Evolutionary clade-guided consensus redesign of IsPETase: A computational framework for enhancing thermodynamic...

The global accumulation of PET is a significant environmental problem, underscoring the need for thermostable PET-degrading enzymes for industrial applications. In this work, a clade-informed consensus design approach was employed to engineer a thermostable PETase, and its structural robustness, catalytic architecture, and thermodynamic properties were analyzed using integrated computational techniques. Catalytic pocket analysis demonstrated an enlarged volume for Con PETase (496 ų) compared to WT IsPETase (427 ų), suggesting greater potential for PET substrate accommodation. Molecular docking analysis revealed increased PET binding in Con PETase (-5.2 kcal mol⁻¹) relative to the WT (-4.9 kcal mol⁻¹), as confirmed by strengthened catalytic triad interactions via one additional H-bond and optimized hydrophobic contacts. Molecular dynamics simulations (40-100°C) showed superior thermodynamic stability for Con PETase, retaining the α/β-hydrolase fold with reduced RMSD, SASA, and Rg, driven by an enhanced salt-bridge interaction network, a more compact hydrophobic core, and improved surface electrostatics. Analyses of RMSD, SASA, and Rg values showed an approximately 40°C increase in the thermodynamic stability window compared to WT PETase. MM-PBSA analyses also confirmed better binding thermodynamics, as Con PETase retained more negative binding energies at 40, 60, and 80°C (-14.32 ± 0.3371, -12.67 ± 0.2997, and -9.57 ± 0.1628 kJ mol⁻¹) than observed for the WT PETase (-11.88 ± 0.2174, -4.58 ± 0.1419, and -3.60 ± 0.135 kJ mol⁻¹). The findings demonstrated that consensus ensemble reconstruction reconfigured the structural and energetic landscape of PETase, resulting in a thermally stable enzyme-substrate complex.