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Structural Characterization of Enpa D,L-Endopeptidase from Enterococcus Faecalis Prophage Provides Insights Into Substrate Specificity of M23 Peptidases

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Structural Characterization of Enpa D,L-Endopeptidase from Enterococcus Faecalis Prophage Provides Insights Into Substrate Specificity of M23 Peptidases International Journal of Molecular Sciences Article Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases Piotr Henryk Małecki 1,†, Paweł Mitkowski 1,†, Elzbieta˙ Jagielska 1, Karolina Trochimiak 1, Stéphane Mesnage 2 and Izabela Sabała 1,* 1 International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland; [email protected] (P.H.M.); [email protected] (P.M.); [email protected] (E.J.); [email protected] (K.T.) 2 Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK; s.mesnage@sheffield.ac.uk * Correspondence: [email protected] † These authors contributed equally to this work. 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 Citation: Małecki, P.H.; Mitkowski, EnpACD lyses bacterial cells live with very high efficiency demonstrating great bacteriolytic potential, P.; Jagielska, E.; Trochimiak, K.; though limited to a low ionic strength environment. We have solved the structure of the EnpACD Mesnage, S.; Sabała, I. Structural H109A inactive variant and analyzed it in the context of related peptidoglycan hydrolases structures Characterization of EnpA to reveal the bases for the specificity determination. All M23 structures share a very conserved D,L-Endopeptidase from Enterococcus β-sheet core which constitutes the rigid bottom of the substrate-binding groove and active site, faecalis Prophage Provides Insights while variable loops create the walls of the deep and narrow binding cleft. A detailed analysis of into Substrate Specificity of M23 the binding groove architecture, specificity of M23 enzymes and D,L peptidases demonstrates that Peptidases. Int. J. Mol. Sci. 2021, 22, the substrate groove, which is particularly deep and narrow, is accessible preferably for peptides 7136. https://doi.org/10.3390/ijms composed of amino acids with short side chains or subsequent L and D-isomers. As a result, the 22137136 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 Academic Editor: Alexande Baykov the substrates is based on their conformations allowed only for polyglycine chains and alternating Received: 19 May 2021 chirality of the amino acids. Accepted: 29 June 2021 Published: 1 July 2021 Keywords: peptidoglycan hydrolase; M23 peptidase; Enterococcus faecalis; endopeptidase Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Peptidoglycan (PG) is a major component of bacterial cell walls providing cell shape and resistance to the internal turgor pressure. Furthermore, it serves as a scaffold for the attachment of proteins and cell wall anionic polymers, such as teichoic acids. The PG network consists of repeating units of disaccharides, stem peptides and cross-bridges Copyright: © 2021 by the authors. (Figure1A). Stem peptides are made of both L- and D-amino acids, and their sequence is Licensee MDPI, Basel, Switzerland. usually conserved among the genera. They are cross-linked directly in most Gram-negative This article is an open access article and in some cases of Gram-positive bacteria such as Streptococcus oralis or Aerococcus distributed under the terms and viridans. In Gram-positive bacteria length and composition of the interpeptides vary; conditions of the Creative Commons e.g., in S. aureus they consist of five glycines, while in E. faecalis there are two L-alanine Attribution (CC BY) license (https:// residues [1]. creativecommons.org/licenses/by/ 4.0/). Int. J. Mol. Sci. 2021, 22, 7136. https://doi.org/10.3390/ijms22137136 https://www.mdpi.com/journal/ijms Int.Int. J. J.Mol. Mol. Sci. Sci. 20212021, 22, 22, x, FOR 7136 PEER REVIEW 2 of2 of 17 17 Figure 1. Schematic representations of E. faecalis peptidoglycan (A) and EnpA modular organization Figure 1. Schematic representations of E. faecalis peptidoglycan (A) and EnpA modular organiza- (B). (A) Cross-linked peptidoglycan monomers (black) and other elements of PG (gray); EnpACD tion (B). (A) Cross-linked peptidoglycan monomers (black) and other elements of PG (gray); cleavage site is marked with red dash arrow. (B) Domain organization of EnpA; numbers represent EnpACD cleavage site is marked with red dash arrow. (B) Domain organization of EnpA; numbers amino acid positions in a full-length protein. represent amino acid positions in a full-length protein. Most bacterial genomes encode a large number of hydrolytic enzymes that target virtu- Most bacterial genomes encode a large number of hydrolytic enzymes that target vir- ally every bond present in PG [2]. These enzymes are not only required for the remodeling tually every bond present in PG [2]. These enzymes are not only required for the remod- of normal cell walls during growth and division but also are used as weapons to compete eling of normal cell walls during growth and division but also are used as weapons to with other bacterial strains living in the same ecological niche. Peptidoglycan hydrolases competeare used with by bacteriophagesother bacterial tostrains enter living bacterial in the cells same and ecological to release theniche. progeny Peptidoglycan at the end hydrolasesof the phage are lifeused cycle. by bacteriophages Consequently, to they enter are bacterial acquiring cells increasing and to release attention the as progeny a poten- attial the weaponend of the against phage pathogenic life cycle. Consequent bacteria, particularlyly, they are thoseacquiring resistant increasing to antibiotics attention [3 as–9 ]. a Amongpotential peptidoglycan weapon against hydrolases pathogenic are bacteria endopeptidases, particularly belonging those resistant to the M23to antibiotics MEROPS [3–9].family Among [10], such peptidoglycan as lysostaphin hydrolases and LytM thatare cleaveendopeptidases pentaglycine belonging cross-bridges to the present M23 in MEROPSstaphylococcal family [10], PG. Memberssuch as lysostaphin of the M23 and family LytM are that metallopeptidases cleave pentaglycine containing cross-bridges a zinc presention in theirin staphylococcal active site, coordinated PG. Members by twoof the histidine M23 family and oneare metallopeptidases aspartic acid residues contain- in the ingconserved a zinc ion motifs: in their HXXXD active site, and coordinated HXH [11–15 by]. two Some histidine members and of one this aspartic family acid have resi- also duesshown in the to beconserved potent antimicrobial motifs: HXXXD agents and that HX couldH [11–15]. be applied Some to members eliminate of staphylococci, this family haveparticularly also shown antibiotic-resistant to be potent antimicrobial strains [3,4,16 agents,17]. that could be applied to eliminate staphylococci,Enzymatic particularly activity of antibiotic-resistant the M23 domain strains from E. [3,4,16,17]. faecalis protein called EnpA was re- portedEnzymatic previously activity [18 of]. theE. faecalis M23 domainis a Gram-positive from E. faecalis bacterium protein called inhabiting EnpA was the reported intestinal previouslytract of healthy [18]. E. humansfaecalis is anda Gram-positive animals. As bacterium an opportunistic inhabiting pathogen, the intestinal it can tract cause of healthy various humansinfectious and diseases, animals. suchAs an as opportunistic urinary infections, pathogen, bacteremia it can cause and various endocarditis infectious [19– 21diseases,]. EnpA suchis a as prophage urinary infections, encoded 1721 bacteremia residues and protein endocarditis composed [19–21]. of three EnpA domains is a prophage (Figure encoded1B); the 1721N-terminal residues domainprotein composed (1350 amino of three acids) doma sharesins (Figure homology 1B); withthe N- phageterminal tail domain tape measure (1350 aminoproteins. acids) The shares two homology domains located with phage towards tail tape the C-terminusmeasure proteins. of EnpA The are two homologous domains lo- to cateddomains towards belonging the C-terminus to PG hydrolases of EnpA are with ho lyticmologous transglycosylase to domains activitybelonging (cleaving to PG hydro- glycan laseschains) with andlytic M23transglycosylase peptidase activity activity (cleaving(cleaving glycan PG peptides). chains) and The M23 functional peptidase analysis activity of (cleavingthe EnpA PG catalytic peptides). domain The functional (EnpACD )analysis revealed of its the hydrolytic EnpA catalytic activity domain on the (EnpA D-Ala–L-AlaCD) re- vealedbond, its which hydrolytic is characteristic activity on the for E.D-Ala–L-Ala faecalis peptidoglycan, bond, which is and characteristic proposed thefor minimumE. faecalis Int. J. Mol. Sci. 2021, 22, 7136 3 of 17 substrate of EnpCD to consist of three residues of the donor stem and the amino acids of the cross-bridge [18]. Herein we report a crystal structure of M23 D,L-endopeptidase EnpACD, and discuss its place among other characterized M23 peptidases in terms of the structural–functional relationship.
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