ni2+-NTA) and extensive washing with 20 mM
potassium phosphate buffer, pH 7.4, containing 0.1%
-mercaptoethanol, 0.5 mM MgCl2, 300 mM NaCl, and 20-100 mM imidazole to remove
impurities; elution of His6-GlmU with imidazole (100-300
mM) added to washing buffer; dialysis of
His6-GlmU eluate against 100 volumes of the same phosphate buffer supplemented with 10% glycerol. The His6-tagged
GlmU enzymes prepared in this manner were all at least 90% pure, as
estimated by SDS-PAGE. Protein concentrations were determined by the
method of Bradford, with bovine serum albumin as a standard (30).
Extraction and Quantitation of Peptidoglycan
Precursors--
Cells of JM83(pFP3-Tr331) were grown at 37 °C in
2YT medium (1-liter cultures). At OD = 0.1 (~6 × 107 cells·ml
1), IPTG was added
to one culture at a final concentration of 1 mM. As soon as
the first effects on cell growth were observed in induced cells (~2 h
later, final OD = 0.7), cultures were stopped by rapid chilling to
4 °C, and cells were harvested in the cold. Cultures of JM83 cells
carrying the pTrcHis30 vector were made in parallel as a
control. The extraction of peptidoglycan nucleotide precursors as well
as the analytical procedure used for their quantitation were as
described previously (31). UDP-GlcN was purified from cell extracts by
first using the same two-step chromatographic procedure that is
commonly used to purify the peptidoglycan nucleotide precursors: a gel
filtration on Sephadex G-25 followed by HPLC on a column of µBondapak
C18 (7.8 × 300 mm), where it is eluted in mixture
with UDP-GlcNAc (31). The separation of UDP-GlcN and UDP-GlcNAc was
then achieved by a second step of HPLC on the same column, using this
time an elution with 50 mM triethylammonium formate, pH
4.75, at a flow rate of 3 ml·min
1 (their
retention times were 9 and 21 min, respectively).
Isolation of Sacculi and Quantitation of
Peptidoglycan--
Cells of JM83(pFP3-Tr331) and
JM83(pTrcHis30) were grown and induced with IPTG as
described above (0.5-liter cultures). Harvested cells were washed with
a cold 0.85% NaCl solution and centrifuged again. Bacteria were then
rapidly suspended under vigorous stirring in a hot (95-100 °C)
aqueous 4% sodium dodecyl sulfate (SDS) solution (20 ml) for 30 min.
After standing overnight at room temperature, the suspensions were
centrifuged for 30 min at 200,000 × g in a Beckman
TL100 centrifuge and the pellets were washed several times with water.
Final suspensions were made in 2 ml of water, and aliquots (100 µl)
were hydrolyzed and analyzed with a Biotronik model LC2000 amino acid
analyzer. The peptidoglycan content of the sacculi was expressed in
terms of its muramic acid content (31, 32).
Enzymatic Assays--
Assays for both activities of GlmU were
performed as described previously (17), after appropriate dilutions of
the enzyme in 20 mM potassium phosphate buffer, pH 7.4, containing 1 mg·ml
1 BSA, 0.5 mM
MgCl2, and 0.1%
-mercaptoethanol. One unit of enzyme activity was defined as the amount that catalyzed the formation of 1 µmol of product/min. UDP-GlcN, which is used as an alternative substrate of GlmU in some acetyltransferase assays, was synthesized enzymatically by using UDP-glucose pyrophosphorylase, as described by
Gehring et al. (18). This compound was purified by HPLC as described above, and its authenticity was confirmed by determination of
its hexosamine content after acid hydrolysis.
Estimation of Molecular Weight--
Pure samples of the
different truncated GlmU proteins (50 µl of 1 mg·ml
1 solutions) were applied onto a
column of Superdex 200 HR 10/30 connected to a fast pressure liquid
chromatography apparatus (Amersham Pharmacia Biotech). The column was
equilibrated with 50 mM potassium phosphate buffer, pH 7.4, containing 150 mM NaCl and 0.1%
-mercaptoethanol, and
samples were eluted at 0.5 ml·min
1.
Calibration was carried out with cytochrome c, myoglobin,
-chymotrypsinogen A, ovalbumin, and BSA, and the void and total
volumes were determined with blue dextran 2000 and tyrosine,
respectively. Elution was followed by measurement of the absorbance at
280 nm.
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RESULTS |
Construction of Various Truncated Forms of GlmU and Their
Activity--
As attempts to crystallize the full-length GlmU protein
remained unsuccessful, crystallization of individual domains was
therefore envisaged. This prompted us to more precisely define the size of the two putative autonomous domains. Plasmids for high-level overexpression of GlmU proteins truncated either in the N- (del constructs) or in the C-terminal (Tr constructs) region were
constructed (Fig. 1). All of these
proteins were expressed in a His6-tagged form (N-terminal
Met-His6-Gly-Ser extension), allowing their convenient one-step purification. Most of them were successfully overproduced in a
soluble form and were purified to near homogeneity (Fig. 2). Others (del26, del182, del250, and
Tr227) either appeared very poorly produced, due probably to structural
instability and rapid intracellular degradation, or formed insoluble
inclusion bodies.

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Fig. 1.
Schematic structure of various truncations of
the GlmU protein generated in the present study. Truncated
proteins are represented by cross-hatched regions, and
numbers indicate amino acid residue numbers within the
wild-type protein sequence. All truncated proteins were expressed with
a N-terminal His6 tag extension, as shown for the
full-length protein.
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Fig. 2.
SDS-polyacrylamide gel electrophoresis of
purified truncated forms of the E. coli GlmU
protein. Full-length and truncated GlmU proteins were overproduced
in E. coli cells in the His6-tagged form
(N-terminal Met-His6-Gly-Ser extension). Their one-step
purification on Ni2+-NTA was performed as described in the
text, and aliquots (2 µg) were analyzed by SDS-PAGE. Lane
A, full-length GlmU; lane B, del78;
lane C, del130; lane D,
Tr331; lane E, del227; lane
F, del233; lane G, Tr250. The position
and molecular mass (kilodaltons) of marker proteins are indicated on
the left.
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However, none of these different plasmids could complement the
thermosensitive glmU mutant strain UGS83, indicating that
the engineered truncations had resulted in the loss of at least one of
the two activities of GlmU. This was confirmed by assays of the pure
proteins for acetyl- and uridyltransferase activities (Table
I). The uridyltransferase activity of
proteins truncated in the N-terminal region (del78, del130, del227, and
del233) was decreased by a factor of at least 1000, but surprisingly a
residual and almost invariant activity (kcat = 0.1-0.3 s
1) was retained by all of them. As
discussed below, this residual activity was due to contaminating
wild-type GlmU enzyme (originating from chromosomal gene expression)
that could form heterotrimers with these His6-tagged
truncated proteins. The deletion of the first 130 N-terminal amino acid
residues of GlmU resulted in only a 50% decrease of its
acetyltransferase activity. Truncation of 100 more residues (del233)
was accompanied by a more important decrease (98%) of activity, but
the residual acetyltransferase activity remained relatively high with a
kcat of 25 s
1. Gehring
et al. (18) also previously constructed a truncated form of
GlmU (glutathione S-transferase fusion), deleted in that case of the first 179 residues. Its acetyltransferase activity was
150-fold reduced as compared with full-length GlmU with a kcat of 0.5 s
1. As
shown in Table I, an inverse pattern of activities was observed for
proteins carrying deletions in the C-terminal region; Tr331 and Tr250
proteins lacking the last 125 and last 206 amino acid residues,
respectively, had undetectable acetyltransferase activity but retained
significant uridyltransferase activity (42% and 2.5% of wild-type
enzyme activity, respectively).
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Table I
Enzymatic activities and oligomerization state of truncated GlmU
proteins
Full-length and truncated GlmU proteins were overproduced in E. coli cells in the His6-tagged form (N-terminal
Met-His6-Gly-Ser extension). They were purified on
Ni2+-NTA and assayed for both enzyme activities of GlmU. The
values represent the means of three determinations. Their
oligomerization state was determined by gel filtration as described
under "Experimental Procedures." Yields of these different
truncated proteins were similar, from 2.5 to 10 mg of protein per 0.5 liter of culture.
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Trimer Organization--
As reported previously, chromatography of
full-length GlmU protein on gel filtration was consistent with a
homotrimer arrangement (13). The oligomerization state of the different
truncated GlmU fragments generated here was then investigated (Table
I). The ability of the proteins to trimerize was clearly correlated to the presence of at least the initial part of the C-terminal domain. The
fact that the two del227 and Tr331 proteins were trimers suggested that
residues involved in the oligomerization process might be located
between these two sites of truncation. With the exception of the Tr250
protein, which turned out to be a monomer, all other truncated proteins
generated in the present work consisted of trimers.
As shown above, a very low but detectable uridyltransferase activity of
0.1-0.3 s
1 was consistently detected with
all preparations of proteins carrying either partial or complete
deletions of the N-terminal domain (del78 to del233). It should be
noted that these proteins, which carry only one of the two activities
of GlmU, could not complement the glmU mutant strain UGS83
and have consequently been expressed in a wild-type E. coli
strain. The simplest interpretation was the presence in these
preparations of contaminating full-length (non-His-tagged) GlmU enzyme
originating from chromosomal expression. That the wild-type enzyme
could not be eliminated by extensive washings of the
His6-tagged truncated proteins adsorbed on
Ni2+-NTA with buffers containing 300 mM NaCl
and 20-100 mM imidazole suggested a very tight association
(formation of heterotrimers) between the full-length and the truncated
proteins. It was effectively controlled that the pure wild-type
(non-His-tagged) GlmU protein had by itself no particular affinity for
the Ni2+-NTA matrix, i.e. was detected
essentially in the pass-through fraction and had been completely
eliminated from the column by washing with 20 mM imidazole
containing buffers, as confirmed by SDS-PAGE analysis and enzymatic
assays (data not shown).
Toxic Effects of the Overproduction of GlmU-Tr331 on E. coli Cell
Growth--
JM83 cells carrying the plasmid pFP3-Tr331 were grown in
rich medium and expression of the truncated gene was induced with 1 mM IPTG. It resulted in a significant overproduction of the truncated protein, as observed by SDS-PAGE. Interestingly, after 2 h of induction, a rapid slow down of cell growth occurred which was
followed by cell lysis about 1 h later (Fig.
3). Observation of cells by optical
microscopy showed morphological changes characteristic of an arrest of
peptidoglycan synthesis: progressive change of cell shape from rods to
greatly enlarged ovoids and cell lysis, as indicated by the presence of
many ghosts in the culture. It was reminiscent of the effects
previously observed in a thermosensitive glmU mutant strain
when grown under restrictive conditions. Cell lysis was clearly due to
an arrest of peptidoglycan synthesis as the peptidoglycan content of
induced JM83(pFP3-Tr331) cells appeared about 40% lower than that of
noninduced cells (at the time where the first effects on cell growth
were observed) (Table II). An analysis of
the pool levels of peptidoglycan precursors revealed the significant
reduction of the pools of UDP-GlcNAc and UDP-MurNAc-pentapeptide in
induced cells (Table II), consistent with the inhibition of an early
step leading to the formation of UDP-GlcNAc (6, 7). Interestingly, the
significant accumulation of a compound which was subsequently
identified as UDP-glucosamine was observed in these cells (see below).
The morphology, peptidoglycan content, and levels of precursors in
noninduced JM83(pFP3-Tr331) cells were similar to those of control
JM83(pTrcHis30) cells (Table II).

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Fig. 3.
Effects of the overproduction of the
truncated GlmU-Tr331 protein on E. coli cell
growth. Cells of JM83(pFP3-Tr331) were grown at 37 °C in
2YT-ampicillin medium. At the time indicated by an arrow
(optical density = 0.1), the overproduction of the Tr331 protein
was induced with IPTG (1 mM) and growth of induced
(open symbols) and not induced (filled
symbols) cells was monitored at 600 nm. Growth of cells
carrying as a control the plasmid vector pTrcHis30 was
unaffected by IPTG and paralleled that of noninduced JM83(pFP3-Tr331)
cells (data not shown).
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Table II
Effects of the overproduction of the truncated GlmU-Tr331 protein on
peptidoglycan synthesis and activities of the GlmU enzyme in
wild-type E. coli cells
Cells were grown in 2YT-ampicillin medium at 37 °C. At OD = 0.1, IPTG (1 mM) was eventually added and incubation was
continued until the first effects on cell growth were observed, about
2 h later (Fig. 3). At this time, peptidoglycan and its precursors
were extracted and quantitated as described in the text. Crude protein
extracts were also prepared and were assayed for GlcN-1-P
acetyltransferase and GlcNAc-1-P uridyltransferase activities. The
values are means of determinations obtained in two independent
experiments.
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The levels of the acetyltransferase and uridyltransferase activities of
GlmU were determined in crude extracts from cells of JM83(pFP3-Tr331)
grown in the absence or presence of IPTG. In the absence of IPTG,
JM83(pFP3-Tr331) cells contained about 8-fold more uridyltransferase
activity but 30-fold less acetyltransferase activity than control
JM83(pTrcHis30) cells. The overproduction of
uridyltransferase was due to basal expression from the plasmid promoter
of the truncated form of GlmU (which has a normal uridyltransferase activity). The concomitant dramatic decrease of acetyltransferase activity clearly indicated that the activity of newly synthesized wild-type GlmU molecules originating from the chromosomal gene could
only be partially detected, due probably to the formation of inactive
heterotrimers. When IPTG was added, this effect was exacerbated as a
200-fold increase of uridyltransferase activity but a null
acetyltransferase activity were measured in induced JM83(pFP3-Tr331)
cells. The loss of acetyltransferase activity and the arrest of
UDP-GlcNAc and consequently peptidoglycan biosynthesis were clearly
correlated features. It should be noted that the effects on cell
morphology and peptidoglycan metabolism were observed only in the
presence of IPTG. The fact that the 30-fold decrease of
acetyltransferase activity detected in noninduced cells had no apparent
effect on cell growth was consistent with the previous demonstration
that this activity of GlmU was in great excess in E. coli
cells as compared with its specific requirements (13).
Formation of a mixture of four different heterotrimers
Wt(3), Wt(2)-Tr(1),
Wt(1)-Tr(2), and Tr(3) is expected
to occur in vivo, whose proportions theoretically reflect
relative abundance of wild-type (Wt) and truncated (Tr) monomers. In
conditions where the truncated monomer is largely predominant (at least
a 200: 1 ratio could be estimated in IPTG-induced cells from the
increase of uridyltransferase activity), Wt(3) and
Wt(2)-Tr(1) species are most probably very
rarely generated, and wild-type monomers should be exclusively in the
Wt(1)-Tr(2) form (model shown in Fig.
4). The fact that the acetyltransferase
activity of the wild-type enzyme was completely undetectable in these
conditions therefore suggested that more than one full-length monomer
per trimer was required for exhibition of the latter activity. However,
the question of the acetyltransferase activity of the two
Wt(2)-Tr(1) and
Wt(1)-Tr(2) species remained. To generate
significant amounts of these two heterotrimers, JM83 cells were
transformed by two plasmids, pFP1kan and pFP3-Tr331, for
concomitant expression of the wild-type (Wt) and the
His6-tagged truncated (Tr) proteins, respectively. Analysis of crude extracts prepared from these cells confirmed the large accumulation of the two proteins (data not shown). Purification on
Ni2+-NTA was then performed as described above, using
extensive washing steps for complete elimination of the non-His-tagged
Wt(3) form. The resulting purified material appeared
composed of both Wt and Tr proteins in roughly equivalent amounts, as
judged by SDS-PAGE (data not shown). Unfortunately, attempts to
separate the different His-tagged heterotrimers present in this mixture
by gel filtration techniques failed, due to their very close molecular
masses, 111, 123, and 135 kDa for Tr(3),
Wt(1)-Tr(2), and
Wt(2)-Tr(1), respectively. However, this
mixture, which theoretically contains the two
Wt(1)-Tr(2) and
Wt(2)-Tr(1) heterotrimers, exhibited a high
uridyltransferase activity (120 s
1) but had
no detectable acetyltransferase activity, as observed for the pure
Tr(3) homotrimer. This finding was thus consistent with the
hypothesis (discussed below) that three full-length monomers should be
present in the trimer for expression of the latter activity.

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Fig. 4.
Ribbon diagram of the GlmU-Tr331 trimer
viewed perpendicular to the L H axis. The
additional coils have been modeled (dark gray)
only in one subunit and contain residues Pro328 to
Gly424.
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Overexpression of other truncated proteins described in the present
work had no effects on cell growth. It should be noted that most of
them exhibited significant acetyltransferase activity. In fact, the
only other engineered protein with undetectable acetyltransferase activity, Tr250, appeared unable to oligomerize and consequently could
not trap the full-length GlmU enzyme into inactive heterotrimers.
Interestingly, IPTG-induced JM83(pFP3-Tr331) cells were shown to
accumulate large amounts of a compound, which was identified as
UDP-GlcN (Table II). This unexpected finding was clearly correlated with the depletion of the acetyltransferase activity of GlmU in these
cells. It suggested that in the absence of the latter enzyme activity
GlcN-1-P molecules had been transformed into UDP-GlcN by the still
present (and highly overproduced) uridyltransferase activity of GlmU.
GlmU-catalyzed uridylyltransfer to glucosamine-1-P was previously
reported to be undetectable (<0.0001 s
1)
(18). In our hands, however, the pure GlmU enzyme could catalyze the
synthesis of UDP-GlcN from GlcN-1-P and UTP with a very good efficiency
(kcat = 23 s
1, as
compared with 330 s
1 when GlcNAc-1-P is used
as the substrate), a finding consistent with the in vivo
accumulation of this compound.
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DISCUSSION |
Data obtained in the present study confirm that the bifunctional
GlmU protein is organized in two autonomously folding and functional
domains. As judged by the specific activities exhibited by the various
truncated forms of GlmU described here, the size of the two individual
domains might be roughly equivalent, each one representing about half
of the protein. In the light of the recently established crystal
structure of a truncated form of GlmU (Tr331), the connection between
the two domains is achieved by a long
-helical arm located between
residues Asn228 and Ala250 (20). It suggests
that this bacterial protein evolved by fusion of two uridyltransferase
and acetyltransferase fragments. In eukaryotes, the biosynthesis of
UDP-GlcNAc occurs by a slightly different route (via GlcNAc-6-P) in
which GlcN-6-P acetyltransferase and GlcNAc-1-P uridyltransferase
activities are carried by two distinct monofunctional enzymes (33-37).
The selective advantage (if any) conferred to bacteria by this unique
bifunctional enzyme remains an enigma. In particular, there is no
apparent requirement for a common regulation of the component
activities at the level of transcription or translation. It was
previously shown that both activities were in great excess in cells as
compared with specific requirements in UDP-GlcNAc molecules of the
peptidoglycan and lipopolysaccharide pathways (7, 13). As demonstrated
by Plumbridge et al. (38), the glmU gene is
cotranscribed with the downstream GlcN-6-P synthase glmS
gene in E. coli and seems to be expressed at a high
constitutive level whatever the growth conditions used. The
construction of various mutated forms of GlmU enzyme affected in either
of the two activities also showed that the ratio of acetyltransferase
and uridyltransferase activities (which is about 5 for wild-type
enzyme) could be greatly modified in vivo without detectable
effect on the functioning of this pathway. Additionally, there is no
apparent advantage for GlmU to be a bifunctional protein in terms of
reaction mechanism. By using radiolabeled substrates, Gehring et
al. (18) demonstrated that GlcNAc-1-P was released by the enzyme
before being used as substrate by the second enzyme activity. In
addition, the thermosensitive glmU mutant strain UGS83 was
shown to accumulate large amounts of GlcNAc-1-P when grown at the
restrictive temperature (7). These results were a priori not
consistent with the hypothesis of a concerted action of the two enzyme activities.
Gehring et al. (18) reported previously that the GlmU enzyme
was unable to catalyze uridylyltransfer from UTP to GlcN-1-P but could
catalyze acetyltransfer from acetyl-CoA to UDP-GlcN, although at a
12-fold reduced rate. It was one of the arguments why these authors
conclude that acetyltransfer precedes uridylyltransfer in the two-step
formation of UDP-GlcNAc by GlmU. We effectively confirmed here that
GlmU could catalyze acetyltransfer to UDP-GlcN and the
kcat value we determined was 170 s
1 (9-fold lower as compared with 1500 s
1 for GlcN-1-P). However, we here observed
that GlmU could also efficiently catalyze uridylyltransfer to GlcN-1-P,
at a 15-fold reduced rate (kcat = 23 s
1) as compared with that with GlcNAc-1-P
(350 s
1). In our hands, the GlmU enzyme
therefore appears theoretically capable to catalyze a two-step
synthesis of UDP-GlcNAc in which uridylyltransfer precedes
acetyltransfer but the greatly reduced kinetic parameters confirmed the
previous assumption that these two reactions occur in the inverse order
under normal physiological conditions (13, 18). As shown in the present
report, a significant accumulation of UDP-GlcN was observed in cells in
which the acetyltransferase activity of GlmU has been inhibited. This
finding confirmed that GlmU could effectively catalyze the
uridylyltransfer from UTP to GlcN-1-P in vivo, but it is
clear that this only occurred because of very particular physiological
conditions in which the availability of the preferred substrate
GlcNAc-1-P was impaired.
GlmU and its truncated derivative Tr331 are trimeric proteins, as
judged by their behavior on gel filtration. The recently obtained
crystal structure of GlmU-Tr331 also showed a trimeric arrangement
around the long dimension of the L
H prism (20). The trimeric
association of the particular L
H domain is highly conserved between
GlmU-Tr331 and other previously characterized bacterial acetyl- or
acyltransferases, namely LpxA, PaXAT, DapD, and Cam (20-24). However,
it is not known whether a trimeric organization of these proteins is
absolutely required for expression of their acetyl- or acyltransferase
activities. This also raised the question of the activity of the
various heterotrimers that could be generated from a mixture of
wild-type and truncated monomers. To answer this question, we followed
the growth of a wild-type E. coli strain during the
overexpression of GlmU-Tr331, a truncated protein with null
acetyltransferase activity but still able to associate in trimers. Its
overproduction by a factor of about 200-fold as compared with the
wild-type protein level resulted in the complete disappearance of
acetyltransferase activity in cells, which was followed by deleterious
effects on peptidoglycan biosynthesis and cell growth. This finding
strongly suggested that the wild-type enzyme could not exhibit
acetyltransferase activity under a monomer form in vivo. It
also indicated that the activity of this wild-type protein originating
from chromosomal expression could no more be detected once trapped
within heterotrimers formed in presence of the predominantly expressed
truncated protein (model shown in Fig. 4). An organization in trimer is
therefore required for exhibition of the acetyltransferase activity but
the data here obtained could be interpreted in several ways. (i) Each
monomer carries a complete catalytic site, the active conformation of
which is formed only during trimer assembly; (ii) each catalytic site
is made of specific complementary regions belonging to more than one
monomer, suggesting that one to three catalytic site(s) could exist per
trimer unit. To date, only the crystal structure of the truncated form
of GlmU has been determined (20) and the exact position and number of
binding-sites of the substrate acetyl-CoA are not known. It should be
noted that, in the homologous structures of acetyltransferases DapD and
PaXAT (21, 22), three binding sites for the substrate acetyl-CoA were
detected, each one being located between two subunits on the exterior
face of the trimeric L
H domains. This positioning could also be
adopted for GlmU. The finding that a mixture of Tr(3),
Wt(1)-Tr(2), and
Wt(2)-Tr(1) heterotrimers has undetectable acetyltransferase activity further suggests that the catalytic site(s)
of the active trimer Wt(3) should be formed by adjacent and
complementary regions from three full-length monomers. The confirmation
of this hypothesis now requires the elucidation of the
three-dimensional structure of the entire GlmU protein. Trimerization is clearly not essential for expression of the uridyltransferase activity of GlmU. The latter activity was retained by the Tr250 protein, which is unable to associate in trimers. However, the 40-fold
reduced uridyltransferase activity of this protein suggests that
trimerization or at least interactions between regions of the two
domains may participate in the folding and stability of the N-terminal
domain. In the crystal structure, the only observed contacts between
the two domains were van der Waals interactions between the surface
loop Ala31-Gly32 in the N-terminal domain and
the Arg263 side chain in the C-terminal domain (20).
Residues within the long
-helical arm
(Asn228-Ala250) also established numerous
interactions with residues in the two domains (20). The abolishment of
at least the van der Waals interactions in the Tr250 protein could
partially account for the decreased activity of this protein. However,
the positioning of the long
-helical arm seems to play an important
role for the uridyltransferase activity with the helix dipole aligned
with the position of the key positively charged Arg18
residue. Whether an incorrect positioning of the
-helical
linker in the Tr250 protein could explain for its decreased activity remains to be elucidated.
As mentioned above, the pathway for UDP-GlcNAc biosynthesis appears
significantly different in eukaryotes. In the latter, acetyltransfer
occurs on GlcN-6-P and not on GlcN-1-P, and, most importantly,
acetyltransferase and uridyltransferase activities are carried by two
distinct monofunctional enzymes that show little sequence homology with
GlmU (35-37). The GlmU enzyme, which is essential and specific of the
bacterial world, should therefore be considered as an interesting
target for the search of new antibiotics. Biochemical and
crystallographic investigations are now developed to gain more
information on active sites of this bifunctional enzyme. The present
demonstration that a trimer organization is absolutely required for
acetyltransferase activity of GlmU could also open the way for a search
of inhibitors based on the inhibition of the oligomerization process.