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 PETaseD186Nimproved both the activity and thermostability with an increase inTmby 8.89 °C. The PET degradation product concentrations were 1.86 and 3.69 times higher than those obtained with PETaseWTat 30 and 40 °C, respectively. The most stable PETaseD186Vshowed an increase inTmof 12.91 °C over PETaseWT. 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.
危险品化学品经营许可证（带存储）