Rational Engineering of Multiple Active Sites in an Ester Hydrolase

Area of Science:

• Biocatalysis and Enzyme Engineering
• Computational Chemistry and Molecular Modeling

Background:

• Modifying active sites in homogeneous enzymatic catalysts is well-documented.
• Increasing the number of active sites within a single enzyme, analogous to heterogeneous catalysis, remains an underexplored area due to challenges in accommodating catalytic residues.

Purpose of the Study:

• To investigate the feasibility of creating an additional catalytic active site within an existing enzyme.
• To engineer a serine ester hydrolase with two distinct functional reactive groups.

Main Methods:

• Utilized the Protein Energy Landscape Exploration (PELE) software to identify potential binding pockets for additional catalytic triads.
• Introduced specific mutations to convert an identified binding pocket into a functional catalytic site.
• Characterized the substrate specificity, preference, and catalytic activity of the engineered enzyme.

Main Results:

• A potential binding pocket capable of accommodating an extra catalytic triad and oxyanion hole contacts was identified using PELE.
• Two mutations successfully transformed the binding pocket into a secondary catalytic site.
• The engineered enzyme displayed distinct substrate specificity, preference, and catalytic activity compared to the wild-type enzyme and other hydrolases due to altered active site architecture.

Conclusions:

• Converting a binding pocket into an additional catalytic active site is a viable strategy for creating multifunctional enzymes.
• The study successfully generated a serine ester hydrolase with two functional reactive groups.
• Results highlight the predictive power of computational modeling in enzyme engineering and open avenues for designing enzymes with multiple catalytic environments.