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J. Biol. Chem., Vol. 282, Issue 42, 30414-30422, October 19, 2007

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Unexpected Inhibition of Peptidoglycan LD-Transpeptidase from Enterococcus faecium by the -Lactam Imipenem^*

From the ^aINSERM, U872, LRMA Pôle 4, Equipe 12 F-75006 Paris, France, ^bCentre de Recherche des Cordeliers, Université Pierre et Marie Curie, UMR S872, F-75006, Paris, France, ^cUniversité Paris Descartes, UMR S872, F-75006 Paris, France, ^dAssistance Publique-Hospitaux de Paris, Hôpital Européen Georges Pompidou, F-75015 Paris, France, ^eCNRS, UMR 5153, F-75231 Paris, France, ^fINSERM, U565, F-75231 Paris, France, ^gMuseum National d'Histoire Naturelle, Mass Spectrometry Facility, USM 0503, F-75231 Paris, France, ^hMuséum National d'Histoire Naturelle, USM0502, Plateforme de Spectrométrie de Masse et de Protéomique du Muséum, Département Recherche Développement et Diversité Moléculaire, F-75005 Paris, France, ⁱCNRS, UMR 8041, F-75005 Paris, France, and ^jMedical and Research Services, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106

Received for publication, May 24, 2007 , and in revised form, July 10, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The -lactam antibiotics mimic the D-alanyl⁴-D-alanine⁵ extremity of peptidoglycan precursors and act as "suicide" substrates of the DD-transpeptidases that catalyze the last cross-linking step of peptidoglycan synthesis. We have previously shown that bypass of the DD-transpeptidases by the LD-transpeptidase of Enterococcus faecium (Ldt_fm) leads to high level resistance to ampicillin. Ldt_fm is specific for the L-lysyl³-D-alanine⁴ bond of peptidoglycan precursors containing a tetrapeptide stem lacking D-alanine⁵. This specificity was proposed to account for resistance, because the substrate of Ldt_fm does not mimic -lactams in contrast to the D-alanyl⁴-D-alanine⁵ extremity of pentapeptide stems used by the DD-transpeptidases. Here, we unexpectedly show that imipenem, a -lactam of the carbapenem class, totally inhibited Ldt_fm at a low drug concentration that was sufficient to inhibit growth of the bacteria. Peptidoglycan cross-linking was also inhibited, indicating that Ldt_fm is the in vivo target of imipenem. Stoichiometric and covalent modification of Ldt_fm by imipenem was detected by mass spectrometry. The modification was mapped into the trypsin fragment of Ldt_fm containing the catalytic Cys residue, and the Cys to Ala substitution prevented imipenem binding. The mass increment matched the mass of imipenem, indicating that inactivation of Ldt_fm is likely to involve rupture of the -lactam ring and acylation of the catalytic Cys residue. Thus, the spectrum of activity of -lactams is not restricted to transpeptidases of the DD-specificity, as previously thought. Combination therapy with imipenem and ampicillin could therefore be active against E. faecium strains having the dual capacity to manufacture peptidoglycan with transpeptidases of the LD- and DD-specificities.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The peptidoglycan of Enterococcus faecium is generated by polymerization of a disaccharide-peptide subunit composed of 1–4-linked N-acetylglucosamine and N-acetylmuramic acid, a linear stem pentapeptide (L-Ala¹-D-iGln²-L-Lys³-D-Ala⁴-D-Ala⁵) linked to the lactoyl group of N-acetylmuramic acid by an amide bond, and a side-chain D-isoasparagynyl or D-isoaspartyl (D-iAsx)² residue linked to the amino group of L-Lys³ (1, 2). The final steps of peptidoglycan synthesis involve polymerization of the glycan strands by glycosyltransferases (3) and cross-linking of peptide stems by DD-transpeptidases (4). The peptide bond formed by the latter enzyme links the carbonyl of D-Ala⁴ to the amino group of D-iAsx. The two-step reaction for the formation of these D-Ala⁴D-iAsx-L-Lys³ cross-links starts by the nucleophilic attack of the carbonyl of D-Ala⁴ of the acyl donor substrate by the oxygen of the catalytic serine leading to the release of D-Ala⁵ and to the formation of a covalent adduct (acylenzyme) (5, 6). In the second step, the carbonyl of D-Ala⁴ is attacked by the amino group of D-iAsx of the acceptor substrate leading to formation of the D-Ala⁴D-iAsx-L-Lys³ cross-link and to the release of the DD-transpeptidase. The -lactam antibiotics are structural analogues of the D-Ala⁴-D-Ala⁵ extremity of the pentapeptide stem and act as "suicide" substrates of the DD-transpeptidases (see Fig. 1). Opening of the -lactam ring, which mimics the rupture of the D-Ala⁴-D-Ala⁵ peptide bond in the first step of the DD-transpeptidation reaction, leads to inactivation of the DD-transpeptidases. Because the acylenzymes typically have half-lives in the order of several hours, inactivation of the DD-transpeptidases by -lactams is essentially irreversible in the scale of the generation time of bacteria. The DD-transpeptidases, which are highly redundant enzymes, are collectively the killing target of -lactams, because peptidoglycan cross-linking is essential to the integrity of the cell wall. These enzymes belong to a large family of active-site serine acyl-transferases that bind penicillin covalently and are thus referred to as penicillin-binding proteins (PBPs) (4).

The -lactams are one of the oldest and still the most broadly used class of antibiotics for the treatment of severe infections despite the development of several resistance mechanisms, including the enzymatic detoxification of the drug by -lactamases (7), the modification of the transport of the antibiotic in Gram-negative bacteria (8), and modifications of the target, the DD-transpeptidases, that result in reduced acylation of these enzymes by -lactams (4). In E. faecium, intrinsic resistance to moderate levels of ampicillin involves the production of one such low affinity PBP, designated PBP5_fm (9, 10). Acquisition of higher levels of resistance to this drug in clinical isolates results from overproduction of PBP5_fm and from amino acid substitutions in the DD-transpeptidase domain of the protein that further decrease the interaction of the protein with ampicillin (11, 12). We have previously described a novel mechanism of -lactam resistance in a mutant of E. faecium selected in vitro in five consecutive steps on increasing concentrations of ampicillin (13, 14). Analysis of the peptidoglycan structure of the resulting mutant, E. faecium M512, indicated that ampicillin resistance was due to target substitution, because the D-Ala⁴D-iAsx-L-Lys³ cross-links generated by the DD-transpeptidases (PBPs) were replaced by L-Lys³D-iAsx-L-Lys³ cross-links generated by an LD-transpeptidase (13). The "LD" designation refers to cleavage of a peptide bond between amino acids of the L and D configurations prior to peptide bond formation, as opposed to DD-transpeptidases (PBPs) that act on the D-Ala⁴-D-Ala⁵ bond of the donor (Fig. 1A). The DD- and LD-transpeptidases use the same amino group in the acyl acceptor for peptide bond formation, corresponding to the -amino group of D-iAsx in E. faecium (15).

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Structural similarity between -lactams and the peptidyl-L-Lys3-D-Ala4-D-Ala5 extremity of peptidoglycan precursors. A, L-Lys3(D-Asx)D-Ala4-D-Ala5 extremity of the peptidoglycan precursors of E. faecium showing the position of the nucleophilic attacks of the carbonyls of D-Ala4 and of L-Lys3 by the catalytic Ser and Cys residues of the DD-transpeptidases (PBP) and LD-transpeptidases (Ldt), respectively. B, structure of the penam ampicillin. C, structure of the cephem ceftriaxone. D, structure of carbapenems. E, acylation of the active site serine of a PBP by imipenem. The analogy with D or L amino acids is indicated for relevant chiral carbons of -lactams. Note that ampicillin is a structural analogue of D-Ala-D-Ala despite the fact that one of the carbon atoms of the antibiotic has an L-configuration.

The -lactam antibiotics are thought to have a unique type of target consisting of members of the active-site serine acyltransferase family of proteins characterized by a common fold and the presence of conserved amino acid motifs, S*XXK, SXN (or analogue), and KTG (or analogue) (4). Because Ldt_fm functions on a different peptide bond (L-Lys³-D-Ala⁴ instead of D-Ala⁴-D-Ala⁵) and is structurally unrelated to the PBPs (16, 17), it was expected that the lack of inhibition by -lactams, as shown for ampicillin (16), should also concern all drugs of this family. In this report, we have examined this notion in a critical manner and unexpectedly found that a specific class of -lactams, the carbapenems (Fig. 1D), is highly active against the mutant M512. We show that Ldt_fm is the target of the carbapenems in vivo and that these drugs inactivate the enzyme by acylation of the catalytic cysteine. These results extend the diversity of the targets of -lactams to include active-site cysteine peptidase of the LD-specificity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bacterial Strains, Growth Conditions, and -Lactam Susceptibility Testing—E. faecium D344S derives from D344, a clinical isolate of E. faecium (10), by a spontaneous deletion of pbp5 encoding the low affinity PBP5. E. faecium M512 is a spontaneous mutant of D344S obtained by five serial selection steps on agar containing increasing concentrations of ampicillin (14). All cultures were performed at 37 °C in brain heart infusion broth or agar (Difco Laboratories). Antibiotic susceptibility was tested by the disk-diffusion assay for representatives of the four main classes of -lactams, including penams (penicillin, ampicillin, amoxicillin, amoxicillin plus clavulanate, piperacillin, and oxacillin), cephems (cefamandole, cefepime, cefixime, cefoperazone, cefotiam, cefoxitin, cefsulodin, ceftazidime, ceftriaxone, and cefuroxime), monobactams (aztreonam), and carbapenems (imipenem, meropenem, and ertapenem). Minimal inhibitory concentrations (MICs) were determined by the agar dilution method (13) for a representative antibiotic of each class of -lactams, including ampicillin (Bristol-Myers), ceftriaxone (Roche Applied Science), and aztreonam (Sanofi-Synthélabo). For carbapenems, MICs were determined for the three molecules in therapeutic use, including imipenem (Merck Sharpe and Dhome-Chibret), meropenem (AstraZeneca), and ertapenem (Merck Sharpe and Dhome-Chibret).

Inhibition of the LD-Transpeptidase Activity of Ldt_fm by Ampicillin, Ceftriaxone, and Imipenem—Domains I and II of Ldt_fm (residues 217–466) fused to a C-terminal His₆ tag was purified by affinity, anion exchange, and size-exclusion chromatographies as previously described (17). Inhibition of Ldt_fm by -lactams was studied in an exchange reaction, which assays for the capacity of the enzyme to cleave the L-Lys-D-Ala peptide bond of the model donor dipeptide Ac₂-L-Lys-D-Ala and to form a peptide bond between Ac₂-L-Lys and D-[¹⁴C]Ala (0.15 mM, 2.0 GBq/mmol, ICN Pharmaceuticals) (14). Ldt_fm (1 µg) was preincubated for 20 min at 37 °C with 2-fold dilutions of ampicillin, ceftriaxone, and imipenem in 25 µl of sodium phosphate (25 mM, pH 6.5) containing Triton X-100 (0.1%, v/v). The exchange reaction was started by adding 25 µl of a solution containing Ac₂-L-Lys-D-Ala and D-[¹⁴C]Ala in the same buffer (final concentrations 5 and 0.15 mM, respectively). Aliquots were taken at 0, 10, 20, and 30 min, boiled at 100 °C for 3 min to stop the reaction, and Ac₂-L-Lys-D-[¹⁴C]Ala was determined by rp-HPLC as previously described (14). The EC₅₀ was defined as the antibiotic concentration that inhibited formation of Ac₂-L-Lys-D-[¹⁴C]Ala by 50%. The EC₅₀ was defined as the -lactam concentration that inhibited the reaction by 50% and was deduced from v_i/v_o = f[I] plots where v_i and v_o are the velocity in the presence and absence of -lactam, respectively, and [I] is the final -lactam concentration. Linear regression of experimental values was performed with Sigma Plot (version 9.0).

Expression of the Pbp5_fm Gene in E. faecium M512 and Peptidoglycan Structure Analysis—The pbp5_fm gene of E. faecium D344 has been previously cloned into the shuttle expression vector pNJ2 (20). The resulting plasmid pAA20(pbp5_fm) was introduced into E. faecium M512 by electroporation (21), and clones were selected in brain heart infusion agar containing spectinomycin (60 µg/ml) and chloramphenicol (5 µg/ml). The structure of the peptidoglycan of the resulting strain, E. faecium M512/pAA20(pbp5_fm), was determined by mass spectrometry as previously described (20). Briefly, the strain was grown at 37 °C to an optical density of 0.7 in 250 ml of brain heart infusion broth containing spectinomycin (120 µg/ml), imipenem (8 µg/ml) plus spectinomycin (120 µg/ml), and ampicillin (128 µg/ml) plus spectinomycin (120 µg/ml). Peptidoglycan was extracted with boiling SDS and digested with mutanolysin and lysozyme (Sigma-Aldrich) (20). The resulting muropeptides were cleaved under alkaline conditions to remove the disaccharide. The resulting lactoyl-peptides were separated by rp-HPLC, identified by mass spectrometry, and sequenced by tandem mass spectrometry, as previously described (20).

Detection of Ldt_fm-Carbapenem Adducts by Electrospay Mass Spectrometry—Ldt_fm (34 µM) was incubated with imipenem (0, 3.4, 6.8, 17, 27.2, and 92 µM) for 1 h at 37 °C in 50 mM Tris-HCl (pH 7.5). The reaction mixture was dialyzed against water for 30 min and conserved at –20 °C. Formation of adducts between Ldt_fm and meropenem or ertapenem was tested in the same conditions. For electrospray mass spectrometry, 5 µl of the reaction mixture was mixed extemporaneously with 5 µl of acetonitrile and 1 µl of 1% formic acid. The mixture (11 µl) was directly injected into the mass spectrometer (Qstar Pulsar I, Applied Biosystems) using rp-HPLC pumps at a flow rate of 0.05 ml/min (acetonitrile 50%, water 49.5%, formic acid 0.5%, per volume). The data were acquired in the positive mode with a capillary voltage of 5200 V and a declustering potential of 20 V. The mass scan range was from m/z 400–2500, and the scan cycle was 1 s. The average mass of proteins and protein-carbapenem adducts was deduced from a total of nine pairs of peaks obtained in three independent experiments, and results were expressed as means and standard deviations. Digestion with trypsin (Proteomics Grade, Sigma-Aldrich) was performed for 18 h at 37 °C at a trypsin to Ldt_fm ratio of 0.05 per mass. Fragments of Ldt_fm were detected by nanospray mass spectrometry.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
-Lactam Resistance Profile Resulting from Activation of the LD-Transpeptidation Pathway in E. faecium M512—Because ampicillin was the only -lactam studied in previous reports (14, 16), antibiotic susceptibility testing was performed with 22 -lactams belonging to the four main classes of -lactams by the disk-diffusion assay and with representatives of each class by the agar dilution method (Fig. 2A and data not shown). Unexpectedly, activation of LD-transpeptidation pathway in E. faecium M512 did not result in resistance to imipenem, and the other carbapenems (meropenem and ertapenem) retained substantial residual activity. The mutant M512 was resistant to all other -lactams, although residual activity was observed for third generation cephalosporins such as ceftriaxone (MIC 128 µg/ml). Finally, M512 remained resistant to the monobactam aztreonam, which has no anti-enterococcal activity (data not shown).

In Vitro Inhibition of the LD-Transpeptidase Activity of Ldt_fm by -Lactams—The effective concentration of imipenem inhibiting Ldt_fm by 50% (EC₅₀) was 0.077 ± 0.003 µg/ml, and full inhibition was detected at 0.5 µg/ml (Fig. 2B). Thus, inhibition of Ldt_fm by imipenem could account for the antimicrobial activity of this drug (MIC 0.5 µg/ml). The low antibacterial activity of ceftriaxone against M512 (MIC 128 µg/ml) also correlates with inhibition of Ldt_fm, because the EC₅₀ of ceftriaxone was 19 ± 7 µg/ml, and complete inhibition of the enzyme was obtained with 200 µg/ml. The EC₅₀ of ampicillin was >3200 µg/ml in agreement with the lack of antibacterial activity of this antibiotic (MIC > 2000 µg/ml).

Ldt_fm Is the Target of Imipenem in Vivo—Inhibition of the formation of the L-Lys³D-iAsx-L-Lys³ cross-links by Ldt_fm could not be tested in E. faecium M512, because this mutant does not produce low affinity PBP5_fm and cannot therefore be grown in the presence of imipenem. To circumvent this problem, we introduced in M512 plasmid pAA20, which encodes PBP5_fm and allowed growth of the mutant in the presence of 8 µg/ml of imipenem. In the absence of -lactam, peptidoglycan manufactured by the resulting strain, M512/pAA20(pbp5_fm), contained both D-Ala⁴D-iAsx-L-Lys³ cross-links generated by DD-transpeptidation and L-Lys³D-iAsx-L-Lys³ cross-links generated by LD-transpeptidation (Table 1 and Fig. 3A). In the presence of ampicillin (128 µg/ml), all multimers contained L-Lys³D-iAsx-L-Lys³ cross-links generated by Ldt_fm indicating that all PBPs, including PBP5_fm, were inhibited by this high drug concentration (Table 1 and Fig. 3B). In the presence of imipenem (8 µg/ml), cross-links were exclusively generated by the DD-transpeptidase activity of PBP5_fm (Table 1 and Fig. 3C). These results indicate that the susceptibility of M512 to imipenem is due to inhibition of the cross-linking activity of Ldt_fm by this antibiotic.

View this table: [in this window] [in a new window]   TABLE 1 Composition of the peptidoglycan manufactured by E. faecium M512/pAA20(pbp5fm) in the presence or absence of -lactams Ampicillin and imipenem were added to the culture medium at a concentration of 128 and 8 µg/ml to inhibit the DD-transpeptidase activity of the PBPs and the LD-transpeptidase activity of Ldtfm, respectively. The lactoyl-peptides purified by rp-HPLC were identified by mass spectrometry and sequenced by tandem mass spectrometry as previously described (20).

View larger version (23K): [in this window] [in a new window]   FIGURE 2. -Lactam susceptibility and inhibition of Ldtfm. A, MICs of ampicillin, ceftriaxone, imipenem, meropenem, and ertapenem against E. faecium M512 and the parental strain D344S. MICs were determined by the agar dilution method after 48 h of incubation at 37 °C. B, in vitro inhibition of the LD-transpeptidase activity of Ldtfm by -lactams. Ldtfm was incubated with various concentrations of ampicillin (Amp), ceftriaxone (Cro), and imipenem (Imi) for 20 min at 37 °C and residual enzyme activity was assayed by determining formation of Ac2-L-Lys-D-[14C]Ala from Ac2-L-Lys-D-Ala and D-[14C]Ala.

To purify the adduct, Ldt_fm was incubated with a 10-fold molar excess of imipenem and purified by size-exclusion chromatography. The adduct and the native protein eluted both as monomers. Electrospray mass spectrometry revealed that the major protein peak corresponded to the protein adduct with only minor amounts of native protein (relative intensity 7%). The latter peak was not increased upon further incubation of the purified protein adduct for 2, 7, 24, and 48 h. These results indicate that the link between imipenem and Ldt_fm is stable in solution.

The average mass of the Ldt_fm-imipenem adduct matched the average mass of the protein incremented by the average mass of imipenem (Table 2). Adducts matching increments of the average mass of meropenem and ertapenem were also detected. For each of the three carbapenems, binding of a single molecule of drug to Ldt_fm was observed up to a drug to protein ratio of 10 that corresponds to the highest ratio that was tested. The observed mass increments are compatible with non-covalent binding of the carbapenems to Ldt_fm or with formation of a covalent bond involving opening of the -lactam ring (Fig. 1E). To distinguish between these two possibilities, Ldt_fm was incubated with carbapenems and digested with trypsin, and the fragments were analyzed by nano-spray mass spectrometry. Digestion of Ldt_fm was expected to generate 14 fragments greater than three residues that were all detected in the trypsin digest of the native protein. Upon addition of imipenem and meropenem, the fragment containing the catalytic Cys residue of Ldt_fm (GSHGCINTPPSVMK) was replaced by fragments incremented by the monoisotopic mass of the corresponding carbapenems (Table 3). These results establish that imipenem and meropenem bind covalently to the same fragment of Ldt_fm. The fact that the observed mass increments are equal to the mass of the drugs indicates that formation of the covalent adduct involves opening of the -lactam ring. Substitution of the catalytic Cys⁴⁴² by Ala (16) totally abolished drug binding. Thus, inactivation of the LD-transpeptidase by carbapenems required a catalytically active protein and is likely to involve formation of a thioester with the catalytic Cys residue (Fig. 1E).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (33K): [in this window] [in a new window]   FIGURE 3. rp-HPLC profiles of lactoyl-peptides from E. faecium M512 pNJ2(pbp5fm). Peptidoglycan was extracted from bacteria grown in the absence of -lactam (A) and in presence of ampicillin (128 µg/ml) (B) or imipenem (8 µg/ml) (C). Peptidoglycan was digested with muramidases and treated with ammonium hydroxide, and the resulting lactoyl-peptides were separated by rp-HPLC; mAU, absorbance unit x 103 at 210 nm. Peaks labeled with roman numbers are uncross-linked monomers. Peaks labeled with letters are multimers generated by the DD-transpeptidase activity of PBPs. Peaks labeled with Arabic numbers are multimers generated by the LD-transpeptidase activity of Ldtfm. D and E, structure of major dimers generated by DD- and LD-transpeptidation, respectively.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Detection of an Ldtfm-imipenem adduct by mass spectrometry. Imipenem was incubated with Ldtfm (34 µM) at drug to enzyme molar ratios of 0 (A), 0.1 (B), 0.2 (C), 0.5 (D), 0.8 (E), and 3.0 (F) and analyzed by electrospray mass spectrometry. Peaks at m/z of 1,001.34 and 1,037.08 correspond to the [M+29H]29+ and [M+28H]28+ ions of the native protein, respectively (deduced average mass of 29,009.70). Peaks at m/z of 1,011.68 and 1,047.80 correspond to the [M+29H]29+ and [M+28H]28+ ions of the Ldtfm-imipenem adduct (deduced average mass of 29,308.61). The relative intensity of the peaks matches the drug to Ldtfm ratio very closely with the saturation of the protein.

To determine whether formation of the adduct is catalyzed by Ldt_fm, imipenem was incubated with the same concentration of enzyme or of a synthetic peptide with the same sequence (³³⁸GSHGCINTPPSVMK³⁵¹) as the tryptic fragment containing the catalytic Cys⁴⁴² residue (underlined). Mass spectrometry analyses (data not shown) indicated that full modification of Ldt_fm occurred in 30 min, whereas no modification of the synthetic peptide was observed in 60 min under the same conditions. Thus, formation of the covalent bond between Ldt_fm and imipenem was catalyzed by the enzyme.

The mass of the Ldt_fm-imipenem adduct was equal to the mass of the free antibiotic plus the mass of the free enzyme (Tables 2 and 3). Opening of the -lactam ring is therefore the most likely mechanism for formation of the covalent adduct (Fig. 1E), because formation of bonds involving amines, hydroxyls, or carboxyls would lead to the elimination of a water molecule. Site-directed mutagenesis identified the sulfur atom of the catalytic Cys⁴⁴² residue as the nucleophile for attack of the carbonyl of the -lactam ring, because the Cys⁴⁴² Ala substitution totally abolished formation of the adduct. Thus, Ldt_fm commits suicide by forming a thioester between Cys⁴⁴² and carbapenems in a reaction similar to the acylation of the active site serine of DD-transpeptidase by -lactams (4, 5).

Because the initial proposal of Tipper and Strominger (22), -lactams are regarded as molecular mimics of the N-acyl-D-alanyl-D-alanine extremity of peptidoglycan precursors, which accounts for the fact that they act as suicide substrates of the DD-transpeptidase module of high molecular weight penicillin-binding proteins. However, this could not be confirmed by direct co-crystallization of the DD-transpeptidases with their natural substrates or with the -lactams, because co-crystals contained the reaction products (23). For this reason, the analogy is only supported by modeling of the conformation of the two types of substrates (6). As discussed by Goffin and Ghuysen (6), the structural analogy relies on the spatial disposition of the carbonyl and carboxylate groups, but the C–CON–C–COOH bonds are far from being isosteric in the -lactams and in the terminal D-Ala⁴-D-Ala⁵ dipeptide. Considering the classic alignment of the D-Ala⁴-D-Ala⁵ backbone with -lactams (Fig. 1), the C of D-Ala⁵ at the C terminus of the natural substrate (Fig. 1A) has a D configuration as the analogous carbon atoms of penams (ampicillin, Fig. 1B). The corresponding carbon is not tetrahedral in cephems (ceftriaxone, Fig. 1C) and carbapenems (Fig. 1D). The carbon atoms mimicking the C of D-Ala⁴ have opposite configuration in carbapenems and in all other -lactams. This chiral center can be superimposed to an amino acid of the L configuration in ampicillin and ceftriaxone but to an amino acid of the D configuration in carbapenems. Despite the configuration inversion in this carbon, the DD-transpeptidase activity of the PBPs is inhibited by all -lactams, including carbapenems. Thus, for the PBPs, the structural analogy between the suicide substrates (the -lactams) and the natural substrates (the peptidoglycan precursors ending D-Ala⁴-D-Ala⁵) does not depend upon the configuration of the carbon mimicking the carbon of D-Ala⁴. For the LD-transpeptidase, the analogy should be analyzed by aligning the -lactams with the L-Lys³-D-Ala⁴ backbone. Paradoxically, the C of L-Lys³ can be superimposed to an amino acid of the D configuration in carbapenems and of the L configuration in other -lactams, despite the fact that only the former antibiotics inactivate Ldt_fm. In conclusion, the reactivity of -lactams for transpeptidases of the LD- and DD-specificities is not principally determined by the configuration of carbons in the antibiotics that mimic the C carbons in the L-Lys³-D-Ala⁴-D-Ala⁵ extremity of peptidoglycan precursors. It is therefore not surprising that imipenem was reported not to inhibit the LD-carboxypeptidase of E. coli (24).

	FOOTNOTES

 
^* This work was supported by the Fondation pour la Recherche Médicale (Equipe FRM 2006 (Grant DEQ200661107918)) and by NIAID, National Institutes of Health Grant R01 AI45626. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 33-1-43-25-00; Fax: 33-1-43-25-68-12; E-mail: michel.arthur{at}bhdc.jussieu.fr.

² The abbreviations used are: D-iAsx, D-isoasparagynyl or D-isoaspartyl; PBP, penicillin-binding protein; MIC, minimum inhibitory concentration; rp-HPLC, reversed-phase high-performance liquid chromatography; Ldt_fm, LD-transpeptidase from E. faecium.

	ACKNOWLEDGMENTS

 
We thank Jean van Heijenoort for critical reading of the manuscript and Matthieu Fonvielle for helpful comments and the drawing of Fig. 1.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Billot-Klein, D., Shlaes, D., Bryant, D., Bell, D., Legrand, R., Gutmann, L., and van Heijenoort, J. (1997) J. Bacteriol. 179, 4684–4688[Abstract/Free Full Text]
2. Bellais, S., Arthur, M., Dubost, L., Hugonnet, J. E., Gutmann, L., van Heijenoort, J., Legrand, R., Brouard, J. P., Rice, L., and Mainardi, J. L. (2006) J. Biol. Chem. 281, 11586–11594[Abstract/Free Full Text]
3. van Heijenoort, J. (2001) Glycobiology 11, 25R–36R[Abstract/Free Full Text]
4. Goffin, C., and Ghuysen, J. M. (1998) Microbiol. Mol. Biol. Rev. 62, 1079–1093[Abstract/Free Full Text]
5. Jamin, M., Wilkin, J. M., and Frère, J.-M. (1995) Essays Biochem. 29, 1–24[Medline] [Order article via Infotrieve]
6. Goffin, C., and Ghuysen, J. M. (2002) Microbiol. Mol. Biol. Rev. 66, 702–738[Abstract/Free Full Text]
7. Jacoby, G. A., and Munoz-Price, L. S. (2005) N. Engl. J. Med. 352, 380–391[Free Full Text]
8. Nikaido, H. (1994) Science 264, 382–388[Abstract/Free Full Text]
9. Fontana, R., Cerini, R., Longoni, P., Grossato, A., and Canepari, P. (1983) J. Bacteriol. 155, 1343–1350[Abstract/Free Full Text]
10. Williamson, R., Le Bouguénec, C., Gutmann, L., and Horaud, T. (1985) J. Gen. Microbiol. 131, 1933–1940[Abstract/Free Full Text]
11. Klare, I., Rodloff, A. C., Wagner, J., Witte, W., and Hakenbeck, R. (1992) Antimicrob. Agents Chemother. 36, 783–787[Abstract/Free Full Text]
12. Rice, L. B., Bellais, S., Carias, L. L., Hutton-Thomas, R., Bonomo, R. A., Caspers, P., Page, M. G., and Gutmann, L. (2004) Antimicrob. Agents Chemother. 48, 3028–3032[Abstract/Free Full Text]
13. Mainardi, J. L., Legrand, R., Arthur, M., Schoot, B., van Heijenoort, J., and Gutmann, L. (2000) J. Biol. Chem. 275, 16490–16496[Abstract/Free Full Text]
14. Mainardi, J. L., Morel, V., Fourgeaud, M., Cremniter, J., Blanot, D., Legrand, R., Frehel, C., Arthur, M., Van Heijenoort, J., and Gutmann, L. (2002) J. Biol. Chem. 277, 35801–35807[Abstract/Free Full Text]
15. Magnet, S., Arbeloa, A., Mainardi, J. L., Hugonnet, J. E., Fourgeaud, M., Dubost, L., Marie, A., Delfosse, V., Mayer, C., Rice, L. B., and Arthur, M. (2007) J. Biol. Chem. 282, 13151–13159[Abstract/Free Full Text]
16. Mainardi, J. L., Fourgeaud, M., Hugonnet, J. E., Dubost, L., Brouard, J. P., Ouazzani, J., Rice, L. B., Gutmann, L., and Arthur, M. (2005) J. Biol. Chem. 280, 38146–38152[Abstract/Free Full Text]
17. Biarrotte-Sorin, S., Hugonnet, J. E., Delfosse, V., Mainardi, J. L., Gutmann, L., Arthur, M., and Mayer, C. (2006) J. Mol. Biol. 359, 533–538[CrossRef][Medline] [Order article via Infotrieve]
18. Magnet, S., Bellais, S., Dubost, L., Fourgeaud, M., Mainardi, J. L., Petit-Frère, S., Marie, A., Mengin-Lecreulx, D., Arthur, M., and Gutmann, L. (2007) J. Bacteriol. 189, 3927–3931[Abstract/Free Full Text]
19. Cremniter, J., Mainardi, J. L., Josseaume, N., Quincampoix, J. C., Dubost, L., Hugonnet, J. E., Marie, A., Gutmann, L., Rice, L. B., and Arthur, M. (2006) J. Biol. Chem. 281, 32254–32262[Abstract/Free Full Text]
20. Arbeloa, A., Hugonnet, J. E., Sentilhes, A. C., Josseaume, N., Dubost, L., Monsempes, C., Blanot, D., Brouard, J. P., and Arthur, M. (2004) J. Biol. Chem. 279, 41546–41556[Abstract/Free Full Text]
21. Arthur, M., Depardieu, F., Snaith, H. A., Reynolds, P. E., and Courvalin, P. (1994) Antimicrob. Agents Chemother. 38, 1899–1903[Abstract/Free Full Text]
22. Tipper, D. J., and Strominger, J. L. (1965) Proc. Natl. Acad. Sci. U. S. A. 54, 1133–1141[Free Full Text]
23. Macheboeuf, P., Contreras-Martel, C., Job, V., Dideberg, O., and Dessen, A. (2006) FEMS Microbiol. Rev. 30, 673–691[CrossRef][Medline] [Order article via Infotrieve]
24. Templin, M. F., Ursinus, A., and Höltje, J.-V. (1999) EMBO J. 18, 4108–4117[CrossRef][Medline] [Order article via Infotrieve]

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