Abstract
Evolutionary engineering of our previously reported Cp*Rh(III)-linked artificial metalloenzyme was performed based on a DNA recombination strategy to improve its catalytic activity toward C(sp(2))-H bond functionalization. Improved scaffold design was achieved with α-helical cap domains of fatty acid binding protein (FABP) embedded within the β-barrel structure of nitrobindin (NB) as a chimeric protein scaffold for the artificial metalloenzyme. After optimization of the amino acid sequence by directed evolution methodology, an engineered variant, designated NB(HLH1)(Y119A/G149P) with enhanced performance and enhanced stability was obtained. Additional rounds of metalloenzyme evolution provided a Cp*Rh(III)-linked NB(HLH1)(Y119A/G149P) variant with a >35-fold increase in catalytic efficiency (k(cat)/K(M)) for cycloaddition of oxime and alkyne. Kinetic studies and MD simulations revealed that aromatic amino acid residues in the confined active-site form a hydrophobic core which binds to aromatic substrates adjacent to the Cp*Rh(III) complex. The metalloenzyme engineering process based on this DNA recombination strategy will serve as a powerful method for extensive optimization of the active-sites of artificial metalloenzymes.