Journal Article

PUBDB-2025-04143

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Crystal structure, enzymatic and thermodynamic properties of the Thermus thermophilus phage Tt72 lytic endopeptidase with unique structural signatures of thermal adaptation

Dorawa, S. ; Biniek-Antosiak, K. ; Bejger, M. ; Kaczorowska, A.-K. ; Ciuchcinski, K. ; Godlewska, A. ; Płotka, M. ; Hreggvidsson, G. O. ; Dziewit, L. ; Kaczorowski, T. (Corresponding author) ; Rypniewski, W. (Corresponding author)

2025
Elsevier San Diego, Calif.

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Please use a persistent id in citations: doi:10.1016/j.jsb.2025.108230

Abstract: We presents the discovery and molecular characterization of a novel lytic enzyme from the extremophilic Thermus thermophilus MAT72 phage vB_Tt72. The protein of 346-aa (MW = 39,705) functions as phage vB_Tt72 endolysin and shows low sequence identity (<37 %) to members of M23 family of peptidoglycan hydrolases, except for two uncharacterized endopeptidases of T. thermophilus phages: φYS40 (87 %) and φTMA (88 %). The enzyme exhibits lytic activity mainly against bacteria of the genus Thermus and, to a lesser extent, against other Gram-negative and Gram-positive bacteria. The protein is monomeric in solution and is highly thermostable (Tm = 98.3 °C). It retains ∼ 50 % of its lytic activity after 90 min of incubation at 99 °C. Crystallographic analysis, at 2.2 Å resolution, revealed a fold characteristic of M23 metallopeptidases, accounting for 40 % of the structure. The remaining parts of the molecule are folded in a manner that was previously undescribed. The M23 fold contains a Zn2+ ion coordinated by a conserved His-Asp-His triad, and two conserved His residues essential for catalysis. The active site is occupied by a phosphate or a sulfate anion, while the substrate-binding groove contains a ligand, which is a fragment of E. coli peptidoglycan. The common sequence-based criteria failed to identify the protein as (hyper)thermophilic. It is likely that the protein’s thermal stability is owed to peculiar features of its three-dimensional structure. Instead of trimmed surface loops, observed in many thermostable proteins, the catalytic domain contains two long loops that interlace and form an α-helical bundle with its own hydrophobic core.

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Contributing Institute(s):

1. EMBL-User (EMBL-User)

Research Program(s):

1. 6G3 - PETRA III (DESY) (POF4-6G3) (POF4-6G3)

Experiment(s):

1. PETRA Beamline P13 (PETRA III)

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