Substrate scope expansion of 4-phenol oxidases by rational enzyme selection and sequence-function relations

Area of Science:

• Biocatalysis and Enzyme Engineering
• Computational Biology
• Organic Synthesis

Background:

• Enzymes offer high selectivity in organic synthesis but have limited substrate scope.
• Expanding enzyme functionality is crucial for broader biocatalytic applications.
• Identifying and modifying enzymes with desired catalytic activities remains a challenge.

Purpose of the Study:

• To present a streamlined computational approach for selecting enzymes with specific functionalities from large sequence datasets.
• To engineer 4-phenol oxidoreductases (4-PORs) with enhanced oxidase activity.
• To establish robust sequence-function relationships for enzyme optimization.

Main Methods:

• Computational selection of eight oxidase-branch 4-POR enzymes from 292 sequences using the A²CA tool based on catalytic pocket residue properties.
• Exploiting sequence-function correlations derived from residue analysis to guide site-saturation mutagenesis.
• Employing a peroxidase-independent screening method to identify active enzyme variants.

Main Results:

• Successfully selected eight promising 4-POR candidates.
• Generated 16 active enzyme variants through mutagenesis, exhibiting up to 90-fold increased activity compared to wildtypes.
• Achieved activity levels up to 6-fold higher than the best natural variants.
• Validated findings through kinetic experiments and structural modeling.

Conclusions:

• The presented computational and mutagenesis strategy enables rational enzyme selection and engineering.
• The approach successfully expanded the functional scope of 4-PORs, yielding highly active variants.
• This methodology provides a robust framework for discovering and optimizing biocatalysts for diverse synthetic applications.