AsiteDesign: a Semirational Algorithm for an Automated Enzyme Design

Area of Science:

• Computational biology
• Enzyme engineering
• Biocatalysis

Background:

• Advances in protein structure prediction increase the availability of high-quality structural data.
• Structure-based enzyme engineering is crucial for optimizing biocatalysts in industrial applications.

Purpose of the Study:

• To present AsiteDesign, a Monte Carlo-based protocol for structure-based engineering of enzyme active sites.
• To introduce new catalytic residues into binding pockets to create or alter enzymatic activity.
• To enhance enzyme performance for industrial processes.

Main Methods:

• AsiteDesign protocol utilizes Monte Carlo simulations and pyRosetta implementation.
• Incorporates enhanced sampling techniques for efficient exploration of the protein design space.
• Tested on *Pseudomonas fluorescens* esterase (PFE) for active site redesign.

Main Results:

• Successfully designed an alternative catalytic triad in PFE, experimentally verified for activity.
• Mutations F158L/F198A and F125A/F158L significantly enhanced hydrolysis of a bulky chiral substrate (1-phenyl-2-pentyl acetate) by PFE.
• Achieved a reversal of enantioselectivity from (R) to (S)-enantiopreference with 32% and 55% enantiomeric excess (ee), respectively.

Conclusions:

• AsiteDesign is effective in designing alternative catalytic triads and modifying enzyme activity.
• The protocol demonstrates potential for optimizing biocatalysts for specific industrial applications.
• Successful experimental validation confirms the utility of AsiteDesign in protein engineering.