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

Vanessa Delfosse,Ana Arbeloa,Arul Marie,Jean-Emmanuel Hugonnet,Louis B Rice,Martine Fourgeaud,Lionel Dubost,Claudine Mayer,Michel Arthur,Sophie Magnet,Jean-Luc Mainardi

doi:10.1074/jbc.m610911200

Abstract

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 beta-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 (mesoDAP(3)) and L-Lys(3)-L-Ala-L-Ala at the third position of the stem peptide, respectively, instead of L-Lys(3)-d-iAsn in E. faecium. Ldt(fs) differed from Ldt(fm) and Ldt(Bs) by its capacity to hydrolyze the L-Lys(3)-D-Ala(4) 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-Lys(3)-D-iAsn by L-Lys(3)-L-Ala-L-Ala. Likewise, Ldt(Bs) tolerated substitution of mesoDAP(3) by L-Lys(3)-D-iAsn and L-Lys(3)-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.

Highlights

We report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the L,Dtranspeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria
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 Ldtfm and by the penicillin-binding proteins (PBPs) [28]
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 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)

Summary

Introduction

We report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the L,Dtranspeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria. 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-Lys3D-iAsn in E. faecium.

Results

Conclusion