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