Evolutionary Engineering of a Cp*Rh(III) Complex-Linked Artificial Metalloenzyme with a Chimeric β-Barrel Protein...

Area of Science:

• Bioinorganic Chemistry
• Protein Engineering
• Catalysis

Background:

• Artificial metalloenzymes combine the selectivity of enzymes with the reactivity of metal complexes.
• Previous Cp*Rh(III)-linked artificial metalloenzymes showed promise for C(sp2)-H functionalization.
• Enhancing catalytic activity and stability remains a key challenge in artificial metalloenzyme development.

Purpose of the Study:

• To improve the catalytic activity and stability of Cp*Rh(III)-linked artificial metalloenzymes.
• To develop an engineered chimeric protein scaffold for enhanced metalloenzyme performance.
• To investigate the structure-activity relationship of the optimized artificial metalloenzyme.

Main Methods:

• DNA recombination strategy to create a chimeric protein scaffold (nitrobindin with fatty acid binding protein domains).
• Directed evolution methodology for amino acid sequence optimization.
• Kinetic studies and molecular dynamics (MD) simulations to analyze enzyme-substrate interactions.

Main Results:

• An engineered variant, NB^HLH1(Y119A/G149P), exhibited enhanced performance and stability.
• The optimized metalloenzyme showed a >35-fold increase in catalytic efficiency for oxime-alkyne cycloaddition.
• MD simulations revealed a hydrophobic core formed by aromatic residues, facilitating substrate binding near the Cp*Rh(III) complex.

Conclusions:

• The DNA recombination and directed evolution strategy is effective for optimizing artificial metalloenzymes.
• The engineered chimeric scaffold provides a robust platform for enhancing catalytic efficiency and stability.
• This approach offers a powerful method for extensive active-site optimization in artificial metalloenzymes.