Cleavage Specificity of Enterococcus faecalis EnpA (EF1473), a Peptidoglycan Endopeptidase Related to the LytM/Lysostaphin Family of Metallopeptidases.

Enterococcus faecalis EnpA (EF1473) is a 1721-residue predicted protein encoded by prophage 03 that displays similarity to the staphylolytic glycyl–glycyl endopeptidases lysostaphin and LytM. We purified a catalytically active fragment of the protein, EnpAC, comprising residues 1374–1505 and showed that the recombinant polypeptide efficiently cleaved cross-linked muropeptides generated by muramidases, but was poorly active in intact sacculi. Analysis of the products of digestion of purified dimers by mass spectrometry indicated that EnpAC cleaves the d-Ala-l-Ala bond formed by the d,d-transpeptidase activity of penicillin-binding proteins in the last cross-linking step of peptidoglycan synthesis. Synthetic d was identified as the minimum substrate of EnpAC indicating that interaction of the enzyme with the donor peptide stem of cross-linked dimers is sufficient for its activity. Peptidoglycan was purified from various bacterial species and digested with mutanolysin and EnpAC to assess enzyme specificity. EnpAC did not cleave direct cross-links, but tolerated extensive variation in cross-bridges with respect to both their length (one to five residues) and their amino acid sequence. Recognition of the donor stem of cross-linked dimers could account for the substrate specificity of EnpAC, which is significantly broader in comparison to endopeptidases belonging to the lysostaphin family.