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Article

Molecular Characterization of a Novel Lytic Enzyme LysC from Clostridium intestinale URNW and Its Antibacterial Activity Mediated by Positively Charged N-Terminal Extension

1
Laboratory of Extremophiles Biology, Department of Microbiology, Faculty of Biology, University of Gdansk, 80-822 Gdansk, Poland
2
SARomics Biostructures, SE-223 81 Lund, Sweden
3
Collection of Plasmids and Microorganisms, Faculty of Biology, University of Gdansk, 80-308 Gdansk, Poland
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(14), 4894; https://doi.org/10.3390/ijms21144894
Received: 19 June 2020 / Revised: 7 July 2020 / Accepted: 7 July 2020 / Published: 11 July 2020
(This article belongs to the Special Issue Creation of New Antimicrobial Peptides)
Peptidoglycan hydrolytic enzymes are considered to be a promising alternative to conventional antibiotics in combating bacterial infections. To identify novel hydrolytic enzymes, we performed a database search with the sequences of two thermostable endolysins with high bactericidal activity, studied earlier in our laboratory. Both these enzymes originate from Thermus scotoductus bacteriophages MAT2119 and vB_Tsc2631. A lytic enzyme LysC from Clostridium intestinale URNW was found to have the highest amino acid sequence similarity to the bacteriophage proteins and was chosen for further analysis. The recombinant enzyme showed strong activity against its host bacteria C. intestinale, as well as against C. sporogenes, Bacillus cereus, Micrococcus luteus, and Staphylococcus aureus, on average causing a 5.12 ± 0.14 log reduction of viable S. aureus ATCC 25923 cells in a bactericidal assay. Crystallographic studies of the protein showed that the catalytic site of LysC contained a zinc atom coordinated by amino acid residues His50, His147, and Cys155, a feature characteristic for type 2 amidases. Surprisingly, neither of these residues, nor any other of the four conserved residues in the vicinity of the active site, His51, Thr52, Tyr76, and Thr153, were essential to maintain the antibacterial activity of LysC. Therefore, our attention was attracted to the intrinsically disordered and highly positively charged N-terminal region of the enzyme. Potential antibacterial activity of this part of the sequence, predicted by the Antimicrobial Sequence Scanning System, AMPA, was confirmed in our experimental studies; the truncated version of LysC (LysCΔ2–23) completely lacked antibacterial activity. Moreover, a synthetic peptide, which we termed Intestinalin, with a sequence identical to the first thirty amino acids of LysC, displayed substantial anti-staphylococcal activity with IC50 of 6 μg/mL (1.5 μM). This peptide was shown to have α-helical conformation in solution in the presence of detergents which is a common feature of amphipathic α-helical antimicrobial peptides. View Full-Text
Keywords: bacteriophage; endolysin; autolysin; antimicrobial peptide; peptidoglycan; Staphylococcus aureus; thermophiles bacteriophage; endolysin; autolysin; antimicrobial peptide; peptidoglycan; Staphylococcus aureus; thermophiles
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MDPI and ACS Style

Plotka, M.; Szadkowska, M.; Håkansson, M.; Kovačič, R.; Al-Karadaghi, S.; Walse, B.; Werbowy, O.; Kaczorowska, A.-K.; Kaczorowski, T. Molecular Characterization of a Novel Lytic Enzyme LysC from Clostridium intestinale URNW and Its Antibacterial Activity Mediated by Positively Charged N-Terminal Extension. Int. J. Mol. Sci. 2020, 21, 4894. https://doi.org/10.3390/ijms21144894

AMA Style

Plotka M, Szadkowska M, Håkansson M, Kovačič R, Al-Karadaghi S, Walse B, Werbowy O, Kaczorowska A-K, Kaczorowski T. Molecular Characterization of a Novel Lytic Enzyme LysC from Clostridium intestinale URNW and Its Antibacterial Activity Mediated by Positively Charged N-Terminal Extension. International Journal of Molecular Sciences. 2020; 21(14):4894. https://doi.org/10.3390/ijms21144894

Chicago/Turabian Style

Plotka, Magdalena, Monika Szadkowska, Maria Håkansson, Rebeka Kovačič, Salam Al-Karadaghi, Björn Walse, Olesia Werbowy, Anna-Karina Kaczorowska, and Tadeusz Kaczorowski. 2020. "Molecular Characterization of a Novel Lytic Enzyme LysC from Clostridium intestinale URNW and Its Antibacterial Activity Mediated by Positively Charged N-Terminal Extension" International Journal of Molecular Sciences 21, no. 14: 4894. https://doi.org/10.3390/ijms21144894

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