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

Piotr Henryk Małecki,Izabela Sabała,Karolina Trochimiak,Paweł Mitkowski,Stéphane Mesnage,Elżbieta Jagielska

doi:10.3390/ijms22137136

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.

Highlights

Introduction iationsPeptidoglycan (PG) is a major component of bacterial cell walls providing cell shape and resistance to the internal turgor pressure
All EnpACD molecules in the asymmetric unit (ASU) are similar with root-mean-square deviation (RMSD) values of 0.35 Å–0.65 Å and β-factors for chains A–E ranging from 87 to
We evaluated the bacteriolytic activity of EnpACD on 32 different bacterial strains of the diverse composition of peptidoglycans and other components of the cell walls (Table S2)

Summary

Introduction

Peptidoglycan (PG) is a major component of bacterial cell walls providing cell shape and resistance to the internal turgor pressure. It serves as a scaffold for the attachment of proteins and cell wall anionic polymers, such as teichoic acids. Stem peptides are made of both L- and D-amino acids, and their sequence is usually conserved among the genera. They are cross-linked directly in most Gram-negative and in some cases of Gram-positive bacteria such as Streptococcus oralis or Aerococcus viridans. In Gram-positive bacteria length and composition of the interpeptides vary;

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Discussion

Conclusion