Crystal structure, enzymatic and thermodynamic properties of the Thermus thermophilus phage Tt72 lytic endopeptidase with unique structural signatures of thermal adaptation

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 (T_m = 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.