Creating highly active fluoroacetate dehalogenases via gate-based synergetic chain design.

Area of Science:

• Enzyme engineering
• Protein dynamics
• Biocatalysis

Background:

• Gate residues and distal networks regulate enzymatic catalysis.
• Understanding these dynamics is key to protein engineering.

Purpose of the Study:

• Investigate gate-based allostery in fluoroacetate dehalogenase RPA1163.
• Develop a generalizable design principle for enzyme engineering.
• Establish a biocatalytic platform for synthesizing chiral carboxylic acids.

Main Methods:

• Conformational dynamics analysis
• Network engineering of gate residues
• Structural and molecular dynamics simulations
• Biocatalytic synthesis and characterization

Main Results:

• Identified a gate-based allosteric pair (K181-W185) in RPA1163 governing substrate access.
• Engineered a double mutant with significantly enhanced turnover number (> 2 × 10⁵).
• Demonstrated universality of gate-based dynamic rewiring across multiple dehalogenases.
• Achieved high productivity (> 3.7 × 10⁶ turnover number) for chiral carboxylic acid synthesis.

Conclusions:

• Gate-residue allostery is a powerful, generalizable concept in protein engineering.
• This framework bridges conformational dynamics with practical biocatalysis.
• Enables efficient, stereoselective synthesis of pharmaceutically relevant compounds.