From the Institut de Biologie Structurale Jean-Pierre
Ebel, CEA-CNRS, Laboratoire de Cristallographie Macromoléculaire,
41 rue Jules Horowitz, F-38027 Grenoble Cedex 1, France and
§ Institut de Biochimie et Biophysique Moléculaire et
Cellulaire, UMR 8619 CNRS, Laboratoire des Enveloppes
Bactériennes et Antibiotiques, Université Paris-Sud,
Bâtiment 430, 91405 Orsay Cedex, France
Received for publication, October 27, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate
ligase is a cytoplasmic enzyme that catalyzes the
addition of meso-diaminopimelic acid to nucleotide
precursor
UDP-N-acetylmuramoyl-L-alanyl-D-glutamate in the biosynthesis of bacterial cell-wall peptidoglycan. The crystal
structure of the Escherichia coli enzyme in the presence of
the final product of the enzymatic reaction,
UDP-MurNAc-L-Ala- Peptidoglycan, the polymeric mesh of the bacterial cell wall,
plays a critical role in protecting bacteria against osmotic lysis. It
consists of linear repeating disaccharide chains cross-linked by short
peptide bridges. During the cytoplasmic steps involved in the
biosynthesis of the peptidoglycan precursor, four ADP-forming ligases
(namely the Mur ligases) catalyze the assembly of the peptide moiety by
the successive addition of L-alanine,
D-glutamate, diaminopimelic acid, or L-lysine,
and, finally, dipeptide D-alanyl-D-alanine to
UDP-N-acetylmuramic acid (1, 2). Because all these enzymes are essential for cell viability, they are attractive targets for
antibacterial chemotherapy. In Escherichia coli, these
ligases are the products of the murC, murD,
murE, and murF genes, located in the
mra region (3). Sequence comparison of the four E. coli Mur ligases shows several homologous regions, suggesting that these enzymes may be evolutionarily related and may use similar enzymatic mechanisms (4-6). In earlier publications, we reported the
structure of
UDP-N-acetylmuramoyl-L-alanine:D-glutamate
ligase (MurD),1 both in the
native form and complexed with substrates (7-9). MurD consists
of three domains with topologies reminiscent of a nucleotide-binding
fold; the N- and C-terminal domains have a dinucleotide-binding fold
(the Rossmann fold), and the central domain displays a
mononucleotide-binding fold, also seen in ATP-binding proteins. A
comparison of six MurD structures reveals that large C-terminal
rotation, loop rearrangement, and subdomain movements occur upon
substrate binding (9). In addition, several potentially important
residues for substrate binding and/or catalysis have been identified
(10). Recently, the x-ray structure of the
folylpoly- In E. coli, MurE catalyzes the addition of
meso-diaminopimelic acid
(meso-A2pm) to the nucleotide precursor,
UDP-MurNAc-L-Ala-D-Glu (UMAG), according to the
reaction: UMAG + meso-A2pm + ATP -D-Glu-meso-A2pm, has been solved to 2.0 Å resolution. Phase information was obtained by
multiwavelength anomalous dispersion using the K shell edge of
selenium. The protein consists of three domains, two of which have a
topology reminiscent of the equivalent domain found in the already
established three-dimensional structure of the
UDP-N-acetylmuramoyl-L-alanine: D-glutamate-ligase (MurD) ligase, which catalyzes the immediate previous step of incorporation of D-glutamic acid in the
biosynthesis of the peptidoglycan precursor. The refined model reveals
the binding site for
UDP-MurNAc-L-Ala-
-D-Glu-meso-A2pm,
and comparison with the six known MurD structures allowed the
identification of residues involved in the enzymatic mechanism.
Interestingly, during refinement, an excess of electron density was
observed, leading to the conclusion that, as in MurD, a carbamylated
lysine residue is present in the active site. In addition, the
structural determinant responsible for the selection of the amino acid
to be added to the nucleotide precursor was identified.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-L-glutamate synthetase
(FGS) of Lactobacillus casei
has been reported (11). Despite low sequence identity, FGS and MurD are
clearly both members of the Mur ADP-forming ligase superfamily (9,
12).
UDP-MurNAc-L-Ala-
-D-Glu-meso-A2pm (UMT) + ADP + Pi. As established with other Mur ligases,
the MurE reaction presumably proceeds by phosphorylation of the
C-terminal carboxylate of UMAG by the
-phosphate of ATP to form an
acyl phosphate intermediate, followed by nucleophilic attack by the
-amino group of A2pm to produce UMT (Fig.
1), ADP, and inorganic phosphate
(13-15). This mechanism is supported by the three-dimensional structures of MurD and MurD complexes. Some bacteria (such as E. coli and Bacillus subtilis) contain
meso-A2pm, and others (such as
Streptococcus pneumoniae and Staphylococcus
aureus) contain L-lysine at the third position of the
peptide side chain of cell wall peptidoglycan. In each case, the MurE
enzymes have been shown to efficiently discriminate between these two
amino acids in vitro, because they are only able to catalyze
the addition of either meso-A2pm or
L-lysine to UMAG (16, 17). Because these two amino acids
effectively coexist in bacterial cells, the high specificity of the
MurE enzymes acts as a gatekeeper to ensure that only the specific
substrate is incorporated in the peptidoglycan presursor. However, this
difference in specificity is not clearly reflected in the protein
sequence, because only 28 and 32% identity is seen between the
E. coli and S. pneumoniae or E. coli
and B. subtilis sequences, respectively.
View larger version (8K):
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Fig. 1.
UMT, the final product of the
reaction.
This report describes the crystal structure of E. coli MurE
in the presence of the UMT reaction product. The structure reveals that
the enzyme has a three-domain topology and allows the localization of
the active site and the identification of the residues involved in UMT
binding. In addition, we compare and discuss the two structurally characterized ligases, MurD and MurE.
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EXPERIMENTAL PROCEDURES |
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Materials-- DNA restriction enzymes and synthetic oligonucleotides were obtained from Eurogentec or New England Biolabs. Polymerase chain reaction amplification of DNA was performed in a Thermocycler 60 apparatus (Bio-med) using Taq polymerase (Appligene), and DNA fragments were purified using the Wizard purification system. meso-[14C]A2pm was obtained from the Commissariat à l'Energie Atomique (Saclay, France). UMAG was synthesized from UDP-N-acetylmuramoyl-L-Ala, using purified MurD (18), and UMT was prepared as previously described (19).
Bacterial Strains, Plasmid Vectors, and Growth
Conditions--
E. coli strains JM83 (ara [lac-proAB] rpsL thi
80
dlacZ
M15) (20) and B180
(
metA::Cmr) (21) were used as
plasmid hosts and for the preparation of the overproduced
His6-tagged MurE enzyme. The pTrcHis60 plasmid, a pTrc99A (Amersham Pharmacia Biotech) derivative for the
production of C-terminal His6-tagged proteins, has been
recently described (22). 2YT medium (23) or M9 minimal medium (24) was
used for growing cells, with growth being monitored at 600 nm with a
Shimadzu UV-1601 spectrophotometer. For strains carrying drug resistance genes, the antibiotics used were ampicillin (100 µg·ml
1) and chloramphenicol (25 µg·ml
1).
General DNA Techniques and E. coli Cell Transformation-- Small and large scale plasmid isolations were carried out by the alkaline lysis method, and standard procedures were used for endonuclease digestions, ligation, and agarose electrophoresis (24). E. coli cells were made competent and transformed with plasmid DNA using the method of Dagert and Ehrlich (25).
Construction of Plasmids--
A plasmid suitable for high level
overproduction of MurE (C-terminal His6-tagged form) was
constructed as follows. Polymerase chain reaction primers were designed
to incorporate a NcoI site (shown in boldface type)
5' to the initiation codon (underlined) of murE,
5'-GGGACCCATGGCAGATCGTAATTTGCGCGAC-3',
and a BglII site (in boldface type) 3' to the gene without
its stop codon, 5'-TACGCAGATCTTGCAATCACCCCCAGCAG-3'.
These primers were used to amplify the murE gene from
the E. coli chromosome, and then the resulting material was
treated with NcoI and BglII and ligated between
the same sites of vector pTrcHis60, resulting in pMLD117, a
plasmid allowing expression of the gene under the control of the
strong isopropyl--D-thiogalactopyranoside-inducible trc promoter.
Preparation of Crude Protein Extracts and Enzyme
Purification--
JM83 (pMLD117) cells were grown exponentially at
37 °C in 2YT-ampicillin medium (18 liters of culture in a
fermenter). At an optical density of 0.1, isopropyl--D-thiogalactopyranoside was added at a final
concentration of 1 mM, and growth was continued for 3 h (final optical density was 1.3). The cells were harvested at 4 °C
(about 45 g of wet weight), and the cell pellets were washed with
cold 20 mM potassium phosphate buffer, pH 7.4, containing 1 mM
-mercaptoethanol and 0.5 mM
MgCl2 (buffer A) and then stored at
20 °C. Cells from
one-fifth of the preparation were suspended in 9 ml of buffer A and
disrupted by sonication at 4 °C using a Bioblock Vibracell 72412 sonicator. The resulting suspension was centrifuged at 4 °C for 20 min at 200,000 × g, and the pellet was discarded.
SDS-polyacrylamide gel electrophoresis analysis showed that MurE
accounted for about 20% of the crude protein soluble extract (data not shown).
For the preparation of the MurE protein in which all methionine
residues were replaced by selenomethionine (SeMurE), E. coli methionine-auxotrophic strain 180 was transformed with plasmid pMLD117, and the resulting strain was cultured in M9 medium (2 liter
culture) supplemented with 0.4% glucose, ampicillin, and all the usual
amino acids (100 µg·ml
1), except that
L-selenomethionine was substituted for
L-methionine. Expression of murE was induced at
mid-log phase (optical density was 0.6) with 1 mM
isopropyl-
-D-thiogalactopyranoside, and cell growth was
allowed to continue for a further 3 h (final optical density was
1.8). The cells were then harvested, washed in buffer A, and disrupted
by sonication, and the crude protein extract was prepared as described above.
The His6-tagged proteins (MurE and SeMurE) were purified on
Ni2+-nitrilotriacetate-agarose essentially following the
manufacturer's recommendations (Qiagen): binding to the resin, washing
with buffer A containing 20 mM imidazole and 300 mM NaCl, and elution of the adsorbed proteins by a
discontinuous gradient of 20-300 mM imidazole (MurE and
SeMurE behaved similarly and eluted at a concentration of imidazole of
about 150 mM). The pooled fractions were dialyzed against
20 mM HEPES buffer, pH 7.4, containing 5 mM
dithiothreitol and 200 mM NaCl and then concentrated on
PM10 membranes (Millipore) to ~12 mg·ml1 for use in
crystallization experiments. SDS-polyacrylamide gel electrophoresis
analysis of proteins performed on 12% polyacrylamide gels (26) showed
that the His6-tagged proteins were at least 90% pure.
Protein concentrations were determined by the Bradford method (27),
using bovine serum albumin as a standard. Typically, 15-20 mg of pure
protein (MurE or SeMurE) were obtained per liter of culture. Amino acid
and mass spectrometry (matrix-assisted laser
desorption/ionization) analyses confirmed the composition and
molecular mass of the two proteins as well as the 100%
selenomethionine substitution in SeMurE.
Enzyme Activity--
The meso-A2pm adding
activity was assayed by measuring the formation of radioactive UMT. The
reaction mixture (0.1 M Tris-HCl buffer, pH 8.6, 0.1 M MgCl2, 5 mM ATP, 0.2 mM UMAG, and 0.1 mM meso-[14C]A2pm (1.5 KBq); total
volume, 75 µl) was incubated for 30 min at 37 °C. The reaction was
stopped by addition of 10 µl of glacial acetic acid and the
radioactive substrate and product were separated by reverse-phase high
pressure liquid chromatography on a Nucleosil 5C18
column (4.6 × 150 mm; Alltech France, Templemars, France) using
50 mM ammonium formate buffer, pH 3.9, at a flow rate of 0.6 ml·min1. Detection was performed with a radioactive
flow detector (model LB506-C1; EG&G Wallac/Berthold, Evry, France)
using the Quicksafe Flow 2 scintillator (Zinsser Analytic, Maidenhead,
UK), and quantification was carried out using the Winflow software
(EG&G Wallac/Berthold). The activity of the pure preparation of
His6-tagged MurE enzyme was 625 nmol of
meso-A2pm incorporated in UMT per min and mg of protein.
Crystallization and Data Collection--
MurE, expressed as a
C-terminal His6-tagged protein, consists of 502 amino acids
(molecular mass was 54,278.5 Da). Crystals of native and
selenomethionyl MurE were grown in the presence of the reaction
product, UMT. Drops of 2 µl of protein solution (10 mg·ml1 of purified enzyme, 20 mM HEPES, pH
7.5, 200 mM NaCl, 5 mM dithiothreitol, and 1 mM UMT) and 2 µl of reservoir buffer (0.1 M
HEPES, pH 7.5, 13% polyethylene glycol monomethyl ether 5,000, 0.5 M LiCl, 10% isopropanol, and 5 mM
dithiothreitol) were equilibrated against 1 ml of reservoir buffer. The
crystals, space group C2221, had unit cell dimensions of
a = 93.27 Å, b = 99.51 Å, and
c = 234.34 Å, with two molecules in the asymmetric
unit. Multiwavelength anomalous dispersion data collection was
carried out on a single flash cooled crystal at three wavelengths on a
BM14 beam line (European Synchrotron Radiation Facility,
Grenoble, France). The long c axis was aligned so that it
was almost coincident with the spindle axis, and data were collected at
2.8 Å resolution using a MAR ccd detector. The range of data
collection was determined using STRATEGY (28). Data were processed
using DENZO (29). The relevant statistics are given in Table
I. Data at 2 Å resolution were
collected using a MAR ccd on the ID14-EH2 beam line (European Synchrotron Radiation Facility, Grenoble, France), a 110° sweep being
made in increments of 0.25°. Data were processed using XDS (30).
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Multiwavelength Anomalous Dispersion Phasing, Model Building, and
Refinement--
Multiwavelength anomalous dispersion data were input
into the program SOLVE (31). All 24 selenium positions in the
asymmetric unit were found, and experimental phases were calculated
from these using a multiple isomorphous approach. The
noncrystallographic symmetry was defined using FINDNCS (32). Density
modification (33) was then used to extend the experimental
phases to 2 Å using noncrystallographic symmetry averaging, solvent
flattening, and histogram matching. The resulting experimental map was
of excellent quality. The majority of the model was traced
automatically using wARP (34). Refinement to 2 Å was carried out by
sequential use of the Crystallography and NMR Systems program
(35), interspersed with computer graphics model building using O (36).
The final model consists of two molecules of MurE (992 visible
residues, of which 10 have been modeled with double conformations), 141 ligand atoms, and 394 water molecules. The stereochemistry of the final
model was evaluated using the PROCHECK program (37). The coordinates of
the MurE structure have been deposited with the Brookhaven Protein Data
Bank (accession code 1e8c) and will be released 1 year after publication.
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RESULTS |
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Quality of the Refined Model--
The final model, which contains
992 residues, two UMT molecules, and 394 water molecules, had a
crystallographic R factor of 20.2%
(Rfree = 23.0%; Ref. 38) for all 72,674 reflections in the resolution range 46.7-2.0 Å (Table
II). The root mean square (rms)
deviations were 0.006 Å from ideal bond lengths and 1.3° from ideal
bond angles. After density modification, the experimental density map
was of good quality. Fig. 2 shows the
chemical modification of Lys224 by covalent binding to
three atoms (see "Discussion"). The asymmetric unit contains two
molecules (A and B) of MurE, essentially identical in conformation.
After superimposition, the rms deviation between 495 pairs of
equivalent C was 0.45 Å. No symmetry was seen between the two
molecules. The Ramachandran plot (39) for the present model showed all
the nonglycine residues to be in allowed regions. The average
temperature factors were slightly different for molecules A and B (29.9 and 31.4 Å2, respectively) but identical for the two UMT
molecules (39.5 Å2). A few residues at either end of the
polypeptide chain had no visible electron density and were therefore
not included in the model. However, 25 and 20 residues (molecule A and
B, respectively) clearly showed holes in the electron density map. The
damage was produced by third generation synchrotron radiation (40), the most frequent being decarboxylation of acidic residues. The side chains
were built into the electron density, and a partial occupancy was
assigned for the side chain atoms based on difference Fourier maps.
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Overall Protein Structure--
MurE consists of three globular
domains formed from contiguous segments in the amino acid sequence
(Fig. 3).
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Domain 1 comprises residues 1-88 and consists of a five-stranded
-sheet surrounded by two helices (Fig.
4a). Comparison of its
structure with the data base of known protein structures carried out
using the DALI server (41) revealed no homology with known protein
structures; the structure showing the greatest similarity is a fragment
of the transferrin receptor (42) with a rms deviation of 3.9 Å for the
70 structurally equivalent C
atoms.
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Domain 2 (Fig. 4b) comprises residues 90-338 and consists
of a central six-stranded parallel -sheet surrounded by seven
-helices. The fold of the central
-sheet is similar to the
classic "mononucleotide-binding fold" found in many ATP-binding
proteins, and domain 2 will therefore be referred to as the ATP-binding
domain. As expected, using DALI, the structures with the highest Z
scores were the central domain of MurD (7) (Protein Data Bank code
1uag; Z score = 20.0) and the N-terminal domain of FGS (11)
(Protein Data Bank code 1fgs; Z score = 9.7). Superposition of
domain 2 on the corresponding domain of the two ligases gave an rms
deviation of 3.1 Å for the 193 structurally equivalent C
s for MurD
and 3.5 Å for the 168 C
s for FGS. The loop between
6 and
4 of
the ATPase domain (also referred to as the P loop) is probably involved
in ATP binding.
Domain 3 (340) contains a six-stranded -sheet with parallel
strands (
17,
18,
19,
20, and
21) and an antiparallel
strand (
16), and five surrounding
-helices (Fig. 4c).
Surprisingly, as already observed for the C-terminal domains of MurD
and FGS, domain 3 contains a Rossmann fold (Z score = 10.9 and
10.8 for MurD and FGS, respectively). Moreover, superposition of domain 3 on the corresponding domain of the two ligases gave an rms deviation of 2.7 Å for the 119 structurally equivalents C
s for MurD and 2.4 Å for the 107 C
s for FGS. The large insertion (464) between strands
22 and
23 has no structural equivalent in MurD and
FGS.
UMT Binding Site--
The product, UMT, binds in the cleft between
the three domains. The experimental electron density map showed almost
all of both UMT molecules. In the early stage of refinement, only the A2pm density was difficult to interpret. As shown in Fig.
5, the bound UMT makes many polar
interactions with the protein. The domain 1 residues involved in UMT
binding are located in the two loops connecting 1 with
2 and
2
with
2. The geometry of the uridine-ribose moiety of the two UMT
molecules is C2'-endo for the ribose ring pucker
(
=
114.5°) and an anti-orientation about the
glycosyl bond. The uracyl ring forms two hydrogen bonds with
Ser28, these being between O2 and the main
chain nitrogen and between N3 and O
. In addition, it is
inserted between a salt bridge (between Asp27 and
Arg29) and Tyr50. Interestingly, C5
and C6 of the pyrimidine ring are exposed to the solvent,
explaining how dihydrouridine can replace uridine in the nucleotide
substrate with little effect on the Km (97 µM versus 55 µM for the natural
substrate) (43).
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The pyrophosphate of UMT interacts with the loop connecting 2 with
2 (residues 42-47). The protein-phosphate interactions are made
mainly through hydrogen bonds with main chain nitrogens, the
-phosphate oxygens forming hydrogen bonds with Gln44 and
Ala45, and the
-phosphate oxygens with
His43. One of the
-phosphate oxygens forms hydrogen
bonds with ND1 of His43; the latter is the only residue to
balance the negative charges of the pyrophosphate.
The N-acetylmuramic acid ring bridges the gap between domain
1 and the ATP-binding domain. Two groups of hydrogen bonds are important; O1 of Asn156 interact with
O4', and Gln190 and Arg192 are in contact with
the acetyl group. Interestingly, Arg192 forms three other
hydrogen bonds with the carbonyl oxygen of L-Ala and
O
1 of D-Glu. In the ATP-binding
domain, three loops are close to the product (
8-
5,
9-
6, and
6-
10). Finally, the A2pm-containing region of UMT
forms five hydrogen bonds with the C-terminal domain, the atoms
involved being located in two loops connecting secondary structure
elements (
19-
14) and (
21-
22). These very important
interactions are discussed below.
Sequence Alignment of MurE Sequences--
An alignment analysis
using the 20 MurE sequences currently available shows that 24 amino
acid residues are strictly conserved; none of these are found in the
N-terminal domain, whereas 14 occur in the ATP-binding domain and 10 are in the C-terminal domain. Fig. 6
shows the sequence alignment; for clarity, only four representative MurE ligase sequences were used (E. coli, B. subtilis, S. aureus, and S. pneumoniae). Of
the 24 strictly conserved residues, only three (Thr158,
Ser185, and Arg192) interact with the product,
UMT. Eight others (Gly115, Gly118,
Lys119, Glu182, His210,
Asn310, Arg341, and Asp356) are
invariant residues conserved in all the members of the Mur ligase
family (MurC, MurD, MurE, MurF, and Mpl; Mpl protein
(UDP-N-acetylmuramate:L-alanyl--D-glutamyl-meso-diaminopimelateligase) catalyzes the formation of UDP-Mur- NAc-tripeptide by addition of
L-Ala-
-D-Glu-meso-A2pm
to UDP-MurNAc (44)). Putative roles for these residues in
reaction mechanism or structure of Mur ligases have been proposed
(5-10). Excluding the seven glycine and proline residues, which may
play a structural role, the remaining six of the 24 conserved amino
acids are Thr114, Thr116, Tyr220,
His359, Arg443, and Lys465. The
first two form part of the P loop, found in the Mur ligase family, that
has the characteristic finger print
Xaa114-Gly-Xaa-Xaa-Gly-Lys119. A large anion
hole is formed by the loop, which accommodates the phosphates of ATP
(8). In MurE, the loop consists of residues 115-119 and connects
6
to
4. Tyr220 makes two hydrogen bonds, one with
O
of Thr116 and the other with the
carbamylated Lys224 (see below). The hydrophobic part of
A2pm lies against the ring of His359.
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Finally, two buried salt bridges, located in the C-terminal domain,
play a very important role. The first, between Arg443 and
Asp482 (in some orthologs, Asp482 is replaced
by a glutamate), links the first two amino acids of 15 and
16,
stabilizing the domain. The second is between Lys465 and
Glu468. In addition, Lys465 makes two hydrogen
bonds, one with the almost buried side chain of Asn414 and
one with the main chain oxygen of Tyr480.
Asn414 and Glu468 are functionally conserved in
MurE sequences and are involved in the interaction with
A2pm (see below).
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DISCUSSION |
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Comparison with MurD--
An interesting comparison can be made
between the structures of the MurD·UMAG and MurE·UMT complexes.
Superposition of the central domain -sheet of MurD and MurE clearly
shows a well conserved ATP-binding domain (Fig.
7). In addition, the two C-terminal
domains are almost in the same position. As expected from the domain
topology, the two N-terminal domains share no secondary structure
elements. Interestingly, the uridine has a completely different
orientation, and O4 has moved 9.5 Å. Another striking
detail in the comparison is the positional overlap between
enzymatically important residues in both ligases (Table
III). Most of these have already been
identified in the Mur ligase family (5, 6, 8, 9). However,
superposition of the two active sites reveals three new positions,
these being Asp182, Thr321, and
Lys348 in MurD, corresponding to Asp209,
His359, and Arg389 in MurE. His359
is strictly conserved in all MurE sequences, whereas Asp209
and Arg389 are replaced in some orthologs by functionally
equivalent amino acids (Glu and Lys, respectively). In the MurE·UMT
complex, the Arg389 side chain does not interact with
Asp209, in contrast to the situation in MurD, in which a
salt bridge is formed between Lys348 and
Asp182. However, in MurE, the side chain of
Arg389 can move to interact with Asp209. In
conclusion, all important residues in MurD have a corresponding residue
in MurE. This suggests that MurD and MurE have the same catalytic
machinery.
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At this point, it is important to underline the role of
Lys224. This amino acid is not conserved in the Mur ligase
family, because MurC and Mpl have a phenylalanine or tyrosine at the
equivalent position. The experimental density clearly shows chemical
modification of Lys224 in MurE. The same modification was
also observed in all the "closed" structures of MurD for
Lys198 (8). Chemical interpretation of the electron density
form (Fig. 2) and the noncovalent interactions with neighboring atoms of the modified lysine led to the conclusion that Lys224 is
carbamylated. As observed in the
MurD·UDP-N-acetylmuramoyl-L-Ala·ADP·Mg2+
complex (8), this modification is probably crucial for Mg2+
binding and, consequently, for the -phosphate positioning of ATP.
Structural Implications for Catalysis-- In general, ATP-dependent amide-forming enzymes are believed to share a common mechanism involving an initial phosphorylation of the acid carboxylate. Subsequently, the acyl phosphate is attacked by the amino group, producing a tetrahedral intermediate, which ultimately collapses to the final product and inorganic phosphate (14). For ligation to occur between UMAG and A2pm, MurE must (i) bring together the UMAG and ATP, (ii) correctly orientate UMAG and ATP for acyl-phosphate intermediate formation, (iii) orient A2pm for nucleophilic attack, and (iv) stabilize the tetrahedral intermediate, thereby lowering the activation barrier and accelerating catalysis. The structural determination of MurE has confirmed the location of the active site and identified the protein residues involved in the catalytic mechanism (Table III). The similar location of the 11 structurally conserved amino acids in MurE and MurD strongly supports the previous reaction mechanism proposed for MurD (8).
MurE Ligases and A2pm Structural Determinant--
The
primary structures of peptidoglycans display a number of variations,
mainly in the peptide moiety. Depending on the bacterium, MurE ligases
add different amino acids at the third position, the most common being
meso-A2pm, L-lysine,
L-ornithine, and LL-A2pm (45). Mur ligase
specificity has been studied in ten microorganisms; five use
A2pm at the third position, whereas the others use
L-Lys (16). MurE ligases will only add A2pm or
L-Lys to UMAG, whereas MurF, which catalyzes the addition
of D-Ala-D-Ala to UMT, is less specific for the
occurrence of A2pm or L-Lys in the tripeptide moiety. The substrate specificity of E. coli MurE ligase has
been analyzed in detail using A2pm and various analogs (17)
in vitro, the relative specific activity of incorporation
being 100, <0.1, and 2.6 for meso-A2pm,
L-Lys, and LL-A2pm, respectively. Expression in
E. coli cells of the S. aureus MurE enzyme, which
catalyzes the addition of L-Lys, results in 50%
incorporation of L-Lys into the peptidoglycan (46);
interestingly, transpeptidation cannot occur if the acceptor unit bears
an L-lysine residue. Of the 20 known MurE sequences, four
belong to Gram-positive microorganisms (Enterococcus
faecalis, S. aureus, S. pneumoniae, and
Streptococcus pyogenes) and incorporate
L-Lys at the third position. Of the four MurE enzymes shown
in Fig. 6, the first two catalyze the addition of
meso-A2pm, and the last two catalyze the
addition of L-Lys. The amino acids in E. coli
MurE interacting with the free end of A2pm are shown in
Fig. 8. Asp413,
Asn414, and Arg416 are located on a loop
connecting 19 to
14, and Gly464 and
Glu468 are on a loop linking
21 to
22. Because
Arg416 is conserved in all A2pm-adding enzymes
and is replaced by Ala or Asn in the four L-Lys-adding
enzymes, it should be the main structural determinant for
A2pm selection.
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Conclusion--
By solving the crystal structure of E. coli MurE, we have obtained a high resolution model of a closed
active form of the enzyme. MurE shares the same three domain topology
and a similar active site architecture with MurD. Comparison of the
structures of MurE and MurD complexed with the final reaction product
has allowed (i) the identification of key residues potentially involved in the enzymatic mechanism, one of these being, as in MurD, a carbamylated lysine and (ii) the explanation of the presence of a new
fold in the N-terminal domain that is able to accommodate the extension
of the UDP substrate. Finally, an arginine residue seems to be the main
structural determinant involved in selection of the amino acid inserted
at the third position in the peptide moiety of peptidoglycan precursors.
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ACKNOWLEDGEMENTS |
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We thank Gordon Leonard and Sean McSweeney for assistance in data collection and Didier Blanot (Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Orsay) for the helpful discussions and mass spectrometry analyses.
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FOOTNOTES |
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* This work was supported by Grant UMR 8619 from the Center National de la Recherche Scientifique and a grant from "Microbiologie Fondamentale."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.
¶ To whom correspondence should be addressed. Tel.: 33-476-88-56-09; Fax: 33-476-88-54-94; E-mail: otto@ibs.fr.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M009835200
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ABBREVIATIONS |
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The abbreviations used are:
MurD, UDP-N-acetylmuramoyl-L-alanine: D-glutamate
ligase;
FGS, folylpoly--L-glutamate synthetase;
A2pm, diaminopimelic acid;
SeMurE, selenomethionyl-MurE;
UMAG, UDP-N-acetylmuramoyl-L-Ala-D-Glu;
UMT, UDP-N-acetylmuramoyl-L-Ala-
-D-Glu-meso-A2pm;
rms, root mean square.
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