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J. Biol. Chem., Vol. 282, Issue 18, 13151-13159, May 4, 2007

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Specificity of L,D-Transpeptidases from Gram-positive Bacteria Producing Different Peptidoglycan Chemotypes^*

Sophie Magnet^¶, Ana Arbeloa^¶, Jean-Luc Mainardi^¶||, Jean-Emmanuel Hugonnet^¶, Martine Fourgeaud^¶, Lionel Dubost^**, Arul Marie^**, Vanessa Delfosse^¶, Claudine Mayer^¶, Louis B. Rice, and Michel Arthur^¶1

From the INSERM, U655-LRMA F-75006 Paris France, Université Pierre et Marie Curie-Paris6, Centre de Recherches Biomédicales des Cordeliers, F-75006 Paris, France, ^¶Université Paris-Descartes, Facultéde Médecine René Descartes, Centre de Recherches Biomédicales des Cordeliers, F-75006 Paris, France, ^||AP-HP, Hôpital Européen Georges Pompidou, F-75015 Paris, France, ^**Plateforme de Spectrométrie de Masse et de Protéomique, Muséum National d'Histoire Naturelle, Département Recherche Développement et Diversité Moléculaire, F-75005 Paris, France, and Medical and Research Services, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106

Received for publication, November 27, 2006 , and in revised form, January 18, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
We report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the L,D-transpeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria. The lone functionally characterized member of this new family of active site cysteine peptidases, Ldt_fm from Enterococcus faecium, was previously shown to bypass the D,D-transpeptidase activity of the classical penicillin-binding proteins leading to high level cross-resistance to glycopeptide and -lactam antibiotics. Ldt_fm homologues from Bacillus subtilis (Ldt_Bs) and E. faecalis (Ldt_fs) were found here to cross-link their cognate disaccharide-peptide subunits containing meso-diaminopimelic acid (mesoDAP³) and L-Lys³-L-Ala-L-Ala at the third position of the stem peptide, respectively, instead of L-Lys³-D-iAsn in E. faecium. Ldt_fs differed from Ldt_fm and Ldt_Bs by its capacity to hydrolyze the L-Lys³-D-Ala⁴ bond of tetrapeptide (L,D-carboxypeptidase activity) and pentapeptide (L,D-endopeptidase activity) stems, in addition to the common cross-linking activity. The three enzymes were specific for their cognate acyl acceptors in the cross-linking reaction. In contrast to Ldt_fs, which was also specific for its cognate acyl donor, Ldt_fm tolerated substitution of L-Lys³-D-iAsn by L-Lys³-L-Ala-L-Ala. Likewise, Ldt_Bs tolerated substitution of mesoDAP³ by L-Lys³-D-iAsn and L-Lys³-L-Ala-L-Ala in the acyl donor. Thus, diversification of the structure of peptidoglycan precursors associated with speciation has led to a parallel evolution of the substrate specificity of the L,D-transpeptidases affecting mainly the recognition of the acyl acceptor. Blocking the assembly of the side chain could therefore be used to combat antibiotic resistance involving L,D-transpeptidases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The bacterial cell wall peptidoglycan is a net-like macromolecule that surrounds the cytoplasmic membrane (1). The polymer is essential because it supplies the cell with mechanical protection against the osmotic pressure of the cytoplasm. The peptidoglycan subunit contains -1,4-linked N-acetylglucosamine (GlcNAc)² and N-acetylmuramic acid (MurNAc) substituted by a peptide stem (Fig. 1A) (2). Assembly of the subunit at the cell surface is performed by glycosyltransferases that polymerize the glycan strands by formation of -1,4 bonds and D,D-transpeptidases that cross-link glycan strands (2). The latter reaction is catalyzed by penicillin-binding proteins (PBPs) that cleave the D-Ala⁴-D-Ala⁵ bond of a donor stem pentapeptide and link the carbonyl of D-Ala⁴ to the amino group of the side chain carried by the third residue of an acceptor stem peptide (Fig. 1B). This two-step reaction involves formation of a covalent adduct between the -hydroxyl of the active site serine of the PBPs and the carbonyl of D-Ala₄ of the donor stem (3). The glycosyltransferase and D,D-transpeptidase activities of the multimodular peptidoglycan polymerases are the targets of the two major classes of antibiotics available to treat severe infections due to Gram-positive bacteria, the -lactams and the glycopeptides, that act by different mechanisms. The -lactams are structural analogues of D-Ala⁴-D-Ala⁵ and act as suicide substrates of the D,D-transpeptidase module of the PBPs. The glycopeptides bind to the peptidyl-D-Ala⁴-D-Ala⁵ extremity of peptidoglycan precursors and block by steric hindrance both the transglycosylation and transpeptidation reactions (4).

The assembly pathway of peptidoglycan precursors and its mode of polymerization are generally highly conserved in eubacteria. Variations in the structure of peptidoglycan subunits involve mainly the fifth (C-terminal) and third positions of the pentapeptide stem (5). The specificity of peptidoglycan cross-linking enzymes for the donor is the bottleneck that limits emergence of resistance to glycopeptides because the modifications of the precursors that prevent drug binding should be tolerated by these enzymes (4, 6). Successful modifications that have spread in Gram-positive pathogens under the selective pressure of glycopeptides include incorporation of D-lactate or D-Ser instead of D-Ala at the fifth position of pentapeptide stems (7). Strikingly, production of D-lactate-ending precursors can also have an impact on the activity of -lactams in the enterococci and staphylococci, presumably because low affinity PBPs responsible for -lactam resistance cannot function with modified precursors (6, 8). Total elimination of D-Ala⁵ by hydrolysis of the C-terminal residue of pentapeptide stems is an alternative mechanism of glycopeptide resistance in mutants of Enterococcus faecium selected in laboratory conditions (9). Because PBPs cannot function with tetrapeptide donors, peptidoglycan cross-linking in these mutants requires an L,D-transpeptidase (Ldt_fm) that cleaves the L-Lys³-D-Ala⁴ peptide bond of the donor and links the carboxyl of L-Lys³ to the side chain amine of the acceptor (Fig. 1B). This mode of peptidoglycan cross-linking has been originally identified as a bypass of the PBPs that confers high level -lactam resistance (10).

Variation at the third position of the peptide stem concerns both the nature of the diamino acid present at this position, e.g. L-Lys or meso-diaminopimelic acid (mesoDAP), and the presence or absence of a side chain comprising from one to five amino acids (5) (Fig. 1A). Glycine and L-amino acids are incorporated into the side chain of peptidoglycan precursors by transferases of the Fem family that use aminoacyl-tRNAs as the substrate (11). D-Aspartic acid is activated as -aspartyl-phosphate and ligated to the precursors by ATP-dependent ligases belonging to the ATP-Grasp superfamily (12). Synthesis of complete side chains is essential for -lactam resistance mediated by low affinity PBPs in Gram-positive bacteria, in particular methicillin resistance mediated by PBP2a in Staphylococcus aureus (13, 14) and penicillin resistance-mediated PBP2X in Streptococcus pneumoniae (15). For this reason, Fem transferases are considered as attractive targets for the development of novel antibiotics active against -lactam-resistant pathogens (16, 17). The antibacterial activity of such Fem inhibitors will ultimately depend upon the incapacity of cross-linking enzymes to use precursors with incomplete side chains.

Interaction of the peptidoglycan cross-linking enzymes with their substrates has not been extensively investigated, despite its pivotal role for drug development and for our understanding of the mechanisms of resistance to glycopeptides and -lactams. In this report, the specificity of Ldt_fm and of the PBPs was compared based on heterospecific expression of a Fem transferase of E. faecalis in E. faecium and search for modified stem peptides containing L-Lys³-L-Ala instead of L-Lys³-D-iAsn in the donor and acceptor positions of dimers generated in vivo by L,D-transpeptidation and D,D-transpeptidation. The specificity of Ldt_fm was also studied in vitro by directly testing the cross-linking of peptidoglycan fragments isolated from three bacterial species (E. faecium, Bacillus subtilis, and E. faecalis) representative of the structural variability at the third position of the stem peptides (Fig. 1A). Finally, Ldt_fm homologues (Fig. 1C) were purified from E. faecalis and B. subtilis to compare the specificity of enzymes from bacteria producing peptidoglycan of different chemotypes. This analysis represents the first direct assessment of the specificity of peptidoglycan cross-linking enzymes because the PBPs are generally inactive in vitro. In addition, characterization of Ldt_fs revealed that members of the L,D-transpeptidase family can also display L,D-carboxypeptidase and L,D-endopeptidase activities.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Production and Purification of L,D-Transpeptidases—A catalytically active fragment of Ldt_fm from E. faecium M512 (residues 119–466) was produced in E. coli and purified by affinity, anion exchange, and size exclusion chromatographies, as previously described (18). A fragment of the open reading frame encoding residues 136–474 of the L,D-transpeptidase of E. faecalis strain JH2-2 (19), Ldt_fs, was amplified with primers 5'-AACCATGGGGAGTATCCGTCGAGGCAATGG-3' and 5'-AAGGATCCTACTTCTTCGCCGTAATCTA-3'. The PCR product was digested with NcoI and BamHI (underlined) and cloned into pET2818, a derivative of pET2816 (18) lacking the sequence specifying the thrombin cleavage site. The resulting plasmid, pET2818ldt_fs, was introduced in E. coli BL21(DE3) harboring pREP4GroESL (20), and bacteria were grown at 37 °C to an optical density at 600 nm of 0.8 in brain heart infusion broth (Difco, Elancourt, France) containing ampicillin (100 µg/ml). Isopropyl--D-thiogalactopyranoside was added to a final concentration of 0.5 mM, and incubation was continued for 17 h at 16 °C. The cells were disrupted by sonication in 50 mM Tris-HCl, pH 7.5, containing 300 mM NaCl, and cell debris were removed by centrifugation. Ldt_fs was purified from the resulting clarified lysate by affinity chromatography on Ni²⁺-nitrilotriacetate-agarose resin (Qiagen GmbH, Hilden, Germany). Proteins eluted with 200 mM imidazole were dialyzed against 50 mM Tris-HCl, pH 8.0, containing 60 mM NaCl, loaded onto an anion exchange column (MonoQ HR5/5, Amersham Biosciences) equilibrated with the same buffer. Ldt_fs, eluting at 300 mM NaCl, was further purified by size exclusion chromatography on a Superdex HR10/30 column (Amersham Biosciences) equilibrated with 50 mM Tris-HCl, pH 7.5, containing 300 mM NaCl. The protein was obtained with an overall yield of 3 mg/liter of culture, as estimated by the Bio-Rad protein assay using bovine serum albumin as a standard. The protein was stored at –80 °C in 50 mM Tris-HCl, pH 7.5, containing 300 mM NaCl.

The open reading frame encoding the L,D-transpeptidase of B. subtilis strain 168, Ldt_Bs, previously named ykuD (21), was amplified with primers 5'-AACCATGGGGCTGCTTACGTACCAGGTGAAGC-3' and 5 '-TTGGATCCCCGGTTAATCGTGACTCTCGT-3'. The PCR product digested with NcoI and BamHI (underlined) was cloned into pET2818, and Ldt_Bs was produced in E. coli BL21(DE3)/pREP4GroESL using the inducing conditions described above for the L,D-transpeptidase of E. faecalis. Ldt_Bs was purified in one step by affinity chromatography on Ni²⁺-nitrilotriacetate-agarose resin (Qiagen), dialyzed against 50 mM Tris-HCl, pH 7.5, containing 100 mM NaCl, and stored at –20 °C in the same buffer. 10 mg of protein were obtained per liter of culture.

L,D-Transpeptidase Assays—The source of the disaccharide-peptides used as substrates was as follows. The disaccharide-tetrapeptide substituted by a D-iso-asparagine residue (L-Lys³-D-iAsn) was purified from the peptidoglycan of E. gallinarum strain SC1 (22). The disaccharide-pentapeptide and the disaccharide-tetrapeptide substituted by an L-Ala-L-Ala side chain (L-Lys³-L-Ala-L-Ala) were purified from E. faecalis JH2-2 (19) and from a derivative of strain JH2-2 harboring the vanA glycopeptide resistance gene cluster (23), respectively. The disaccharide-tetrapeptide containing meso-diaminopimelic acid (mesoDAP³) was purified from E. coli strain ATCC 25113. The procedures used for peptidoglycan preparation, digestion with muramidases, and reduction of MurNAc to muramitol with sodium borohydride have been previously described for enterococci (24) and E. coli (25). The resulting muropeptides were separated by RP-HPLC in acetonitrile gradients containing trifluoroacetic acid (24) and identified by mass spectrometry (MS). The concentration of the muropeptides was estimated by amino acid analysis after acidic hydrolysis with a Hitachi autoanalyzer (26).

In vitro formation of muropeptide dimers was tested in 10 µl of phosphate buffer (20 mM, pH 7.0) containing the L,D-transpeptidase (7 µM) from E. faecium (Ldt_fm), E. faecalis (Ldt_fs), or B. subtilis (Ldt_Bs) and a combination of three reduced disaccharide-tetrapeptides (200 µM each) containing L-Lys³-D-iAsn, L-Lys³-L-Ala-L-Ala, or mesoDAP³ at the third position of a tetrapeptide stem. The reaction was incubated for 2 h at 37°C, desalted using a micro column (ZipTipC₁₈; Millipore, Saint Quentin-en-Yvelines, France), and analyzed by nanoelectrospray MS in the positive mode (Qstar Pulsar I; Applied Biosystem, Courtaboeuf, France). The sequence of the cross-links in dimers generated in vitro was determined by tandem mass spectrometry (MS/MS). Briefly, dimers were generated in vitro as described above except that the disaccharide-peptides used as substrates were not reduced. The reaction mixture was treated with ammonium hydroxide, desalted using a micro column (ZipTipC₁₈, Millipore), and the resulting lactoyl-peptides were analyzed by nanoelectrospray MS/MS using N₂ as the collision gas (24).

The L,D-transpeptidase activity of Ldt_fs was also tested by using the dipeptides L-Ala-L-Ala and D-Ala-D-Ala as acyl acceptors. The assay performed in 10 µl of 20 mM potassium phosphate buffer, pH 7.0, contained Ldt_fs (7 µM), the cognate disaccharide-tetrapeptide containing an L-Ala-L-Ala side chain as the acyl donor (200 µM), and 1 mML-Ala-L-Ala or D-Ala-D-Ala (Sigma). The products of the reactions were identified by MS and MS/MS, as previously described (18).

Expression of the bppA1 Gene of E. faecalis in E. faecium M512 and Analysis of Peptidoglycan Structure—The bppA1 gene of plasmid pDA15 (27) was subcloned under the control of the inducible promoter of pJEH4 (12) using XbaI and KpnI. The resulting plasmid, pJEH6(bppA1), was introduced by electroporation into E. faecium M512 (10). The recombinant strain was grown in brain heart infusion broth or agar (Difco) containing spectinomycin (120 µg/ml) to counter select loss of pJEH6(bppA1). Induction of the bppA1 gene was performed with 0.3 mM isopropyl--D-thiogalactopyranoside at an optical density at 600 nm of 0.02. Incubation was continued at 37 °C until the optical density reached 0.6, and bacteria were collected by centrifugation. Peptidoglycan was extracted with boiling SDS and digested with mutanolysin and lysozyme (Sigma-Aldrich) (24). The resulting muropeptides were cleaved under alkaline conditions to generate lactoyl-peptides, separated by RP-HPLC, and analyzed by MS and MS/MS (24).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Probing the Substrate Specificity of Ldt_fm in Vivo—Synthesis of the side chain of peptidoglycan precursors is catalyzed in E. faecalis by two members of the Fem family, BppA1 and BppA2, that sequentially add two L-Ala residues (Fig. 1A) (23). In E. faecium, D-Asp is added to the precursors by the Asl_fm ligase and subsequently partially amidated (12). The bppA1 gene of E. faecalis was cloned under the control of an inducible promoter to generate plasmid pJEH6 and introduced into E. faecium M512 in order to manipulate the structure of the substrate of the cross-linking reaction that can be catalyzed in this mutant by Ldt_fm and by the PBPs (28). The BppA1 transferase efficiently competed with the Asl_fm ligase in E. faecium M512/pJEH6(bppA1) because the main monomers contained L-Ala instead of D-iAsp (Fig. 2, peaks 1 and 2). The free side chains in the major dimers generated by L,D- and D,D-transpeptidation also contained L-Ala, indicating that Ldt_fm, as the PBPs, had catalyzed cross-link formation using donors containing this residue (Fig. 2, peaks 3–5). The cross-links of dimers generated by L,D-transpeptidation exclusively contained D-iAsp, whereas D-iAsp or L-Ala was found in cross-links generated by D,D-transpeptidation. Thus, Ldt_fm tolerated the substitution only in the donor substrate, in contrast to the PBPs that catalyzed peptidoglycan cross-linking with modified donor and acceptor substrates. Modifications of the side chain of peptidoglycan precursors were also shown to be tolerated by PBPs of E. faecalis and S. aureus in previous studies (12, 24).

Ldt_fm Is Also Specific for D-iAsn-substituted Acceptors in Vitro—The specificity of Ldt_fm was analyzed by incubating the enzyme with three disaccharide-tetrapeptides obtained by digestion of peptidoglycan of three different chemotypes with muramidases. The substrates were representative of the main variations found at the third position of peptidoglycan precursors of Gram-positive bacteria including the absence of a side chain (mesoDAP³ in B. subtilis) and presence of a side chain consisting of D and L amino acids (L-Lys³-D-iAsn in E. faecium and L-Lys³-L-Ala-L-Ala in E. faecalis) (Fig. 1A). With these three substrates, the cross-linking reaction can potentially lead to the formation of nine dimers, including three homodimers, if the same disaccharide-peptide is used as the donor and the acceptor substrate, and six heterodimers, if different disaccharide-peptides are used in all possible combinations (Table 1). Mass spectrometry analyses of the reaction products indicated that Ldt_fm catalyzed formation of a homodimer containing D-iAsn-substituted acceptor and donor stem peptides and of a heterodimer containing stem peptides substituted by L-Ala-L-Ala and D-iAsn (Fig. 3). Tandem mass spectrometry was performed to determine whether the L-Ala-L-Ala-substituted disaccharide-peptide had been used as a donor or an acceptor substrate in the formation of the heterodimer (Fig. 4). Fragmentation was performed on lactoyl-peptides obtained by cleavage of the disaccharide-peptides by alkaline treatment because amino acid sequencing of peptidoglycan dimers is more efficient in the absence of the disaccharide moiety of the molecules (24). This treatment also converts D-iAsn into D-iAsp (24). As detailed in Fig. 4, fragmentation of the heterodimer allowed assigning L-Ala-L-Ala to the free side chain of the donor stem and D-iAsp to the cross-link. Thus, Ldt_fm tolerated presence of L-Ala in the donor but not in the acceptor substrate of the cross-linking reaction. The specificity of Ldt_fm observed in vitro in the absence of any other cell wall biosynthesis enzyme accounts for the in vivo selection of the acceptor and donor substrates in the derivative E. faecium M512 producing the BppA1 transferase (above).

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Diversity of the structure of peptidoglycan cross-links and their mode of synthesis in Gram-positive bacteria. A, structure of peptidoglycan subunits from E. faecium, E. faecalis, and B. subtilis. The disaccharide-peptide subunit contains -1–4 linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). The D-lactoyl moiety of MurNAc is substituted by a stem pentapeptide containing L-Lys3 in E. faecium and E. faecalis or meso-diaminopimelic acid (mesoDAP3) in B. subtilis and E. coli. The side chain amino group of L-Lys3 is substituted by a D-iso-asparaginyl or D-iso-aspartyl residue (D-iAsx) in E. faecium and by L-Ala-L-Ala in E. faecalis. The amino group of the acyl acceptor participating in the formation of the cross-links is indicated by an arrow. B, schematic representation of muropeptide dimers generated by D,D- and L,D-transpeptidation. The acceptor stem in the represented dimers is a tripeptide ending in L-Lys3. Additional residues can be found at this position (D-Ala4, Gly4, D-Ala4-D-Ala5) (9). C, domain composition of L,D-transpeptidases from E. faecium (Ldtfm), E. faecalis (Ldtfs), and B. subtilis (LdtBs). Hatched boxes represent hydrophobic regions that could act as membrane anchors in Ldtfm and Ldtfs (35). LdtBs comprises a LysM peptidoglycan binding module linked to the catalytic domain (domain II) (21). For each L,D-transpeptidase, the portion of the proteins that has been produced in E. coli is indicated by a thick line terminated by the sequence of the affinity tag (one-letter code).

View larger version (47K): [in this window] [in a new window]   FIGURE 2. Peptidoglycan composition of E. faecium M512 producing BppA1 from E. faecalis. A, RP-HPLC profile of muropeptides from E. faecium M512/pJEH6(bppA1). Bacteria were grown in the presence of spectinomycin (120 µg/ml) to counter select loss of plasmid pJEH6(bppA1) and of isopropyl--D-thiogalactopyranoside (0.3 mM) to induce the bppA1 gene encoding a transferase of the Fem family for incorporation of L-Ala into the side chain of peptidoglycan precursors. Peptidoglycan was extracted with SDS and treated with ammonium hydroxide, and the resulting lactoyl-peptides were separated by RP-HPLC. mAU, absorbance unit x 103 at 210 nm. B, structure of muropeptides. The relative abundance (%) of the material in peaks 1 to 5 was calculated by integration of the absorbance at 210 nm. The retention time (RT) is indicated for minor muropeptides that could not be assigned to specific peaks because of their low abundance. The structure of lactoyl-peptides was deduced from the observed monoisotopic mass (Mass) and confirmed by tandem mass spectrometry for most monomers and dimers (indicated by a star). The observed and calculated mass differed at the maximum by 0.2. The stem peptide of monomers and the acceptor stem of dimers consisted of the tripeptide L-Ala1-D-iGln2-L-Lys3 (Tri), the tetrapeptide L-Ala1-D-iGln2-L-Lys3-D-Ala4 (Tetra), and the pentapeptide L-Ala1-D-iGln2-L-Lys3-D-Ala4-D-Ala5 (Penta). In certain muropeptides of low abundance, the C-terminal D-Ala4 was replaced by a glycyl residue (Gly C-ter). Cross-links generated by D,D-transpeptidases (DD) contained D-iAsp or L-Ala (D-Ala4D-iAsp-L-Lys3 and D-Ala4L-Ala-L-Lys3). Cross-links generated by L,D-transpeptidation (LD) exclusively contained D-iAsp (L-Lys3D-iAsp-L-Lys3).

The Ldt_fm Homologue from B. subtilis Is Specific for mesoDAP-containing Acceptors but Functions with Various Donors—Having shown that Ldt_fm is specific for its cognate acceptor both in vivo and in vitro, our following aim has been to determine whether Ldt_fm homologues from bacteria producing peptidoglycan of different chemotypes are also specific for their respective acceptor. We have started this analysis with the homologue from B. subtilis, Ldt_Bs, because the crystal structure of this protein has been recently solved in the framework of a structural genomics project that did not include functional investigations (21). The full-length Ldt_Bs (167 residues) contains a putative peptidoglycan-binding N-terminal domain consisting of a single LysM module (residues 5–49, Pfam PF01476) and a C-terminal domain that can be superimposed to the catalytic domain of Ldt_fm with a root mean square deviation of 1.21 Å for the 103 C atoms in common (35). The purified protein produced in E. coli catalyzed formation of homodimers from disaccharide-tetrapeptides containing mesoDAP at the third position of the stem peptides (Fig. 5A). Thus, Ldt_fm and Ldt_Bs both displayed L,D-transpeptidase activity on muropeptides despite limited sequence identity (23% for the catalytic domain) and different domain compositions (Fig. 1C). Ldt_Bs also catalyzed formation of heterodimers containing mesoDAP³ in one stem and L-Lys³-D-iAsn or L-Lys³-L-Ala-L-Ala in the other stem (Fig. 5A). Tandem mass spectrometry indicated that stems containing L-Lys³-D-iAsn and L-Lys³-L-Ala-L-Ala were present in the donor position of the dimers (Fig. 5B). Thus, Ldt_Bs was specific for its cognate mesoDAP-containing acceptor but tolerated variations in the structure of the donor substrate.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Analysis of the products of the cross-linking reaction catalyzed by Ldtfm in vitro. Three reduced disaccharide-tetrapeptides containing mesoDAP3, L-Lys3-D-iAsn, or L-Lys3-L-Ala-L-Ala were incubated with Ldtfm, and the products of the cross-linking reaction were analyzed by nanoelectrospray mass spectrometry. Peaks at m/z 942.38, 1,011.41, and 1,039.46 were assigned to be the [M+H]+ ions of the substrates (reduced disaccharide-tetrapeptides containing mesoDAP3, L-Lys3-D-iAsn, or L-Lys3-L-Ala-L-Ala, respectively). Peaks at m/z 966.93, 977.89, and 985.88 were assigned to be the [M+2H]2+, [M+H+Na]2+, and [M+H+K]2+ ions of the homodimer containing L-Lys3-D-iAsn in the donor and acceptor stems as the deduced monoisotopic mass (1,931.85) matched the calculated mass (1,931.91; Table 1). The monoisotopic mass of 1,959.82, deduced from peaks at m/z 980.93 [M+2H]2+, 991.91 [M+H+Na]2+, 999.89 [M+H+K]2+, matched the calculated monoisotopic mass of a heterodimer containing L-Lys3-D-iAsn and L-Lys3-L-Ala-L-Ala (1,959.94). Minor peaks at m/z 964.74 and 974.06 were assigned to be the [M+2H+K]3+ ions of trimers containing three L-Lys3-D-iAsn (calculated monoisotopic mass of 2,853.33) or two L-Lys3-D-iAsn and one L-Lys3-L-Ala-L-Ala (2,881.36), respectively.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Structure of the heterodimer formed by Ldtfm. Fragmentation was performed on the ion at m/z 1,145.59 obtained by ammonium hydroxide treatment of the heterodimer with a monoisotopic mass of 1,959.82 (Table 1). Boxes indicate ions generated by cleavage at single peptide bonds as indicated in the structure of the dimer. Ions at m/z 604.23 and 1,003.51 labeled with arrows establish that D-iAsp is present in the cross-link whereas L-Ala-L-Ala is located in the free side chain of the donor stem. The other peaks could correspond to additional loss of NH3 and CO plus NH3 and to combinations of fragmentations at two peptide bonds, as previously described (24). D-Lac, D-lactoyl.

Ldt_fs Displays L,D-Carboxypeptidase Activity—MS analysis of the product of the L,D-transpeptidation reaction catalyzed by Ldt_fs revealed the presence of an additional product (observed monoisotopic mass of 1,916.81) differing from the expected dimer (1,987.97; above) by the loss of one alanyl residue (Fig. 6A). Tandem mass spectrometry indicated that this additional product was a dimer generated by L,D-transpeptidation that lacked D-Ala⁴ in the acceptor stem (data not shown). The L,D-carboxypeptidase activity of Ldt_fs could have cleaved the L-Lys³-D-Ala⁴ peptide bond in the acceptor stem of the dimer. In addition or alternatively, Ldt_fs could have cleaved the substrate prior to its utilization as an acceptor in the cross-linking reaction because a monomer containing a tripeptide stem ending in L-Lys³ was also detected in the reaction mixture. The latter observation indicated that the L,D-transpeptidase and L,D-carboxypeptidase activities of Ldt_fs acted in competition because tripeptide stems cannot be used as a donor substrate. In contrast, L,D-carboxypeptidase activity was not detected for Ldt_fm from E. faecium (18) and Ldt_Bs from B. subtilis (data not shown). L,D-carboxypeptidases specific for the L-Lys³-D-Ala⁴ or mesoDAP³-D-Ala⁴ bond of peptidoglycan precursors have been described in E. coli (36), Pseudomonas aeruginosa (37), and Lactococcus lactis (38). These enzymes are unrelated to Ldt_fm and were only shown to have a hydrolytic activity.

View larger version (37K): [in this window] [in a new window]   FIGURE 5. Analysis of the dimers formed by LdtBs. A, MS analysis of the products of the cross-linking reaction catalyzed by LdtBs. Three reduced disaccharide-tetrapeptides containing mesoDAP3, L-Lys3-D-iAsn, or L-Lys3-L-Ala-L-Ala were incubated with LdtBs, and the products of the reaction were analyzed by nanoelectrospray MS. Peaks at m/z 942.41, 964.39, and 980.36 were assigned to be the [M+H]+,[M+Na]+, and [M+K]+ ions of the substrate containing mesoDAP3, respectively. Peaks at m/z 897.92, 908.90, 916.89, 919.88, 927.88, and 935.86 were assigned to be the [M+H+H]2+, [M+H+Na]2+, [M+H+K]2+, [M+Na+Na]2+,[M+Na+K]2+, and [M+K+K]2+ ions of the homodimer containing mesoDAP3 in the donor and acceptor stems (calculated monoisotopic mass of 1,793.77; Table 1). The monoisotopic mass of 1,862.68 deduced from peaks at m/z 932.44 [M+2H]2+, 943.43 [M+H+Na]2+, 951.42 [M+H+K]2+, 954.43 [M+Na+Na]2+, and 962.41 [M+Na+K]2+ matched the calculated monoisotopic mass of a heterodimer containing mesoDAP3 and L-Lys3-D-iAsn (1,862.84). The monoisotopic mass of 1,890.67 deduced from peaks at m/z 946.45 [M+2H]2+, 957.45 [M+H+Na]2+, and 965.43 [M+H+K]2+ matched the calculated monoisotopic mass of a heterodimer containing mesoDAP3 and L-Lys3-L-Ala-L-Ala (1,890.87). B, fragmentation of dimers obtained by ammonium hydroxide treatment. The peaks generated by the cleavage of a single peptide bond are boxed. Arrows indicate peaks used to assign stem peptides to the acceptor and donor positions of heterodimers.

Exchange Reactions Catalyzed by Ldt_fs—The L,D-transpeptidase of E. faecium has been previously shown to cleave the L-Lys³-D-Ala⁴ peptide bond of an acyl donor substrate and to form a peptide bond between the carboxyl of L-Lys³ and free D-amino acids (18). Dipeptides were tested in a similar exchange reaction catalyzed by Ldt_fs because this enzyme catalyzed formation of tripeptide and D-Ala-D-Ala from pentapeptide (above). Ldt_fs used the cognate disaccharide-tetrapeptide and D-Ala-D-Ala as acyl donor and acceptor, respectively, leading to the formation of the corresponding pentapeptide (Fig. 6C). Strikingly, L-Ala-L-Ala was not used as an acyl acceptor although the dipeptide mimics the side chain of the acceptor of the cross-linking reaction. This observation strongly suggests that the dipeptide D-Ala-D-Ala and the L-Ala-L-Ala side chain of muropeptides occupy different subsites in the catalytic cavity of the enzyme. The dipeptide D-Ala-D-Ala may occupy the position of the leaving group of the donor substrate. This would account for the specificity of Ldt_fs for D-Ala-D-Ala in the exchange reaction. A distinct subsite may accommodate the L-Ala-L-Ala side chain of the peptidoglycan precursors. Because the dipeptide L-Ala-L-Ala was not a substrate of Ldt_fs, recognition of the acceptor of the cross-linking reaction appears to involve a portion of the molecule larger than the L-Ala-L-Ala moiety of the molecule. The existence of two subsites in the catalytic cavity of the L,D-transpeptidases is supported by the structure of Ldt_fm from E. faecium (35). The catalytic domain of this enzyme contains two access paths to the putative active cysteine residue that could correspond to the binding sites of the acceptor and donor substrates (35).

View larger version (34K): [in this window] [in a new window]   FIGURE 6. Diversity of the reactions catalyzed by Ldtfs in vitro. A, products generated from a disaccharide-tetrapeptide by the L,D-carboxypeptidase and the L,D-transpeptidase activities of Ldtfs alone or in combination. B, products generated from a disaccharide-pentapeptide by the L,D-endopeptidase and L,D-transpeptidase activities of Ldtfs. C, use of the dipeptide D-Ala-D-Ala as an acyl acceptor to form a pentapeptide from a tetrapeptide.

	FOOTNOTES

¹ To whom correspondence should be addressed: INSERM U655-LRMA, Université Pierre et Marie Curie Centre de Recherches Biomédicales des Cordeliers, 15 Rue de l'Ecole de Médecine, 75270 Paris, Cedex 06, France. Tel.: 33-1-43-25-00-33; Fax: 33-1-43-25-68-12; E-mail: michel.arthur{at}bhdc.jussieu.fr.

² The abbreviations used are: GlcNAc, N-acetylglucosamine; PBP, penicillin-binding protein; D-iAsn, D-iso-asparagine; D-iAsp, D-iso-aspartic acid; mesoDAP, meso-diaminopimelic acid; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MurNAc, N-acetylmuramic acid; RP-HPLC, reverse phase high pressure liquid chromatography.

	ACKNOWLEDGMENTS

 
We thank L. Gutmann for helpful comments on the manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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