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Evolutionary Engineering of a Cp*Rh(III) Complex-Linked Artificial Metalloenzyme with a Chimeric β-Barrel Protein Scaffold
[Image: see text] 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 do...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10119979/ https://www.ncbi.nlm.nih.gov/pubmed/36892401 http://dx.doi.org/10.1021/jacs.3c00581 |
Sumario: | [Image: see text] 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. |
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