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

Shunsuke Kato; Akira Onoda; Ulrich Schwaneberg; Takashi Hayashi

doi:10.1021/jacs.3c00581

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

J Am Chem Soc. 2023 Mar 9;145(15):8285-8290. doi: 10.1021/jacs.3c00581. Online ahead of print.

Authors

Shunsuke Kato¹, Akira Onoda¹, Ulrich Schwaneberg², Takashi Hayashi¹

Affiliations

¹ Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, 565-0871, Japan.
² Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, D-52074 Aachen, Germany.

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(sp²)-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.