Molecular Insights into the Enhanced Activity and/or Thermostability of PET Hydrolase by D186 Mutations

PETase exhibits a high degradation activity for polyethylene terephthalate (PET) plastic under moderate temperatures. However, the effect of non-active site residues in the second shell of PETase on the catalytic performance remains unclear. Herein, we proposed a crystal structure- and sequence-based strategy to identify the key non-active site residue. D186 in the second shell of PETase was found to be capable of modulating the enzyme activity and stability. The most active PETase^D186N improved both the activity and thermostability with an increase in T by 8.89 °C. The PET degradation product concentrations were 1.86 and 3.69 times higher than those obtained with PETase^WT at 30 and 40 °C, respectively. The most stable PETase^D186V showed an increase in T of 12.91 °C over PETase^WT. Molecular dynamics (MD) simulations revealed that the D186 mutations could elevate the substrate binding free energy and change substrate binding mode, and/or rigidify the flexible Loop 10, and lock Loop 10 and Helix 6 by hydrogen bonding, leading to the enhanced activity and/or thermostability of PETase variants. This work unraveled the contribution of the key second-shell residue in PETase in influencing the enzyme activity and stability, which would benefit in the rational design of efficient and thermostable PETase.