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

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, Ldtfm 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 β-lactam antibiotics. Ldtfm homologues from Bacillus subtilis (LdtBs) and E. faecalis (Ldtfs) 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. Ldtfs differed from Ldtfm and LdtBs 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 Ldtfs, which was also specific for its cognate acyl donor, Ldtfm tolerated substitution of l-Lys3-d-iAsn by l-Lys3-l-Ala-l-Ala. Likewise, LdtBs 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.

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 crosslink glycan strands (2). The latter reaction is catalyzed by penicillin-binding proteins (PBPs) that cleave the D-Ala 4 -D-Ala 5 bond of a donor stem pentapeptide and link the carbonyl of D-Ala 4 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-Ala 4 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-Ala 4 -D-Ala 5 and act as suicide substrates of the D,D-transpeptidase module of the PBPs. The glycopeptides bind to the peptidyl-D-Ala 4 -D-Ala 5 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 * This work was supported by the program "ACI Microbiologie 2003" from the Fonds National de la Science (Grant ACIM-4-9), the European Community (COBRA, contract LSHM-CT-2003-503335, 6th PCRD), NIAID, National Institutes of Health (Grant R01 AI45626), and the Fondation pour la Recherche Mé dicale. 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  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-Ala 5 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,Dtranspeptidase (Ldt fm ) that cleaves the L-Lys 3 -D-Ala 4 peptide bond of the donor and links the carboxyl of L-Lys 3 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-Lys 3 -L-Ala instead of L-Lys 3 -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 crosslinking 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
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 5Ј-AACCATGGGGAGTATCCGTCGAGGCAATGG-3Ј and 5Ј-AAGGATCCTACTTCTTCGCCGTAATCTA-3Ј. 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, pET2818⍀ldt 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 g/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 Ni 2ϩnitrilotriacetate-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 ϳ300 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 Ϫ80°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 5Ј-AACCATGGGGCTGCTTACG-TACCAGGTGAAGC-3Ј and 5 Ј-TTGGATCCCCGGTTA-ATCGTGACTCTCGT-3Ј. 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 Ni 2ϩ -nitrilotriacetate-agarose resin (Qiagen), dialyzed against 50 mM Tris-HCl, pH 7.5, containing 100 mM NaCl, and stored at Ϫ20°C in the same buffer. 10 mg of protein were obtained per liter of culture.
L,D-Transpeptidase Assays-The source of the disaccharidepeptides used as substrates was as follows. The disaccharidetetrapeptide substituted by a D-iso-asparagine residue (L-Lys 3 -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-Lys 3 -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 (mesoDAP 3 ) 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 l of phosphate buffer (20 mM, pH 7.0) containing the L,Dtranspeptidase (7 M) from E. faecium (Ldt fm ), E. faecalis (Ldt fs ), or B. subtilis (Ldt Bs ) and a combination of three reduced disaccharide-tetrapeptides (200 M each) containing L-Lys 3 -D-iAsn, L-Lys 3 -L-Ala-L-Ala, or mesoDAP 3 at the third position of a tetrapeptide stem. The reaction was incubated for 2 h at 37°C, desalted using a micro column (ZipTipC 18 ; 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 (ZipTipC 18 , Millipore), and the resulting lactoyl-peptides were analyzed by nanoelectrospray MS/MS using N 2 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 l of 20 mM potassium phosphate buffer, pH 7.0, contained Ldt fs (7 M), the cognate disaccharide-tetrapeptide containing an L-Ala-L-Ala side chain as the acyl donor (200 M), and 1 mM L-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 g/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
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,Dtranspeptidation. 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 (mesoDAP 3 in B. subtilis) and presence of a side chain consisting of D and L amino acids (L-Lys 3 -D-iAsn in E. faecium and L-Lys 3 -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 disaccharidepeptides 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-iAsnsubstituted 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  (35). Ldt Bs 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). 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 crosslink. 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).

L,D-Transpeptidation in Gram-positive Bacteria
Of note, interaction of the D,Dtranspeptidases (PBPs) with their donor and acceptor substrates has not been extensively investigated with purified PBPs, because the enzymes are generally inactive in vitro (29) except in very special cases involving highly reactive substrates (e.g. thioester) (30) and atypical enzymes (e.g. the soluble R61 D,Dpeptidase from Streptomyces spp.) (31,32). Recently, a peptidoglycan polymerization assay has been developed for the purified PBP1a and 1b of E. coli as these bifunctional enzymes catalyze transglycosylation and transpeptidation of the natural substrate, a dissacharidepeptide linked to undecaprenyl lipid carrier by a phosphodiester bond (lipid II) (33,34). This approach has not been yet developed for the PBPs of Gram-positive bacteria that use even more complex precursors due to the presence of an additional side chain. Thus, characterization of Ldt fm in the current study represents the first direct in vitro assessment of the substrate specificity of peptidoglycan cross-linking enzyme.
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 . Bacteria were grown in the presence of spectinomycin (120 g/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 ϫ 10 3 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-Ala 1 -D-iGln 2 -L-Lys 3 (Tri), the tetrapeptide L-Ala 1 -D-iGln 2 -L-Lys 3 -D-Ala 4 (Tetra), and the pentapeptide L-Ala 1 -D-iGln 2 -L-Lys 3 -D-Ala 4 -D-Ala 5 (Penta). In certain muropeptides of low abundance, the C-terminal D-Ala 4 was replaced by a glycyl residue (Gly C-ter). Cross-links generated by D,D-transpeptidases (DD) contained D-iAsp or L-Ala (D-Ala 4 3D-iAsp-

TABLE 1 Calculated monoisotopic mass of the dimers generated by L,D-transpeptidation
Cross-linking of three reduced disaccharide-tetrapeptides containing L-Lys 3 -D-iAsn, mesoDAP 3 , and L-Lys 3 -L-Ala-L-Ala in all possible combinations can lead to a total of nine dimers, including three homodimers and six heterodimers. The calculated monoisotopic mass of the corresponding lactoyl-peptides is indicated in parentheses. Disaccharide-peptides and lactoyl-peptides contain D-iAsn and D-iAsp, respectively.   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 activ-ity 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 mesoDAP 3 in one stem and L-Lys 3 -D-iAsn or L-Lys 3 -L-Ala-L-Ala in the other stem (Fig. 5A). Tandem mass spectrometry indicated that stems containing L-Lys 3 -D-iAsn and L-Lys 3 -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.

L,D-Transpeptidation in Gram-positive Bacteria
Ldt fs from E. faecalis Is Specific for Disaccharide-Peptides Containing an L-Ala-L-Ala Side Chain in both the Acceptor and Donor Substrates-The chromosome of E. faecalis encodes a protein of 474 residues, designated Ldt fs , that is closely related to Ldt fm (37% identity for the catalytic domain). The two proteins display the same domain composition with an overall sequence identity of 29% (Fig. 1C). Ldt fs catalyzed formation of homodimers from the cognate branched disaccharide-tetrapeptide containing L-Lys 3 -L-Ala-L-Ala (observed monoisotopic mass of 1,987.96, see Table 1 for the calculated mass). Muropeptides containing mesoDAP 3 or L-Lys 3 -D-iAsn were not used for dimer formation indicating that Ldt fs is specific for the L-Ala-L-Ala side chain both in the donor and acceptor positions.
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,Dtranspeptidation that lacked D-Ala 4 in the acceptor stem (data not shown). The L,D-carboxypeptidase activity of Ldt fs could have cleaved the L-Lys 3 -D-Ala 4 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-Lys 3 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,Dcarboxypeptidase 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-Lys 3 -D-Ala 4 or mesoDAP 3 -D-Ala 4 bond of peptidoglycan precursors have been described in E. coli (36),     (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 NH 3 and CO plus NH 3 and to combinations of fragmentations at two peptide bonds, as previously described (24). D-Lac, D-lactoyl.
Pseudomonas aeruginosa (37), and Lactococcus lactis (38). These enzymes are unrelated to Ldt fm and were only shown to have a hydrolytic activity.
Muropeptides Containing a Pentapeptide Stem Are Substrates of Ldt fs -Replacement of the disaccharide-tetrapeptide substituted by L-Ala-L-Ala by the corresponding pentapeptide in the cross-linking assay led to the formation of hydrolysis and transpeptidation products (Fig. 6B). Ldt fs displayed endopeptidase activity because the enzyme cleaved the L-Lys 3 -D-Ala 4 peptide bond of the pentapeptide, gener-ating a tripeptide and the dipeptide D-Ala-D-Ala. Ldt fs also formed dimers containing a tripeptide stem and a pentapeptide stem at the donor and acceptor position, respectively. Combination of the L,D-transpeptidase and L,D-endopeptidase activity led to the formation of dimers containing two tripeptide stems. In contrast to Ldt fs , the L,D-transpeptidase from E. faecium functions exclusively with acyl donors containing a tetrapeptide stem (18).
Exchange Reactions Catalyzed by Ldt fs -The L,D-transpeptidase of E. faecium has been previously shown to cleave the L-Lys 3 -D-Ala 4 peptide bond of an acyl donor substrate and to form a peptide bond between the ␣ carboxyl of L-Lys 3 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).    MAY 4, 2007 • VOLUME 282 • NUMBER 18

JOURNAL OF BIOLOGICAL CHEMISTRY 13157
Conclusions-Bypass of the D,D-transpeptidase activity of the PBPs by the L,D-transpeptidase activity of Ldt fm confers high level cross-resistance to ␤-lactams and glycopeptides in E. faecium because these drugs do not interact with the enzyme and its substrate, respectively (9). Ldt fm is the first functionally characterized member of a novel family of cysteine peptidases that are widespread in both Gram-positive and Gram-negative bacteria (18,35). We have characterized two additional members of the family from B. subtilis (Ldt Bs ) and E. faecalis (Ldt fs ) and shown that these enzymes catalyze the cross-linking of purified peptidoglycan fragments in vitro. Comparison of the specificity of Ldt fm , Ldt Bs , and Ldt fs (Table 2) indicated that diversification of the structure of peptidoglycan precursors associated with speciation had led to a parallel evolution of the substrate specificity of members of the Ldt fm protein family. This evolution concerns mainly FIGURE 6. Diversity of the reactions catalyzed by Ldt fs in vitro. A, products generated from a disaccharide-tetrapeptide by the L,D-carboxypeptidase and the L,D-transpeptidase activities of Ldt fs alone or in combination. B, products generated from a disaccharide-pentapeptide by the L,D-endopeptidase and L,Dtranspeptidase activities of Ldt fs . C, use of the dipeptide D-Ala-D-Ala as an acyl acceptor to form a pentapeptide from a tetrapeptide. the acceptor because for this substrate all three L,Dtranspeptidases were specific for their cognate disaccharidepeptides. In contrast, Ldt fm and Ldt Bs tolerated substitutions at the third position of the donor. The specificity of the L,D-transpeptidases for the acceptor stem indicates that blocking the assembly of the side chain of peptidoglycan precursors is a potential strategy to combat resistance involving L,D-transpeptidases.