Structure and Energetics of PET-Hydrolyzing Enzyme Complexes: A Systematic Comparison from Molecular Dynamics...

Area of Science:

• Biocatalysis and Enzyme Engineering
• Polymer Degradation
• Computational Biochemistry

Background:

• The enzyme PETase, discovered in 2016 from Ideonella Sakaiensis, exhibits potent poly(ethylene terephthalate) (PET) hydrolytic activity at ambient temperatures.
• Limited thermostability of wild-type PETase hinders its industrial application in PET degradation.
• Engineered variants aim to enhance PETase stability and activity at elevated temperatures.

Purpose of the Study:

• To systematically compare the structural characteristics and binding affinities of wild-type PETase and four engineered variants (DuraPETase, ThermoPETase, FastPETase, HotPETase).
• To analyze enzyme-substrate interactions at both room temperature (300 K) and elevated temperature (350 K).
• To investigate the binding potential of these enzymes towards the alternative polymer poly(ethylene-2,5-furan dicarboxylate) (PEF).

Main Methods:

• Standard molecular dynamics (MD) simulations were employed to model enzyme-substrate complexes.
• Unbinding free energy calculations were performed to quantify binding affinities.
• Analysis focused on enzyme structural features, active site interactions, and substrate binding dynamics.

Main Results:

• PET4 (tetrameric PET chain) formed stable complexes with all five enzymes at 300 K, with key interactions involving residues W185 and Y87.
• The 'W185 wobbling' phenomenon indicates plasticity in the binding pocket across variants, facilitating substrate recognition at moderate temperatures.
• Binding affinity significantly decreased at 350 K, with only HotPETase maintaining moderate interactions; no persistent interactions were observed with PEF.

Conclusions:

• PETase variants exhibit temperature-dependent binding affinities, with reduced efficacy at higher temperatures.
• The structural flexibility of the active site is crucial for substrate binding and recognition.
• Current PETase variants are not optimized for binding PEF, necessitating further engineering for broader plastic degradation applications.