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Updated: Oct 27, 2025
Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry
Published on: May 21, 2018
Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into
Piotr Henryk Małecki1, Paweł Mitkowski1, Elżbieta Jagielska1
1International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.
Abstract:
The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from Staphylococcus simulans or LytM from Staphylococcus aureus. Recently, enzymes with broad specificities were reported, such as EnpACD from Enterococcus faecalis, that cleaves D,L peptide bond between the stem peptide and a cross-bridge. Previously, the activity of EnpACD was demonstrated only on isolated peptidoglycan fragments. Herein we report conditions in which EnpACD lyses bacterial cells live with very high efficiency demonstrating great bacteriolytic potential, though limited to a low ionic strength environment. We have solved the structure of the EnpACD H109A inactive variant and analyzed it in the context of related peptidoglycan hydrolases structures to reveal the bases for the specificity determination. All M23 structures share a very conserved β-sheet core which constitutes the rigid bottom of the substrate-binding groove and active site, while variable loops create the walls of the deep and narrow binding cleft. A detailed analysis of the binding groove architecture, specificity of M23 enzymes and D,L peptidases demonstrates that the substrate groove, which is particularly deep and narrow, is accessible preferably for peptides composed of amino acids with short side chains or subsequent L and D-isomers. As a result, the bottom of the groove is involved in interactions with the main chain of the substrate while the side chains are protruding in one plane towards the groove opening. We concluded that the selectivity of the substrates is based on their conformations allowed only for polyglycine chains and alternating chirality of the amino acids.
Insights
EnpA_CD, an M23 family enzyme, efficiently lyses live bacterial cells under low ionic strength conditions. Its narrow substrate-binding groove explains specificity for polyglycine chains and alternating D,L-amino acids in peptidoglycan hydrolases.
Area of Science:
- Enzymology
- Structural Biology
- Microbiology
Background:
- The M23 family of metalloenzymes includes well-characterized glycyl-glycine hydrolases like lysostaphin and LytM.
- EnpA_CD from Enterococcus faecalis is a recently identified enzyme with broader specificity, cleaving D,L peptide bonds.
- Previous studies demonstrated EnpA_CD activity only on isolated peptidoglycan fragments.
Purpose of the Study:
- To investigate the conditions under which EnpA_CD exhibits efficient bacteriolytic activity on live bacterial cells.
- To elucidate the structural basis for EnpA_CD's substrate specificity.
- To compare EnpA_CD structure with other M23 peptidoglycan hydrolases.
Main Methods:
- Determined the crystal structure of the EnpA_CD H109A inactive variant.
- Analyzed the structure in the context of related peptidoglycan hydrolases.
- Performed detailed analysis of the substrate-binding groove architecture and its implications for enzyme specificity.
Main Results:
- EnpA_CD demonstrates high efficiency in lysing live bacterial cells under low ionic strength conditions, revealing significant bacteriolytic potential.
- The M23 enzyme family shares a conserved β-sheet core forming the base of the substrate-binding groove, with variable loops defining the cleft.
- The deep and narrow binding groove of EnpA_CD preferentially accommodates peptides with short side chains or alternating L and D-amino acids, interacting primarily with the peptide backbone.
Conclusions:
- EnpA_CD exhibits potent bacteriolytic activity on live cells in specific low ionic strength environments.
- The structural analysis reveals that EnpA_CD's substrate specificity is dictated by its narrow binding groove, favoring polyglycine chains and alternating D,L-amino acid configurations.
- This specificity mechanism is conserved among M23 peptidoglycan hydrolases, highlighting the importance of substrate conformation for enzyme function.
