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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 |
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author | Kato, Shunsuke Onoda, Akira Schwaneberg, Ulrich Hayashi, Takashi |
author_facet | Kato, Shunsuke Onoda, Akira Schwaneberg, Ulrich Hayashi, Takashi |
author_sort | Kato, Shunsuke |
collection | PubMed |
description | [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. |
format | Online Article Text |
id | pubmed-10119979 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-101199792023-04-22 Evolutionary Engineering of a Cp*Rh(III) Complex-Linked Artificial Metalloenzyme with a Chimeric β-Barrel Protein Scaffold Kato, Shunsuke Onoda, Akira Schwaneberg, Ulrich Hayashi, Takashi J Am Chem Soc [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. American Chemical Society 2023-03-09 /pmc/articles/PMC10119979/ /pubmed/36892401 http://dx.doi.org/10.1021/jacs.3c00581 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Kato, Shunsuke Onoda, Akira Schwaneberg, Ulrich Hayashi, Takashi Evolutionary Engineering of a Cp*Rh(III) Complex-Linked Artificial Metalloenzyme with a Chimeric β-Barrel Protein Scaffold |
title | Evolutionary Engineering
of a Cp*Rh(III) Complex-Linked
Artificial Metalloenzyme with a Chimeric β-Barrel Protein
Scaffold |
title_full | Evolutionary Engineering
of a Cp*Rh(III) Complex-Linked
Artificial Metalloenzyme with a Chimeric β-Barrel Protein
Scaffold |
title_fullStr | Evolutionary Engineering
of a Cp*Rh(III) Complex-Linked
Artificial Metalloenzyme with a Chimeric β-Barrel Protein
Scaffold |
title_full_unstemmed | Evolutionary Engineering
of a Cp*Rh(III) Complex-Linked
Artificial Metalloenzyme with a Chimeric β-Barrel Protein
Scaffold |
title_short | Evolutionary Engineering
of a Cp*Rh(III) Complex-Linked
Artificial Metalloenzyme with a Chimeric β-Barrel Protein
Scaffold |
title_sort | evolutionary engineering
of a cp*rh(iii) complex-linked
artificial metalloenzyme with a chimeric β-barrel protein
scaffold |
url | 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 |
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