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PMID:26375388
| Citation |
Plotka, M, Kaczorowska, AK, Morzywolek, A, Makowska, J, Kozlowski, LP, Thorisdottir, A, Skírnisdottir, S, Hjörleifsdottir, S, Fridjonsson, OH, Hreggvidsson, GO, Kristjansson, JK, Dabrowski, S, Bujnicki, JM and Kaczorowski, T (2015) Biochemical Characterization and Validation of a Catalytic Site of a Highly Thermostable Ts2631 Endolysin from the Thermus scotoductus Phage vB_Tsc2631. PLoS ONE 10:e0137374 |
|---|---|
| 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. |
| Links |
PubMed PMC4573324 Online version:10.1371/journal.pone.0137374 |
| Keywords |
Amino Acid Sequence; Bacteriophages/enzymology; Bacteriophages/physiology; Catalytic Domain; Cations, Divalent/pharmacology; Endopeptidases/chemistry; Endopeptidases/metabolism; Enzyme Stability; Models, Molecular; Molecular Sequence Data; Sodium Chloride/pharmacology; Substrate Specificity; Temperature; Thermus/virology |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0051715: cytolysis in other organism |
ECO:0000314: direct assay evidence used in manual assertion |
P |
interacting_taxon:37636 |
In figure 3, T. thermophilus was exposed to different concentrations of Ts2631 endolysin. At 5 minute intervals, for 40 minutes, the number of cells still alive was found by measuring OD600. According to the graph, no matter what concentration of endolysin was added, a significant number of cells died compared to cell death in the control. At the 35 minute mark, all concentration amounts showed over 50% cell death. This means that the endolysin from Ts2631 phage causes the death of the host cell. |
complete | |||
Notes
See also
References
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