From the AFMB-UMR6098, 31 Chemin Joseph Aiguier,
13402 Marseille Cedex 20, France and
Aventis Pharma-Hoechst
Marion Roussel, Infectious Diseases Group, 102 Route de Noisy, 93235 Romainville Cedex, France
Received for publication, December 13, 2000
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ABSTRACT |
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The bifunctional bacterial enzyme
N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU)
catalyzes the two-step formation of UDP-GlcNAc, a fundamental
precursor in bacterial cell wall biosynthesis. With the emergence of
new resistance mechanisms against GlmU catalyzes acetyltransfer from acetyl-coenzyme A
(AcCoA)1 to glucosamine-1-P
with release of GlcNAc-1-P, and subsequently uridyltransfer from UTP to
GlcNAc-1-P in the presence of Mg2+, yielding PPi and the
nucleotide-activated precursor sugar UDP-GlcNAc (1) (see Fig.
1A). UDP-GlcNAc is one of the main cytoplasmic precursors of
the bacterial cell wall, being situated at the branch point of two
important biosynthetic pathways, namely peptidoglycan and lipid A
biosynthesis (2). In eukaryotes, a bifunctional enzyme equivalent to
GlmU is missing, and acetyltransfer and uridyltransfer are accomplished
by two distinct enzymes, both very distantly related in sequence to
GlmU, the latter thus advancing to an attractive target for the
development of new antibiotics.
The crystal structures of a truncated form of Escherichia
coli GlmU (GlmU-Tr) and of a GlmU-Tr·UDP-GlcNAc complex
have been recently reported (3). These structures confirmed that the enzyme is organized in the following two separate domains as proposed previously (4, 5): (i) an N-terminal uridyltransferase (PPase) domain,
comprising Asn-3 to Arg-227, resembling the dinucleotide binding
Rossmann fold, first reported in the lactate dehydrogenase family (6),
and containing the signature motif
G-X-G-T-(R/S)-(X)4-P-K, found
in the majority of pyrophosphorylases, and (ii) a C-terminal acetyltransferase domain, containing the hexapeptide repeat
(L/I/V)-(G/A/E/D)-X2-(S/T/A/V)-X, a
signature of the unusual left-handed Here we present the crystal structures of full-length GlmU from the
pathogenic organism Streptococcus pneumoniae in its unbound form and in complex with AcCoA and both AcCoA and the product UDP-GlcNAc. These structures define the precise location of the acetyltransferase active site, reveal substantial conformational changes occurring both upon AcCoA and UPD-GlcNAc binding, and highlight
the structural elements responsible for substrate recognition and
catalysis in the two distinct active sites of this bifunctional enzyme.
Expression, Purification, and Crystallization--
The coding
region of SpGlmU was amplified from S. pneumoniae strain R
800 DNA by polymerase chain reaction and inserted into the
bacterial expression plasmid PQE30 (Qiagen). Recombinant SpGlmU was
overexpressed in M15 cells and purified to homogeneity by nickel-nitrilotriacetic acid-agarose and gel filtration chromatography. Enzyme activity has been tested and found similar to that of
full-length E. coli GlmU (3). Crystals were grown at
20 °C by the hanging-drop vapor diffusion method by mixing equal
volumes of protein solution (13 mg/ml) with reservoir solution composed
of 26% (v/v) PEG 400, 50 mM NaCl, and 300 mM
CaCl2 at pH 8.0 by TRIS-HCl. Small rhombohedral crystals
with a typical size of 0.1 × 0.1 × 0.1 mm appeared within 1 week. Crystals belong to space group R3 and contain two molecules per
asymmetric unit. As molecular replacement with GlmU-Tr (Protein Data
Bank entry 1FXJ) failed, selenomethionine-substituted enzyme was
produced using the same bacterial strain grown in minimum medium and
supplemented, before induction, with selenomethionine and amino acids
known to inhibit methionine biosynthesis (8). The yield of
selenomethionine substitution was about 50% as judged by
matrix-assisted laser desorption ionization/time of flight mass
spectroscopy analysis. Crystals of bigger dimensions and higher
diffraction quality were obtained for the selenomethionine-substituted enzyme under the same crystallization conditions as adopted for the
native protein. Crystals for the AcCoA complex were obtained by
incubating the enzyme with 20 mM AcCoA prior to
crystallization and lowering the PEG 400 concentration to 18% (v/v).
AcCoA·UDP-GlcNAc complex crystals were obtained by cocrystallization
with 20 mM AcCoA followed by harvesting into a stabilizing
solution made of 30% (v/v) PEG 400, 50 mM NaCl, 300 mM CaCl2 at pH 8.0 by TRIS-HCl and supplemented
with 10 mM UDP-GlcNAc.
Data Collection, Structure Solution, and Refinement--
All
data sets were collected at 100 K on flash-frozen crystals.
Cryosolutions were of the same composition as the
crystallization/harvesting solutions with the addition of an increasing
amount of PEG 400 and supplemented with 5% (v/v) glycerol. A
3-wavelength multiple anomalous dispersion data set for
selenomethionine-substituted SpGlmU was collected on beamline BM14
(European Synchrotron Radiation Facility, Grenoble, France), a
data set for native SpGlmU and data for the AcCoA complex were
collected on beamlines ID14-EH2, and data for the AcCoA·UDP-GlcNAc
complex were collected on beamline ID14-EH3 (European Synchrotron
Radiation Facility, Grenoble, France). Data were indexed and integrated
with DENZO (9), and all further computing was carried out with
the CCP4 program suite (10) unless otherwise stated. Data collection
statistics are summarized in Table I and Table II.
The SpGlmU structure was solved using
the program SOLVE (11). The initial multiple anomalous dispersion
phases had a mean figure of merit of 0.340- to 2.8-Å resolution and
were improved by density modification with the program DM (12) and
extended to the resolution of the native data set (2.3 Å). Because of
the low yield of selenomethionine incorporation only a few of these residues could be located in the experimental electron density maps,
which were of mediocre quality. Non-crystallography symmetry averaging and phase combination techniques were of great help in
overcoming these problems, and a preliminary model could be constructed
for most of the L The crystal structure of full-length SpGlmU was determined by
multiple anomalous dispersion techniques. The apo-SpGlmU, SpGlmU-AcCoA, and SpGlmU-AcCoA·UDP-GlcNAc structures were refined to 2.3, 2.5, and
1.75 Å, respectively, and have good stereochemistry. The apo-SpGlmU structure consists of residues Ser-2 to Val-142 and Val-149 to Glu-447.
The surface loop Arg-143-lactam and glycopeptide
antibiotics, the biosynthetic pathway of UDP-GlcNAc represents an
attractive target for drug design of new antibacterial agents. The
crystal structures of Streptococcus pneumoniae GlmU
in unbound form, in complex with acetyl-coenzyme A (AcCoA) and in
complex with both AcCoA and the end product UDP-GlcNAc, have been
determined and refined to 2.3, 2.5, and 1.75 Å, respectively. The
S. pneumoniae GlmU molecule is organized in two
separate domains connected via a long
-helical linker
and associates as a trimer, with the 50-Å-long left-handed
-helix
(L
H) C-terminal domains packed against each other in a parallel
fashion and the C-terminal region extended far away from the L
H core
and exchanged with the
-helix from a neighboring subunit in the
trimer. AcCoA binding induces the formation of a long and narrow
tunnel, enclosed between two adjacent L
H domains and the
interchanged C-terminal region of the third subunit, giving rise to an
original active site architecture at the junction of three subunits.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helix (L
H) structural motif, typically found in other bacterial acetyl- and acyltransferases (7) (Fig. 1B). Furthermore, the GlmU-Tr·UDP-GlcNAc complex structure identified the precise location of the uridyltransfer reaction, the pyrophosphorylase activity of GlmU-Tr being retained. However, acetyltransferase activity was lost because of spontaneous truncation during purification, confirming that the bifunctional enzyme
possesses indeed two distinct active sites located in separate domains,
with the acetyltransferase activity residing in the C-terminal portion
of the enzyme (4). Although the crystal structure of the E. coli enzyme, coupled to mutagenesis studies, has revealed some
residues crucial for pyrophosphorylase activity (3), the catalytic
machineries responsible for both pyrophosphorylase and acetyltransferase activity remain to be elucidated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
H domain and the core of the N-terminal PPase
domain using the program TURBO-FRODO (13). However, most of the loop
regions, some of the
-helixes in the N-terminal domain, and the last
25 C-terminal residues turned out to be extremely disordered, if
visible at all. A striking improvement of the map quality was observed
for the AcCoA complex. A continuous model could be built comprising
residues Ser-2
Gln-459. A crystal lattice rearrangement occurred upon
soaking of AcCoA complex crystals in the solution containing
UDP-GlcNAc, and the structure was solved by molecular replacement with
the program AMoRe (14). Refinement was carried out with the programs
REFMAC (15) and CNS (16), using the maximum likelihood method and
incorporating bulk solvent corrections, anisotropic
Fobs versus
Fcalc scaling, and non-crystallography symmetry
restraints. 10% of the reflections were set aside during refinement
for cross-validation purposes. Automated correction of the model
and solvent building were performed with the program ARP/wARP
(17). The stereochemistry of the final models was verified with the
program PROCHECK (18). Refinement statistics are summarized in Table
II. Coordinates have been deposited in the Protein Data Bank under
accession reference numbers 1HM0 for apo-SpGlmU and 1HM8 and 1HM9 for
the AcCoA and the AcCoA·UDP-GlcNAc complex, respectively. Fig.
1B was generated with Alscript
(19), and Figs. 2-4 were generated with SPOCK (20) and Raster3D
(21).
MAD data collection and statistics
Data collection and refinement statistics
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Fig. 1.
Catalytic reactions and sequence
alignment of SpGlmU. A, schematic representation of the
two-step reaction catalyzed by GlmU: acetyltransferase (I)
and pyrophosphorylase (II). B, the SpGlmU
sequence is aligned with a consensus sequence calculated on the basis
of 12 known sequences of bacterial GlmU. Invariant residues are
highlighted in white with a black
background. h, s, p,
c, and . denote hydrophobic, small, polar, charged, and any
residues, respectively. Residues buried at the trimer interface
(black circles above sequence), involved in AcCoA
(light gray triangles pointing upwards),
UDP-GlcNAc/Ca2+ (gray triangles pointing
downwards/gray circles), and putative GlcN-1-P
(gray triangles pointing downwards) binding are shown; those
forming the catalytic triad and involved in the PPase activity are
shown as gray circles above sequence and black circles
below sequence, respectively.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Glu-148, located in the pyrophosphorylase domain, and the last 12 residues of the acetyltransferase domain, Tyr-448
Gln-459, could not be built because of lack of electron density. The two complex structures, SpGlmU·AcCoA· and
SpGlmU·AcCoA·UDP-GlcNAc, consist of residue Ser-2 to Gln-459, and
clear unbiased electron density could be observed for both AcCoA and
UDP-GlcNAc prior to the incorporation in the refinement
(Fig. 2a).
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Fig. 2.
Map quality and overall fold of the
SpGlmU structure. A, stereo pair of the 1.75-Å
resolution 2Fo Fc
averaged electron density map, calculated prior to the incorporation of
AcCoA in the refinement and contoured at 1.0 (blue) and 3.0
(black) around an AcCoA molecule. Phases were calculated
after rigid-body refinement based on the two apo SpGlmU molecules
present in the asymmetric unit. B, left, ribbon
model of a SpGlmU subunit, showing the PPase domain
(orange), the
-helical linker (magenta), the
L
H domain (yellow with the unique insertion loop in
orange), and the C-terminal arm (cyan);
right, the SpGlmU trimer with bound AcCoA and UDP-GlcNAc
(gray bonds with red oxygen, blue
nitrogen, green sulfur, and purple phosphorus
atoms) viewed in the same orientation as in the panel on the
left (top) and down the L
H axis
(bottom); for clarity a single subunit is color-coded as in
the panel on the left, with the remaining two
subunits shown in gray. C, stereo view overlay of
the C
trace of apo-SpGlmU (cyan) and SpGlmU·AcCoA
(orange), with the two respective C termini labeled. The
overlap is based on a least squares fit of 440 C
positions.
The SpGlmU molecule assembles into a trimeric arrangement with overall
dimensions of 89 × 85 × 90 Å (Fig. 2b). The
LH domains (Val-252
Ile-437) are tightly packed against each other
in a parallel fashion, an
-helical linker (Arg-229
Met-248) sits on
top of each
-helix and projects the globular pyrophosphorylase
domain (Ser-2
Asn-227) far away from the trimer interface.
The SpGlmU apo-structure, except for the two missing regions
Arg-143Glu-148 and Tyr-448
Gln-459, is highly similar to the SpGlmU·AcCoA complex structure, with a root mean square deviation of
0.450 Å for 440 C
positions (Fig. 2c). The SpGlmU-AcCoA
complex structure, in turn, is almost identical to the
SpGlmU·AcCoA·UDP-GlcNAc complex structure in the acetyltransferase
domain (root mean square deviation of 0.17 Å for 208 C
positions).
However, the two complex structures differ greatly in the
pyrophosphorylase domain, as discussed further below.
The SpGlmU overall fold for residues Ser-2 to His-330 is similar to the
E. coli-truncated enzyme (3). However, the relative arrangement of the pyrophosphorylase and the acetyltransferase domain
differs between the crystal structures of SpGlmU and E. coli
GlmU-Tr (Fig. 3a). Indeed, the
two GlmU structures present a 20° deviation in the direction of the
-helical linker, indicating that this is, in fact, a flexible hinge.
A direct consequence of this deviation are major differences between
GlmU-Tr and SpGlmU occurring in the regions of the pyrophosphorylase
domain neighboring the N-cap of the
-helical linker. These
conformational changes, together with a high overall mobility of the
pyrophosphorylase domain, as opposed to the acetyltransferase domain,
suggest that the presented structures may represent only snapshots of a
highly dynamic system.
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The Pyrophosphorylase Domain-- The SpGlmU PPase domain can be divided into two lobes separated by the active site pocket. The first hundred residues, containing the consensus sequence motif G-X-G-T-(RS)-(X)4-P-K, form the nucleotide binding lobe, whereas the second lobe, responsible for recognition of the sugar moiety, encompasses the remaining residues of the N-terminal domain (Fig. 3B).
Striking differences exist between the PPase domains of apo-SpGlmU and
the SpGlmU·AcCoA·UDP-GlcNAc complex (root mean square deviation of
2.2 Å for 226 C atoms), indicating that the enzyme undergoes a
substantial conformational change upon substrate/product binding. In
the absence of UDP-GlcNAc (apo-SpGlmU and SpGlmU·AcCoA), SpGlmU
adopts an open conformation, whereas in the UDP-GlcNAc complex two
regions within the sugar binding lobe move toward each other giving
rise to a closed conformation (Fig. 3B). Upon product
binding the entire region encompassing residues Thr-132
Lys-166 moves
as a rigid body, making a 20° tilt resulting in a 7-Å movement of
the
5b-
6 surface loop. The melting of the last turn of the
-helix
5, facing the
5b-
6 loop, transforms the following
5-
6 surface loop (Asn-191
Tyr-197) into an extended thumb-shaped
hairpin. These movements bring the two above surface loops close to
each other, such that in the UDP-GlcNAc complex the Ala-192 N
hydrogen bonds Asp-157 OD1 (Fig. 3b), whereas in the unbound
form these two residues are 14 Å apart. This suggests that the two
surface loops function like a pair of tongs, closing up upon substrate binding and anchoring the sugar deep into the active site pocket thereby shielding it from solvent.
The "breathing" of the PPase domain of SpGlmU could not be observed for the E. coli GlmU-Tr enzyme, where the crystal structures reveal a closed conformation for both the apo- and UDP-GlcNAc complexed forms (3). However, analysis of the crystal packing in the E. coli GlmU-Tr structures reveals that the pyrophosphorylase domain is constrained into its closed conformation in both the apo-form and the GlmU-Tr·UDP-GlcNAc complex by the packing environment, whereas no such constraints exist in apo- or complexed SpGlmU crystals.
The interactions of the enzyme with the nucleotide and the sugar are
largely conserved within the complex crystal structures from S. pneumoniae and E. coli GlmU, yet significant
differences reside in the surroundings of the pyrophosphate moiety.
Whereas in the GlmU-Tr·UDP-GlcNAc complex both phosphates are
solvent-exposed, in the SpGlmU·AcCoA·UDP-GlcNAc complex the
-phosphate is stabilized through weak hydrogen bonds to the side
chains of sequence-conserved Arg-15 and Lys-22, located within the
signature motif. Moreover, both phosphate groups interact through a
calcium ion with Asp-102 and Asn-227, situated in the
4-
4 hairpin
and in the N-cap of the long
-helical linker, respectively (Fig.
3c). This calcium ion exhibits the octahedral coordination
geometry characteristic of Mg2+ ions and thus mimics the
catalytically important Mg2+ ion. Arg-15 presents static
disorder, with the conformation of minor occupancy contacting the
-phosphate (Fig. 3B). This disorder, together with the
weak hydrogen bond to Lys-22, indicates that instead of stabilizing the
product Arg-15 and Lys-22 must have a role either in substrate
recognition or transition-state stabilization during the single
displacement reaction (22), consistent with mutagenesis data of the
E. coli GlmU enzyme (3).
Surprisingly in the crystal structure of the E. coli
GlmU-Tr·UDP-GlcNAc complex the functional residues Arg-18, Lys-25,
Asp-105, and Asn-227 are located far away from the pyrophosphate group. These residues are carried by three structural elements in intimate contact with each other, with the -helical arm, and with the acetyltransferase domain of a neighboring subunit. As mentioned above,
the E. coli GlmU-Tr structures differ from the SpGlmU
structures in the relative arrangement of the acetyltransferase and
pyrophosphorylase domains, probably because of enzyme truncation.
Consequently in E. coli GlmU-Tr, the
-helical arm pushes
the signature motif Gly-14
Lys-25 away from the substrate binding
pocket of the pyrophosphorylase domain, which might explain the 2-fold
reduction in the kcat value of E. coli GlmU-Tr, as compared with the wild-type enzyme (3).
The Acetyltransferase Domain--
The C-terminal acetyltransferase
LH domain resembles an equilateral prism, with the three sides
formed by three parallel
-sheets composed of short
-strands (Fig.
2B). The 50-Å long
-helix of full-length SpGlmU consists
of 10 regular coils, whereas the E. coli GlmU-Tr structure
is truncated after the fourth coil. The buried surface area to a 1.6-Å
probe radius of a SpGlmU subunit upon trimer formation is 4690 Å2, a value in the highest range when compared with other
homologous trimeric L
H structures. The regularity of the prism is
interrupted only at the seventh coil by a single insertion loop,
encompassing the sequence-conserved region Asn-385 to Lys-393, which
projects from one of the vertices of the prism and flanks an adjacent
subunit (Fig. 2B). The dominant and striking feature of the
SpGlmU trimeric assembly is the domain exchange of the C-terminal
region. Although this is a novel feature within the family of bacterial
acetyltransferases, such a domain exchange has been reported for a
number of proteins and is referred to as three-dimensional
domain swapping (23). After 10 1/3 complete turns the peptide chain is
exchanged with an adjacent subunit, thus forming the unique
antiparallel
-strand of an additional coil within the L
H domain.
Therefore, together with the
-helical linker sitting on top of the
L
H domain, the C-terminal domain exchange contributes to the
stabilization of the SpGlmU trimeric assembly. At residue Glu-447 the
anti-parallel
-strand reaches the vertex of the prism of a
neighboring subunit. At this point the polypeptide chain inserts
between two neighboring subunits and coils backwards in the direction
of the N terminus, forming two successive 310-helices and
ending in intimate contact with the insertion loop Asn-385
Lys-393 of
an adjacent subunit. An 8-Å long and very narrow tunnel is formed in
this way, enclosing bound AcCoA located at the interface of two
subunits, and closed from the outside by the exchanged C-terminal arm
of the third subunit and the insertion loop Asn-385
Lys-393 (Fig.
4A), revealing that the
trimeric assembly is required for the acetyltransferase activity. To
our knowledge, such an active site architecture located at the junction
of three subunits is novel and exemplifies how an oligomeric assembly,
coupled to a domain exchange, can create a specific binding site. The
C-terminal region past residue Glu-447 could not be observed in the
apo-structure, suggesting that this region is highly flexible and
becomes only structured upon AcCoA binding. No other major structural
rearrangements occur upon AcCoA binding in the acetyltransferase
domain, except maybe for the insertion loop Asn-385
Lys-393, which is
highly disordered in the apo-structure, as indicated by a main chain
average B-factor of 74 Å2 as compared with an average main
chain B-factor of 35 Å2 for the rest of the
acetyltransferase domain.
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Major stabilization of the AcCoA cofactor is ensured by stacking of the
adenine group between the side chains of Ile-437 and Arg-441, hydrogen
bonds from the 3'-phosphate group to Lys-445 NZ and Tyr-448 OH,
and electrostatic interactions with Arg-439. Additional stabilization
arises from hydrophobic interactions, and hydrogen bonds from the
-mercaptoethylamine moiety to main-chain atoms of Asn-385, Ser-404,
and Ala-422. The pyrophosphate is exposed to the solvent and does not
interact with the protein.
Structural comparison of the SpGlmU·AcCoA complex with other related
bacterial acetyltransferases reveals a common location of the AcCoA
binding site. Indeed, AcCoA adopts a conformation very similar to the
fishhook-like conformation observed for CoA in the
tetrahydrodipicolinate N-succinyltransferase (24), bent at
the pyrophosphate group and with an extended pantetheine arm running
parallel to the LH domain (Fig. 4A). Although the
C-terminal domain exchange, from which the AcCoA binding site emerges,
is novel and dissimilar to other related bacterial acetyltransferases, structuring of the C-terminal portion upon CoA binding has been previously reported for tetrahydrodipicolinate
N-succinyltransferase (24).
Implications for Catalysis--
Acetyltransferases utilizing AcCoA
as substrate donor transfer the acetyl group, loosely bound through the
weak thioester linkage, either to a cysteine residue, forming a
covalent acetyl-enzyme intermediate, or directly to the substrate (25,
26). In the light of the first of these two mechanisms, the role of the
four cysteine residues in the E. coli enzyme was
investigated by site-directed mutagenesis studies (27). However, none
of the cysteine residues are conserved between known GlmU sequences,
and acetyltransferase activity was dramatically decreased only by the
Ala mutant of Cys-307, which is disulfide-bridged and points toward the
interior of the LH domain in the E. coli GlmU-Tr
structure. SpGlmU contains only one single cysteine residue, Cys-369,
located 10 Å apart from the active site, excluding thus the hypothesis
of a covalent acetyl-cysteine enzyme intermediate.
Inspection of the SpGlmU active site points rather toward a direct acetyl group transfer, based on a catalytic triad formed by the conserved residues His-362, Glu-348, and Ser-404 (Fig. 4B). His-362 is the only residue located in close proximity of the thioester, which may function as a general base catalyst, activating the C-2 amine of glucosamine-1-P for nucleophilic attack. Hydrogen bonding of His-362 ND1 to Glu-348 OE1 ensures the proper tautomeric form of the imidazole, lacking one proton on NE2. Ser-404, located behind the thioester, is well positioned to stabilize, together with the main-chain nitrogen atom of Ala-379, the negative charge building up on the thioester carbonyl at the transition state. The sequence-conserved Asn-385 residue, within hydrogen bond distance to the sulfur, could have a role in proton transfer at the end of the catalytic cycle. The importance of His-362 is highlighted by a superimposition of SpGlmU with the crystal structure of tetrahydrodipicolinate N-succinyltransferase, which positions SpGlmU His-362 similarly to tetrahydrodipicolinate N-succinyltransferase Asp-141, a residue proposed to function as the general base (24). A histidine, His-79, has been suggested to function as the general base, as well, in the related hexapeptide xenobiotic acetyltransferase from Pseudomonas aeruginosa (28).
In absence of a complex with GlcN-1-P, we have modeled GlcN-1-P into
the small pocket containing the catalytic triad and surrounded by bulky
side chains protruding from two neighboring subunits and the insertion
loop Asn-385Lys-393. The orientation of GlcN-1-P is constrained by a
cluster of sequence-conserved electropositive residues (Arg-332,
Lys-350, and Lys-391), candidates for binding the C-1 phosphate group,
a hypothesis supported by earlier kinetic studies showing that GlcN is
a very poor substrate compared with GlcN-1-P for the acetyltransfer
reaction (4). In our model the acceptor amino group on C-2 is within
hydrogen binding distance from the proposed catalytic base (NE2 of
His-362) and ideally poised to make a nucleophilic attack on the
thioester (Fig. 4B).
The three crystal structures of SpGlmU in unbound and complexed form
described in this paper highlight novel structural features necessary
to achieve the acetyltransferase reaction and define a structural
template to design new antibiotics. A detailed dissection of the two
distinct GlmU catalytic mechanisms must await further crystallographic
investigations of substrate and inhibitor complexes.
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ACKNOWLEDGEMENTS |
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We thank Anne Belaich for the S. pneumoniae cDNA library, the staff of the European Synchrotron Radiation Facility for technical support in data collection, and Dominique Mengin-Lecreulx, Bernard Henrissat, Christian Cambillau, and Gideon Davies for helpful discussion.
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FOOTNOTES |
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* This work was funded in part by a Groupement d'Intérèt Public-Hoechst Marion Roussel grant and the Centre National de la Recherche Scientifique (UMR 6098, Marseille, France).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 atomic coordinates and the structure factors (code 1HM0, 1HM8, and 1HM9) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Former post-doctoral fellow of Hoechst Marion Roussel. Holder of an EMBO fellowship.
¶ Present address: ENSBANA, Département de Microbiologie, 1 Esplanade Erasme, 21000 Dijon, France. Former post-doctoral fellow of Hoechst Marion Roussel.
** To whom correspondence should be addressed. Tel.: 33-4-91-16-45-08; Fax: 33-4-91-16-45-36; E-mail: yves@afmb.cnrs-mrs.fr.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M011225200
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ABBREVIATIONS |
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The abbreviations used are:
AcCoA, acetyl-coenzyme A;
GlmU, N-acetyl-glucosamine-1-phosphate
uridyltransferase;
GlmU-Tr, truncated form of Escherichia
coli GlmU;
PPase, pyrophosphorylase;
LH, left-handed
-helix;
SpGlmU, Streptococcus pneumoniae GlmU;
PEG, polyethylene
glycol.
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REFERENCES |
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