Thermal Adaptation of Extremozymes: Temperature-Sensitive Contact Analysis of Serine Proteases.

Enzyme thermal adaptation reflects a delicate interplay between sequence, structure, and dynamics of proteins, fine-tuning the catalytic activity to environmental demands. Understanding these evolutionary relationships can drive bioengineering advances, including industrial enzyme design, biocatalysts for extreme conditions, and novel therapeutics. This work explores sequence-dynamics connections in subtilisin-like serine protease homologs using a recently developed computational methodology that uses expanded ensemble simulations and temperature-sensitive contact analysis. We reveal that thermophilic enzymes achieve thermal stability through extensive salt bridges and hydrophobic networks, while psychrophilic enzymes rely on targeted interaction stability for cold adaptation. An unsupervised cluster analysis of residue conservation, flexibility, and hydrophobic interactions provides a comprehensive view of residue-specific contributions to thermal adaptation. These findings underscore the coordinated roles of conserved and variable regions in enzyme stability and offer a framework for tailoring enzymes to specific thermal properties for biotechnological applications.