title: Biochemical Characterization and Validation of a Catalytic Site of a Highly Thermostable Ts2631 Endolysin from the Thermus scotoductus Phage vB_Tsc2631.
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 × 10(4) 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.
authors.affiliation: Department of Microbiology, University of Gdansk, Gdansk, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Plotka
authors.affiliation: Collection of Plasmids and Microorganisms, University of Gdansk, Gdansk, Poland.
authors.forename: Anna-Karina
authors.initials: AK
authors.lastname: Kaczorowska
authors.affiliation: Department of Microbiology, University of Gdansk, Gdansk, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Morzywolek
authors.affiliation: Faculty of Chemistry, University of Gdansk, Gdansk, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Makowska
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, Poland.
authors.forename: Lukasz P
authors.initials: LP
authors.lastname: Kozlowski
authors.affiliation: VESO Vikan, Namsos, Norway.
authors.forename: Audur
authors.initials: A
authors.lastname: Thorisdottir
authors.affiliation: Matis, Reykjavik, Iceland.
authors.forename: Sigurlaug
authors.initials: S
authors.lastname: Skírnisdottir
authors.affiliation: ORF genetics, Kopavogur, Iceland.
authors.forename: Sigridur
authors.initials: S
authors.lastname: Hjörleifsdottir
authors.affiliation: Matis, Reykjavik, Iceland.
authors.forename: Olafur H
authors.initials: OH
authors.lastname: Fridjonsson
authors.affiliation: Matis, Reykjavik, Iceland; Faculty of Life and Environmental Sciences, University of Iceland, Reykjavik, Iceland.
authors.forename: Gudmundur O
authors.initials: GO
authors.lastname: Hreggvidsson
authors.affiliation: Prokazyme ehf, Reykjavik, Iceland.
authors.forename: Jakob K
authors.initials: JK
authors.lastname: Kristjansson
authors.affiliation: A&A Biotechnology, Gdynia, Poland.
authors.forename: Slawomir
authors.initials: S
authors.lastname: Dabrowski
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, Poland; Laboratory of Bioinformatics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland.
authors.forename: Janusz M
authors.initials: JM
authors.lastname: Bujnicki
authors.affiliation: Department of Microbiology, University of Gdansk, Gdansk, Poland.
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Kaczorowski
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology* / Q000502:physiology; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000494:pharmacology; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D013824:Thermus / Q000821:virology*
chemical_list: D002413:Cations, Divalent; D012965:Sodium Chloride; D010450:Endopeptidases; C001143:endolysin