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, EnpA(C), 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 EnpA(C) 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.