A Novel Peptidoglycan Cross-linking Enzyme for a β-Lactam-resistant Transpeptidation Pathway

Abstract

The β-lactam antibiotics remain the most commonly used to treat severe infections. Because of structural similarity between the β-lactam ring and the d-alanyl4-d-alanine5 extremity of bacterial cell wall precursors, the drugs act as suicide substrates of the dd-transpeptidases that catalyze the last cross-linking step of cell wall assembly. Here, we show that this mechanism of action can be defeated by a novel type of transpeptidase identified for the first time by reverse genetics in aβ-lactam-resistant mutant of Enterococcus faecium. The enzyme, Ldtfm, catalyzes in vitro the cross-linking of peptidoglycan subunits in a β-lactam-insensitive ld-transpeptidation reaction. The specificity of Ldtfm for the l-lysyl3-d-alanine4 peptide bond of tetrapeptide donors accounts for resistance because the substrate does not mimic β-lactams in contrast to d-alanyl4-d-alanine5 in the pentapeptide donors required for dd-transpeptidation. Ldtfm homologues are encountered sporadically among taxonomically distant bacteria, indicating that ld-transpeptidase-mediated resistance may emerge in various pathogens. The β-lactam antibiotics remain the most commonly used to treat severe infections. Because of structural similarity between the β-lactam ring and the d-alanyl4-d-alanine5 extremity of bacterial cell wall precursors, the drugs act as suicide substrates of the dd-transpeptidases that catalyze the last cross-linking step of cell wall assembly. Here, we show that this mechanism of action can be defeated by a novel type of transpeptidase identified for the first time by reverse genetics in aβ-lactam-resistant mutant of Enterococcus faecium. The enzyme, Ldtfm, catalyzes in vitro the cross-linking of peptidoglycan subunits in a β-lactam-insensitive ld-transpeptidation reaction. The specificity of Ldtfm for the l-lysyl3-d-alanine4 peptide bond of tetrapeptide donors accounts for resistance because the substrate does not mimic β-lactams in contrast to d-alanyl4-d-alanine5 in the pentapeptide donors required for dd-transpeptidation. Ldtfm homologues are encountered sporadically among taxonomically distant bacteria, indicating that ld-transpeptidase-mediated resistance may emerge in various pathogens. More than 60 years after the introduction of penicillin, the class of β-lactam antibiotics remains the most commonly used to treat severe infections despite the emergence of various resistance mechanisms, including drug detoxification by production of β-lactamases (1.Jacoby G.A. Munoz-Price L.S. N. Engl. J. Med. 2005; 352: 380-391Crossref PubMed Scopus (672) Google Scholar), decreased affinity of the target (2.Goffin C. Ghuysen J.M. Microbiol. Mol. Biol. Rev. 1998; 62: 1079-1093Crossref PubMed Google Scholar), and decreased permeability (3.Nikaido H. Science. 1994; 264: 382-388Crossref PubMed Scopus (1271) Google Scholar). The success story of β-lactams involves the development of several generations of β-lactams to defeat the resistance mechanisms and their association with inhibitors of β-lactamases (4.Walsh C. Nature. 2000; 406: 775-781Crossref PubMed Scopus (1199) Google Scholar, 5.Strynadka N.C. Jensen S.E. Johns K. Blanchard H. Page M. Matagne A. Frere J.M. James M.N. Nature. 1994; 368: 657-660Crossref PubMed Scopus (111) Google Scholar). All β-lactams have a common mechanism of action, as they share structural similarity with bacterial cell wall precursors. These drugs act as suicide substrates of the dd-transpeptidase catalytic domain of the penicillin-binding proteins (PBPs) 2The abbreviations used are: PBP, penicillin-binding protein; Ac2-l-Lys-d-Ala, Nα,Nϵ-diacetyl-l-lysyl-d-alanine; iAsn, isoasparaginyl; Ldtfm, ld-transpeptidase of Enterococcus faecium; MurNAc, N-acetylmuramic acid; HPLC, high pressure liquid chromatography; iGlu, isoglutamyl; iGln, isoglutaminyl. responsible for the last cross-linking step of cell wall assembly (Fig. 1A) (2.Goffin C. Ghuysen J.M. Microbiol. Mol. Biol. Rev. 1998; 62: 1079-1093Crossref PubMed Google Scholar, 6.Kelly J.A. Moews P.C. Knox J.R. Frere J.M. Ghuysen J.M. Science. 1982; 218: 479-481Crossref PubMed Scopus (64) Google Scholar, 7.Spratt B.G. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2999-3003Crossref PubMed Scopus (702) Google Scholar). Among the dd-transpeptidases of Enterococcus faecium, the low affinity PBP5 is unessential for growth but responsible for intrinsic low level resistance to ampicillin (8.Fontana R. Cerini R. Longoni P. Grossato A. Canepari P. J. Bacteriol. 1983; 155: 1343-1350Crossref PubMed Google Scholar, 9.Williamson R. Le Bouguénec C. Gutmann L. Horaud T. J. Gen. Microbiol. 1985; 131: 1933-1940PubMed Google Scholar), the first-line drug used at high dosage to treat severe enterococcal infections. In clinical isolates, higher levels of resistance to ampicillin are commonly associated with multiple amino acid substitutions in the transpeptidase domain of PBP5 (10.Klare I. Rodloff A.C. Wagner J. Witte W. Hakenbeck R. Antimicrob. Agents Chemother. 1992; 36: 783-78717Crossref PubMed Scopus (43) Google Scholar, 11.Rice L.B. Bellais S. Carias L.L. Hutton-Thomas R. Bonomo R.A. Caspers P. Page M.G. Gutmann L. Antimicrob. Agents Chemother. 2004; 48: 3028-3032Crossref PubMed Scopus (90) Google Scholar). We have shown previously that even higher levels of resistance to ampicillin can be achieved by a novel mechanism in the absence of PBP5 (12.Mainardi J.L. Legrand R. Arthur M. Schoot B. van Heijenoort J. Gutmann L. J. Biol. Chem. 2000; 275: 16490-16496Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Analysis of the peptidoglycan of the in vitro selected β-lactam-resistant mutant E. faecium M512 revealed that the d-Ala4→d-iAsn-l-Lys3 cross-link had been replaced by a novel type of cross-link between the l-Lys3 carboxyl group of a donor and the side-chain d-iAsn of an acceptor (12.Mainardi J.L. Legrand R. Arthur M. Schoot B. van Heijenoort J. Gutmann L. J. Biol. Chem. 2000; 275: 16490-16496Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) (Fig. 1B). In the presence of ampicillin, the novel l-Lys3→d-iAsn-l-Lys3 cross-links were exclusively found in the peptidoglycan of E. faecium M512, indicating that the dd-transpeptidase activity of the PBPs did not participate in the formation of the cross-links. Selection of E. faecium M512 from E. faecium D344S in five consecutive steps on increasing concentrations of ampicillin led to the gradual activation of the ld-transpeptidation pathway because the proportion of l-Lys3→d-iAsn-l-Lys3 was 3.1% for D344S and increased at each selection step (13.Mainardi J.L. Morel V. Fourgeaud M. Cremniter J. Blanot D. Legrand R. Frehel C. Arthur M. van Heijenoort J. Gutmann L. J. Biol. Chem. 2002; 277: 35801-35807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Activation of the resistance pathway was associated with the production of a dd-carboxypeptidase that generated precursors containing a tetrapeptide stem in the cytoplasm of E. faecium M512 (13.Mainardi J.L. Morel V. Fourgeaud M. Cremniter J. Blanot D. Legrand R. Frehel C. Arthur M. van Heijenoort J. Gutmann L. J. Biol. Chem. 2002; 277: 35801-35807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Based on these observations, we have speculated that precursors containing a tetrapeptide stem are essential for the formation of the l-Lys3→d-iAsn-l-Lys3 cross-links. In the present work, we have identified in E. faecium an ld-transpeptidase as the key enzyme of this alternate transpeptidation pathway and have shown that its substrate does not bear structural similarity to β-lactams. The bypass mechanism could invalidate the β-lactams as the major class of antibiotics. Purification of the ld-Transpeptidase and N-terminal Sequencing— The ld-transpeptidase from E. faecium (Ldtfm) was partially purified in four chromatographic steps using a radioactive exchange assay (13.Mainardi J.L. Morel V. Fourgeaud M. Cremniter J. Blanot D. Legrand R. Frehel C. Arthur M. van Heijenoort J. Gutmann L. J. Biol. Chem. 2002; 277: 35801-35807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) to detect active fractions. Briefly, E. faecium M512 (12.Mainardi J.L. Legrand R. Arthur M. Schoot B. van Heijenoort J. Gutmann L. J. Biol. Chem. 2000; 275: 16490-16496Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) was grown to an A650 of 0.7 in 24 liters of brain heart infusion broth (Difco), harvested by centrifugation, and washed twice in 10 mm sodium phosphate (pH 7.0). Bacteria were disrupted with glass beads in a cell disintegrator (The Mickle Laboratory Engineering Co., Gromshall, UK) for 2 h at 4 °C. The extract was centrifuged (5,000 × g for 10 min at 4 °C) to remove cell debris, and the supernatant was ultracentrifuged at 100,000 × g for 30 min at 4 °C. Soluble proteins (1 g) were loaded onto an anion exchange column (Hi-Load™ 26/10 Q-Sepharose™ HP, Amersham Biosciences) equilibrated with 25 mm sodium cacodylate buffer (pH 7.86) (buffer A). Elution was performed with a linear 0–2 m NaCl gradient in buffer A. Active fractions were pooled (30 mg of proteins), concentrated by ultrafiltration (Centricon YM10, Millipore, Saint-Quentin-en-Yve-lines, France), and loaded onto a gel filtration column (Superdex 75 HR26/60, Amersham Biosciences) equilibrated with buffer A containing 0.3 m NaCl. Active fractions (1 mg of proteins) were loaded onto a weak anion exchange column (HiTrap™ DEAE fast flow™ 1 ml, Amersham Biosciences) equilibrated with buffer A. Proteins (300 μg) eluting between 0.2 and 0.3 m NaCl were concentrated by ultrafiltration (Amicon ultra-4, Millipore) and loaded onto a gel filtration column (Superdex 200 PC 3.2/30, Amersham Biosciences) equilibrated with buffer A containing 0.3 m NaCl. Active fractions (70 μg of proteins) were concentrated (Amicon ultra-4) and analyzed by SDS-PAGE revealing a major 48-kDa protein band that was transferred onto a polyvinylidene difluoride membrane (Problott, Applied Biosystems, Framingham, MA) by passive adsorption (14.Messer M. Griffiths M. Rismiller P.D. Shaw D.C. Comp. Biochem. Physiol. B. 1997; 118: 403-410Crossref PubMed Scopus (43) Google Scholar). N-terminal Edman sequencing was performed on an Applied Biosystems Procise 494HT instrument with reagents and methods recommended by the manufacturer. The open reading frame for the ld-transpeptidase was identified by similarity searches between the N-terminal sequence of the 48-kDa protein (AEKQEIDPVSQNHQKLDTTV) and the partial genome sequence of E. faecium using the software tBLAST at the National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov/). The partially purified 48-kDa protein corresponded to a proteolytic fragment because the sequence encoding its N terminus was not preceded by a translation initiation codon. The upstream sequence contained a single likely translation initiation site (ACTTAAggagTTGTCGATatg), consisting of an ATG codon preceded by a putative ribosome binding site (lowercase). Production of the ld-Transpeptidase in E. coli and Purification of the Protein—A portion of the ldtfm open reading frame of E. faecium M512 was amplified with the primers 5′-TTCCATGGCAGAAAAACAAGAAATAGATCC-3′ and 5′-TTGGATCCGAAGACCAATACAGGCG-3′. The PCR product digested with NcoI and BamHI (underlined) was cloned into pET2818, a derivative of pET2816 (15.Chastanet A. Fert J. Msadek T. Mol. Microbiol. 2003; 47: 1061-1073Crossref PubMed Scopus (112) Google Scholar) lacking the sequence specifying the thrombin cleavage site (our laboratory collection). The resulting plasmid, pET2818Ωldtfm, was introduced into E. coli BL21(DE3) harboring pREP4GroESL (16.Amrein K.E. Takacs B. Stieger M. Molnos J. Flint N.A. Burn P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1048-1052Crossref PubMed Scopus (150) Google Scholar), and bacteria were grown at 37 °C to an A650 of 0.8 in three liters of brain heart infusion broth 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. Ldtfm was purified from a clarified lysate by affinity chromatography on Ni2+-nitrilotriacetate-agarose resin (Qiagen GmbH, Hilden, Germany) followed by anion exchange chromatography (MonoQ HR5/5, Amersham Biosciences) with a NaCl gradient in Tris-HCl, pH 7.5. An additional gel filtration was performed on a Superdex HR10/30 column (Amersham Biosciences) equilibrated with 50 mm Tris-HCl (pH 7.5) containing 300 mm NaCl at a flow rate of 0.5 ml/min. The overall yield was 3 mg/liter of culture. Site-directed mutagenesis was performed according to the QuikChange procedure of Stratagene (La Jolla, CA). Substrates of the ld-Transpeptidase—The dipeptide Nα,Nϵ-diacetyl-l-lysyl-d-alanine (Ac2-l-Lys-d-Ala) was prepared as described previously (13.Mainardi J.L. Morel V. Fourgeaud M. Cremniter J. Blanot D. Legrand R. Frehel C. Arthur M. van Heijenoort J. Gutmann L. J. Biol. Chem. 2002; 277: 35801-35807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Nα,Nϵ-diacetyl-l-lysine-d-alanyl-d-alanine (Ac2-l-Lys-d-Ala-d-Ala), l-Ala-d-iGlu-l-Lys-d-Ala-d-Ala (pentapeptide), and amino acids were purchased from Sigma. d-2-Hydroxy acids were obtained from Acros Organics (Noisy-Le-Grand, France). UDP-N-acetylmuramyl-l-Ala-d-iGlu-l-Lys-d-Ala-d-Ala (UDP-MurNAc-pentapeptide) was prepared from Staphylococcus aureus (17.Billot-Klein D. Shlaes D. Bryant D. Bell D. Legrand R. Gutmann L. van Heijenoort J. J. Bacteriol. 1997; 179: 4684-4688Crossref PubMed Google Scholar). The R39 dd-carboxypeptidase (18.Billot-Klein D. Gutmann L. Collatz E. van Heijenoort J. Antimicrob. Agents Chemother. 1992; 36: 1487-1490Crossref PubMed Scopus (34) Google Scholar) was used to generate UDP-MurNAc-l-Ala-d-iGlu-l-Lys-d-Ala (UDP-MurNAc-tetrapeptide) and l-Ala-d-iGlu-l-Lys-d-Ala (tetrapeptide) from UDP-MurNAc pentapeptide and pentapeptide, respectively. Disaccharide peptide fragments of the peptidoglycan (muropeptides) of Enterococcus gallinarum were prepared by scaling up a previously procedure published previously (19.Grohs P. Gutmann L. Legrand R. Schoot B. Mainardi J.L. J. Bacteriol. 2000; 182: 6228-6232Crossref PubMed Scopus (34) Google Scholar) including GlcNAc-MurNAc-l-Ala-d-iGln-l-(Nϵ-d-iAsn)Lys-d-Ala-d-Ala (GlcNAc-MurNAc-pentapeptide-iAsn), GlcNAc-MurNAc-l-Ala-d-iGln-l-(Nϵ-d-iAsn)Lys-d-Ala (GlcNAc-MurNAc-tetrapeptide-iAsn), GlcNAc-MurNAc-l-Ala-d-iGln-l-(Nϵ-d-iAsn)Lys (GlcNAc-MurNAc tripeptide-iAsn), and GlcNAc-MurNAc-l-Ala-d-iGln-l-Lys-d-Ala (GlcNAc-MurNAc-tetrapeptide). The concentration of muropeptides was estimated after acidic hydrolysis (20.Mengin-Lecreulx D. Falla T. Blanot D. van Heijenoort J. Adams D.J. Chopra I. J. Bacteriol. 1999; 181: 5909-5914Crossref PubMed Google Scholar). The structure of the different substrates was confirmed by mass spectrometry and tandem mass spectrometry as described previously (21.Arbeloa A. Hugonnet J.E. Sentilhes A.C. Josseaume N. Dubost L. Monsempes C. Blanot D. Brouard J.P. Arthur M. J. Biol. Chem. 2004; 279: 41546-41556Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). ld-Transpeptidase Assays—The exchange reaction assays for the capacity of the enzyme to catalyze cleavage of the l-Lys-d-Ala peptide bond of the model donor dipeptide substrate Ac2-l-Lys-d-Ala and formation of a peptide bond between Ac2-l-Lys and d-[14C]Ala (13.Mainardi J.L. Morel V. Fourgeaud M. Cremniter J. Blanot D. Legrand R. Frehel C. Arthur M. van Heijenoort J. Gutmann L. J. Biol. Chem. 2002; 277: 35801-35807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 22.Coyette J. Perkins H.R. Polacheck I. Shockman G.D. Ghuysen J.M. Eur. J. Biochem. 1974; 44: 459-468Crossref PubMed Scopus (29) Google Scholar). The standard assay (50 μl) contained Ac2-l-Lys-d-Ala (5 mm), d-[14C]Ala (0.15 mm; 2.0 GBq/mmol, ICN Pharmaceuticals, Orsay, France), 10 mm sodium cacodylate buffer (pH 6.0), and 0.1% Triton X-100 (v/v). The reaction products were separated by reverse phase HPLC and detected by scintillation with a radioflow detector (LB508, PerkinElmer Life Sciences) coupled to the HPLC device (13.Mainardi J.L. Morel V. Fourgeaud M. Cremniter J. Blanot D. Legrand R. Frehel C. Arthur M. van Heijenoort J. Gutmann L. J. Biol. Chem. 2002; 277: 35801-35807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To assay for inhibition by ampicillin, Ldtfm (3 μg) was incubated with Ac2-l-Lys-d-Ala (0.3 mm), d-[14C]Ala (0.15 mm), and various concentrations of the drug. To different 3 μg of Ldtfm were incubated for 60 min in the that Ac2-l-Lys-d-Ala was replaced by mm), mm), tetrapeptide mm), pentapeptide mm), (1 mm), and (1 To assay for in vitro the ld-transpeptidase (3 μg) was incubated with the muropeptides GlcNAc-MurNAc-l-Ala-d-iGln-l-(Nϵ-d-iAsn)Lys-d-Ala GlcNAc-MurNAc-l-Ala-d-iGln-l-(Nϵ-d-iAsn)Lys (5 and GlcNAc-MurNAc-l-Ala-d-iGln-l-Lys-d-Ala (5 for 2 h at in 25 of mm sodium phosphate buffer (pH The reaction was by the for 3 and the was centrifuged × 2 The formation of was by mass spectrometry on a tandem mass spectrometry the of the reaction was with to the to (21.Arbeloa A. Hugonnet J.E. Sentilhes A.C. Josseaume N. Dubost L. Monsempes C. Blanot D. Brouard J.P. Arthur M. J. Biol. Chem. 2004; 279: 41546-41556Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The for of the resulting with as the were as described previously (21.Arbeloa A. Hugonnet J.E. Sentilhes A.C. Josseaume N. Dubost L. Monsempes C. Blanot D. Brouard J.P. Arthur M. J. Biol. Chem. 2004; 279: 41546-41556Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). of the ld-Transpeptidase of E. faecium M512 We identified the encoding the ld-transpeptidase for the formation of the l-Lys3→d-iAsn-l-Lys3 responsible cross-links in E. faecium M512 by partial of the enzyme sequencing of its N and similarity searches in the partial genome sequence of E. faecium (Fig. A and The partially purified protein was a proteolytic fragment lacking the N-terminal including a putative membrane The portion of the open frame encoding the proteolytic fragment was in coli for protein (Fig. The protein was active in an exchange assay (Fig. and was not by ampicillin (Fig. indicating that the encoding the ld-transpeptidase of E. faecium M512 (Ldtfm) had been of into the activity of Ldtfm, various and acids were as acceptor substrates in an exchange reaction using the model dipeptide substrate Ac2-l-Lys-d-Ala as the donor (Fig. and of with and acids revealed that Ldtfm can catalyze formation of in to peptide containing a as and acid were used as in the transpeptidation and of the of Ac2-l-Lys-d-Ala was not detected in the presence of a acceptor indicating a for Ldtfm, in contrast to the previously in peptidoglycan in E. coli A. J. 1999; PubMed Scopus Google Scholar). Ldtfm was because product was detected was used as donor reaction by Ldtfm between Ac2-l-Lys-d-Ala and various not not in a The Ldtfm specificity for peptide donors was with the exchange assay using d-[14C]Ala as the of radioactive was not with Ac2-l-Lys-d-Ala (Fig. but with the peptidoglycan and with donors containing a tetrapeptide in In product was detected with and containing a pentapeptide in Ldtfm catalyzes peptidoglycan cross-linking exclusively with donors that are in by the β-lactam-insensitive dd-carboxypeptidase according to the pathway in the specificity of Ldtfm for a tetrapeptide donor in accounts for the of inhibition by β-lactams (Fig. because the drugs are structural of the extremity of the pentapeptide stem of peptidoglycan precursors. of in Ldtfm had the for a peptidoglycan cross-linking enzyme, we the formation of l-Lys3→d-iAsn-l-Lys3 cross-links with substrates the peptidoglycan precursors. substrates were prepared from the peptidoglycan of E. as of containing a tetrapeptide stem (19.Grohs P. Gutmann L. Legrand R. Schoot B. Mainardi J.L. J. Bacteriol. 2000; 182: 6228-6232Crossref PubMed Scopus (34) Google Scholar). was with a of three muropeptides to of donors and (Fig. of the reaction products revealed formation of with of donors and and of and in the four The containing an tetrapeptide stem was not used as an indicating that the side-chain is cross-links were not detected in the peptidoglycan of E. faecium M512 (12.Mainardi J.L. Legrand R. Arthur M. Schoot B. van Heijenoort J. Gutmann L. J. Biol. Chem. 2000; 275: 16490-16496Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). To the structure of the obtained in the reaction was up and with to the to are to sequencing by tandem mass spectrometry than (21.Arbeloa A. Hugonnet J.E. Sentilhes A.C. Josseaume N. Dubost L. Monsempes C. Blanot D. Brouard J.P. Arthur M. J. Biol. Chem. 2004; 279: 41546-41556Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). was found to d-iAsn into The (Fig. and the in vitro formation of l-Lys3→d-iAsn-l-Lys3 cross-links by formation not been obtained in the of purified dd-transpeptidase in substrates the from P. M. Frere J.M. R.A. Biochem. J. 2003; PubMed Google for a Ldtfm from the PBPs in its capacity to in a a that could be to for the of cross-linking of Ldtfm activity is present in from the E. faecium mutant M512 and from the D344S (13.Mainardi J.L. Morel V. Fourgeaud M. Cremniter J. Blanot D. Legrand R. Frehel C. Arthur M. van Heijenoort J. Gutmann L. J. Biol. Chem. 2002; 277: 35801-35807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). of from E. faecium D344S and M512 revealed of the of Ldtfm to the protein not The of the to that its sequence was in and in the E. faecium genome These that activation of the ld-transpeptidation pathway (Fig. does not of the activity of Ldtfm but that of the of the tetrapeptide donor substrate for the cross-linking reaction. The of the ld-transpeptidase in E. faecium is of the peptidoglycan structure in E. coli revealed that a proportion of the cross-links is generated by ld-transpeptidation the phase of growth D. J. Bacteriol. 1985; PubMed Google Scholar). An of their the phase was to a of peptidoglycan subunits containing the pentapeptide required for D. J. Bacteriol. 1985; PubMed Google Scholar). Because the peptidoglycan of E. faecium pentapeptide (12.Mainardi J.L. Legrand R. Arthur M. Schoot B. van Heijenoort J. Gutmann L. J. Biol. Chem. 2000; 275: 16490-16496Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), we that Ldtfm may have a in the of peptidoglycan structure because the enzyme can catalyze cross-links of of that Ldtfm is the first of a novel of proteins that is sporadically among taxonomically distant homologues (Fig. were detected in bacteria including and Enterococcus but not in S. aureus and similarity to the terminus of Ldtfm was detected in proteins of from and bacteria, but the and the domain of the proteins were domain is to from in the of the of the but of been and their putative in β-lactam resistance not been of the domain and present at and of Ldtfm (Fig. as catalytic Site-directed mutagenesis of Ldtfm led to an protein for the The mutant protein with the of the activity of the These that could be the catalytic of In the PBPs an essential active site is by their substrate and β-lactams. In Ldtfm is the first of a novel type of peptidoglycan The of Ldtfm homologues that β-lactam resistance by the ld-transpeptidase bypass mechanism can emerge in various The specificity of the Ldtfm for tetrapeptide substrates the of the β-lactam activity of the most used class of that previously to defeat β-lactam resistance mechanisms, in by of the to the β-lactam be to the development of ld-transpeptidase We D. Blanot for of Ac2-l-Lys-d-Ala, J. Cremniter and S. for the production of the Ldtfm and J. van R. and for reading of the