Structure-Guided Interface Engineering of Formate Oxidase to Suppress Aggregation and Enhance Structural Stability

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Protein Biophysics

Background:

• Formate oxidase (FOx) is a promising biocatalyst for agricultural and food industries.
• High enzyme concentrations needed for biosensors and H2O2 generation lead to significant activity loss.
• Understanding FOx deactivation mechanisms is crucial for improving its stability and application scope.

Purpose of the Study:

• To elucidate the deactivation mechanism of formate oxidase at high concentrations.
• To engineer FOx variants with reduced aggregation and improved stability.
• To develop a transferable interface-engineering framework for multimeric enzymes.

Main Methods:

• Biophysical assays and kinetic analysis to study enzyme behavior.
• Molecular dynamics simulations to investigate deactivation pathways.
• Rosetta-based computational design for interface engineering.

Main Results:

• FOx exhibits concentration-dependent aggregation, leading to activity loss, not thermal instability at 25 °C.
• Interface engineering identified 11 variants, with six showing reduced aggregation at 1 mg/mL.
• The A378L variant effectively reduced aggregation while preserving catalytic efficiency by weakening dimer packing.

Conclusions:

• Enzyme aggregation is the primary cause of formate oxidase activity loss at high concentrations.
• Interface engineering is a viable strategy to enhance FOx stability and reduce aggregation.
• The developed engineering framework can be applied to improve other multimeric enzymes.