Structure and function of the Ts2631 endolysin of Thermus scotoductus phage vB_Tsc2631 with unique N-terminal extension used for peptidoglycan binding

Magdalena Plotka,Sebastian Dorawa,Tadeusz Kaczorowski,Lukasz P Kozlowski,Anna-Karina Kaczorowska,Kornelius Zeth,Enea Sancho-Vaello

doi:10.1038/s41598-018-37417-6

Magdalena Plotka, Sebastian Dorawa + Show 5 more

Open Access

PDF Available

https://doi.org/10.1038/s41598-018-37417-6

Copy DOI

Export

Save

Cite

Abstract
Highlights/Summary
Full-Text PDF
Similar Papers

Abstract

Listen

To escape from hosts after completing their life cycle, bacteriophages often use endolysins, which degrade bacterial peptidoglycan. While mesophilic phages have been extensively studied, their thermophilic counterparts are not well characterized. Here, we present a detailed analysis of the structure and function of Ts2631 endolysin from thermophilic phage vB_Tsc2631, which is a zinc-dependent amidase. The active site of Ts2631 consists of His30, Tyr58, His131 and Cys139, which are involved in Zn2+ coordination and catalysis. We found that the active site residues are necessary for lysis yet not crucial for peptidoglycan binding. To elucidate residues involved in the enzyme interaction with peptidoglycan, we tested single-residue substitution variants and identified Tyr60 and Lys70 as essential residues. Moreover, substitution of Cys80, abrogating disulfide bridge formation, inactivates Ts2631, as do substitutions of His31, Thr32 and Asn85 residues. The endolysin contains a positively charged N-terminal extension of 20 residues that can protrude from the remainder of the enzyme and is crucial for peptidoglycan binding. We show that the deletion of 20 residues from the N-terminus abolished the bacteriolytic activity of the enzyme. Because Ts2631 exhibits intrinsic antibacterial activity and unusual thermal stability, it is perfectly suited as a scaffold for the development of antimicrobial agents.

Highlights

Upon completing replication and the assembly of new infectious virions, bacteriophages, viruses that infect prokaryotic cells, often rely on their lytic enzymes to release phage progeny from the host
The two monomers in the asymmetric unit interact with each other via the N-terminus and an interaction of the N-terminal extension with the globular domain (Fig. 2A). We reasoned that this interface might be involved in dimerization, but biochemical data based on analytical ultracentrifugation together with a detailed analysis using PISA software (PISA score: 0.25) pointed towards the monomer as the dominant form in solution (Supplementary Fig. S1)[15]
Studies on novel endolysins are important for the development of effective antimicrobial agents as alternative to traditional antibiotics[2,19,20]

Summary

Introduction

Upon completing replication and the assembly of new infectious virions, bacteriophages, viruses that infect prokaryotic cells, often rely on their lytic enzymes (endolysins) to release phage progeny from the host. Bacteriophage vB_Tsc2631 was isolated from a hot spring of the Hveragerði geothermal area, Iceland[5] This lytic phage can use the thermophilic bacteria Thermus scotoductus MAT2631 and Thermus thermophilus HB8 as host cells for propagation. A comparative in silico analysis of the amino acid sequence of Ts2631 endolysin indicated that this enzyme is a structural homolog of phage T7 lysozyme and belongs to a large superfamily including three families of proteins capable of binding bacterial PGN. The first such family is exemplified by the T7 lysozyme itself, the best studied example of type 2 amidases. Special attention was paid to those residues whose counterparts in PGRPs form the PGN-binding pocket

Methods

Results

Conclusion