Specificity of L,D-transpeptidases from Gram-positive bacteria producing different peptidoglycan chemotypes

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, 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-Lys 3-D-iAsn in E. faecium. Ldtfs differed from Ldt fm and LdtBs by its capacity to hydrolyze the L-Lys 3-D-Ala4 bond of tetrapeptide (L,D-carboxypeptidase activity) and pentapeptide (L,D-endopeptidase activity) stems, in addition to the common crosslinking 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-Lys 3-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.