Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases

The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from Staphylococcus simulans or LytM aureus. Recently, enzymes with broad specificities were reported, EnpACD Enterococcus faecalis, that cleaves D,L peptide bond between stem and a cross-bridge. Previously, activity was demonstrated only on isolated peptidoglycan fragments. Herein we report conditions in which lyses bacterial cells live very high efficiency demonstrating great bacteriolytic potential, though limited to low ionic strength environment. We have solved structure H109A inactive variant analyzed it context related hydrolases structures reveal bases for specificity determination. All share conserved β-sheet core constitutes rigid bottom substrate-binding groove active site, while variable loops create walls deep narrow binding cleft. A detailed analysis architecture, peptidases demonstrates substrate groove, is particularly narrow, accessible preferably peptides composed amino acids short side chains subsequent L D-isomers. As result, involved interactions main chain protruding one plane towards opening. concluded selectivity substrates based their conformations allowed polyglycine alternating chirality acids.