Structural insights into an engineered feruloyl esterase with improved MHET degrading properties.

Area of Science:

• Biochemistry and Molecular Biology
• Enzyme Engineering
• Biocatalysis and Biodegradation

Background:

• Polyethylene terephthalate (PET) is a widely used plastic with significant environmental persistence.
• Mono(2-hydroxyethyl) terephthalate (MHET) esterases (MHETases) are crucial enzymes for PET biodegradation.
• Engineering existing esterases to mimic MHETase activity is a promising strategy for enhanced plastic degradation.

Purpose of the Study:

• To elucidate the structural basis for enhanced MHET degradation by an engineered feruloyl esterase.
• To understand the molecular mechanisms underlying improved catalytic activity and substrate promiscuity.
• To identify key mutations that confer MHETase-like properties to other esterases.

Main Methods:

• Protein engineering of a feruloyl esterase to create an MHETase active site mimic.
• X-ray crystallography to determine the apo- and ligand-bound structures of the engineered enzyme.
• Molecular dynamics simulations to analyze enzyme dynamics, substrate binding, and conformational changes.

Main Results:

• The engineered variant exhibited increased MHET degradation activity compared to the wild-type enzyme.
• Crystal structures revealed a novel hydrogen bond and a trans-to-cis peptide bond isomerization near the catalytic site.
• Simulations showed stabilization of a key loop and expansion of the substrate binding cleft, enhancing substrate accommodation.

Conclusions:

• Specific mutations can effectively engineer esterases for enhanced PET biodegradation.
• The observed structural changes facilitate broader substrate binding, indicating potential for promiscuous biocatalysis.
• This engineered enzyme represents a promising biocatalyst for developing sustainable PET recycling solutions.