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Article

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

1
International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland
2
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editor: Alexande Baykov
Int. J. Mol. Sci. 2021, 22(13), 7136; https://doi.org/10.3390/ijms22137136
Received: 19 May 2021 / Revised: 18 June 2021 / Accepted: 29 June 2021 / Published: 1 July 2021
(This article belongs to the Special Issue Molecular Enzymology: Advances and Applications)
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. View Full-Text
Keywords: peptidoglycan hydrolase; M23 peptidase; Enterococcus faecalis; endopeptidase peptidoglycan hydrolase; M23 peptidase; Enterococcus faecalis; endopeptidase
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MDPI and ACS Style

Małecki, P.H.; Mitkowski, P.; Jagielska, E.; Trochimiak, K.; Mesnage, S.; Sabała, I. Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases. Int. J. Mol. Sci. 2021, 22, 7136. https://doi.org/10.3390/ijms22137136

AMA Style

Małecki PH, Mitkowski P, Jagielska E, Trochimiak K, Mesnage S, Sabała I. Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases. International Journal of Molecular Sciences. 2021; 22(13):7136. https://doi.org/10.3390/ijms22137136

Chicago/Turabian Style

Małecki, Piotr H., Paweł Mitkowski, Elżbieta Jagielska, Karolina Trochimiak, Stéphane Mesnage, and Izabela Sabała. 2021. "Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases" International Journal of Molecular Sciences 22, no. 13: 7136. https://doi.org/10.3390/ijms22137136

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