Biochemical characterization and validation of a catalytic site of a highly thermostable Ts2631 endolysin from the Thermus scotoductus phage vB-Tsc2631

Magdalena Plotka, Anna Karina Kaczorowska, Agnieszka Morzywolek, Joanna Makowska, Lukasz P. Kozlowski, Audur Thorisdottir, Sigurlaug Skírnisdottir, Sigridur Hjörleifsdottir, Olafur H. Fridjonsson, Gudmundur O. Hreggvidsson, Jakob K. Kristjansson, Slawomir Dabrowski, Janusz M. Bujnicki, Tadeusz Kaczorowski

Research output: Contribution to journalArticlepeer-review

26 Citations (Scopus)

Abstract

Phage vB-Tsc2631 infects the extremophilic bacterium Thermus scotoductus MAT2631 and uses the Ts2631 endolysin for the release of its progeny. The Ts2631 endolysin is the first endolysin from thermophilic bacteriophage with an experimentally validated catalytic site. In silico analysis and computational modelling of the Ts2631 endolysin structure revealed a conserved Zn2+ binding site (His30, Tyr58, His131 and Cys139) similar to Zn2+ binding site of eukaryotic peptidoglycan recognition proteins (PGRPs). We have shown that the Ts2631 endolysin lytic activity is dependent on divalent metal ions (Zn2+ and Ca2+). The Ts2631 endolysin substitution variants H30N, Y58F, H131N and C139S dramatically lost their antimicrobial activity, providing evidence for the role of the aforementioned residues in the lytic activity of the enzyme. The enzyme has proven to be not only thermoresistant, retaining 64.8% of its initial activity after 2 h at 95°C, but also highly thermodynamically stable (Tm = 99.82°C, ΔHcal = 4.58 × 104 cal mol-1). Substitutions of histidine residues (H30N and H131N) and a cysteine residue (C139S) resulted in variants aggregating at temperatures ≥75°C, indicating a significant role of these residues in enzyme thermostability. The substrate spectrum of the Ts2631 endolysin included extremophiles of the genus Thermus but also Gram-negative mesophiles, such as Escherichia coli, Salmonella Panama, Pseudomonas fluorescens and Serratia marcescens. The broad substrate spectrum and high thermostability of this endolysin makes it a good candidate for use as an antimicrobial agent to combat Gram-negative pathogens.

Original languageEnglish
Article numbere0137374
JournalPLoS ONE
Volume10
Issue number9
DOIs
Publication statusPublished - 16 Sept 2015

Bibliographical note

Publisher Copyright:
Copyright © 2015 Plotka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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