Residue-level determinants of the thermal stability of the extremophilic Ts2631 endolysin.

In the face of the growing crisis of antibiotic resistance, peptidoglycan-degrading endolysins derived from bacteriophages offer a promising alternative to traditional antimicrobials. Thermostable variants, capable of maintaining their structure and activity under extreme conditions, are of particular interest. Here, we present a comprehensive analysis of endolysin Ts2631 from bacteriophage vB_Tsc2631, which infects Thermus scotoductus in Icelandic hot springs. This type 2 amidase exhibits exceptional thermostability, with a melting point ranging from 99.8 °C to 104.7 °C, depending on the solvent. Structural comparison with its mesophilic counterpart, T7 lysozyme, revealed shorter loops with lower B factors, suggesting reduced conformational flexibility. The Ts2631 sequence contains increased levels of tyrosine, proline, tryptophan, and arginine residues, amino acids commonly associated with thermophilic adaptation. To elucidate the contribution of individual residues, the melting temperatures (Tm) of 55 point mutants were determined. Substitutions at the catalytic Zn²⁺-coordinating residues (H30, H131, and C139) led to significant destabilization. Substitutions at positions Y60 and Y69 also reduced stability. In contrast, the disulfide bond (C80-C90) and arginine-mediated salt bridges showed limited effects. Among the aromatic and proline mutants, R20A, W102A, W109A, P140A, and W145A had significantly reduced Tm values. R20 proved critical for peptidoglycan binding, while the hidden tryptophan and proline contributed significantly to the thermophilic fold. Overall, the data indicate that buried aromatic residues play a key role in maintaining the remarkable thermal resistance of Ts2631. This suggests a future strategy to enhance the stability of mesophilic endolysins with therapeutic potential by replacing non-conserved amino acids with tryptophan or proline residues.