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Ultrahigh-Throughput Directed Evolution of a Metal-Free α/β-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase

[Image: see text] Finding new mechanistic solutions for biocatalytic challenges is key in the evolutionary adaptation of enzymes, as well as in devising new catalysts. The recent release of man-made substances into the environment provides a dynamic testing ground for observing biocatalytic innovati...

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Autores principales: Schnettler, J. David, Klein, Oskar James, Kaminski, Tomasz S., Colin, Pierre-Yves, Hollfelder, Florian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2022
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9853848/
https://www.ncbi.nlm.nih.gov/pubmed/36583539
http://dx.doi.org/10.1021/jacs.2c10673
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author Schnettler, J. David
Klein, Oskar James
Kaminski, Tomasz S.
Colin, Pierre-Yves
Hollfelder, Florian
author_facet Schnettler, J. David
Klein, Oskar James
Kaminski, Tomasz S.
Colin, Pierre-Yves
Hollfelder, Florian
author_sort Schnettler, J. David
collection PubMed
description [Image: see text] Finding new mechanistic solutions for biocatalytic challenges is key in the evolutionary adaptation of enzymes, as well as in devising new catalysts. The recent release of man-made substances into the environment provides a dynamic testing ground for observing biocatalytic innovation at play. Phosphate triesters, used as pesticides, have only recently been introduced into the environment, where they have no natural counterpart. Enzymes have rapidly evolved to hydrolyze phosphate triesters in response to this challenge, converging onto the same mechanistic solution, which requires bivalent cations as a cofactor for catalysis. In contrast, the previously identified metagenomic promiscuous hydrolase P91, a homologue of acetylcholinesterase, achieves slow phosphotriester hydrolysis mediated by a metal-independent Cys-His-Asp triad. Here, we probe the evolvability of this new catalytic motif by subjecting P91 to directed evolution. By combining a focused library approach with the ultrahigh throughput of droplet microfluidics, we increase P91’s activity by a factor of ≈360 (to a k(cat)/K(M) of ≈7 × 10(5) M(–1) s(–1)) in only two rounds of evolution, rivaling the catalytic efficiencies of naturally evolved, metal-dependent phosphotriesterases. Unlike its homologue acetylcholinesterase, P91 does not suffer suicide inhibition; instead, fast dephosphorylation rates make the formation of the covalent adduct rather than its hydrolysis rate-limiting. This step is improved by directed evolution, with intermediate formation accelerated by 2 orders of magnitude. Combining focused, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in nature, resulting in alternative reagents with novel catalytic machineries.
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spelling pubmed-98538482023-01-21 Ultrahigh-Throughput Directed Evolution of a Metal-Free α/β-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase Schnettler, J. David Klein, Oskar James Kaminski, Tomasz S. Colin, Pierre-Yves Hollfelder, Florian J Am Chem Soc [Image: see text] Finding new mechanistic solutions for biocatalytic challenges is key in the evolutionary adaptation of enzymes, as well as in devising new catalysts. The recent release of man-made substances into the environment provides a dynamic testing ground for observing biocatalytic innovation at play. Phosphate triesters, used as pesticides, have only recently been introduced into the environment, where they have no natural counterpart. Enzymes have rapidly evolved to hydrolyze phosphate triesters in response to this challenge, converging onto the same mechanistic solution, which requires bivalent cations as a cofactor for catalysis. In contrast, the previously identified metagenomic promiscuous hydrolase P91, a homologue of acetylcholinesterase, achieves slow phosphotriester hydrolysis mediated by a metal-independent Cys-His-Asp triad. Here, we probe the evolvability of this new catalytic motif by subjecting P91 to directed evolution. By combining a focused library approach with the ultrahigh throughput of droplet microfluidics, we increase P91’s activity by a factor of ≈360 (to a k(cat)/K(M) of ≈7 × 10(5) M(–1) s(–1)) in only two rounds of evolution, rivaling the catalytic efficiencies of naturally evolved, metal-dependent phosphotriesterases. Unlike its homologue acetylcholinesterase, P91 does not suffer suicide inhibition; instead, fast dephosphorylation rates make the formation of the covalent adduct rather than its hydrolysis rate-limiting. This step is improved by directed evolution, with intermediate formation accelerated by 2 orders of magnitude. Combining focused, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in nature, resulting in alternative reagents with novel catalytic machineries. American Chemical Society 2022-12-30 /pmc/articles/PMC9853848/ /pubmed/36583539 http://dx.doi.org/10.1021/jacs.2c10673 Text en © 2022 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Schnettler, J. David
Klein, Oskar James
Kaminski, Tomasz S.
Colin, Pierre-Yves
Hollfelder, Florian
Ultrahigh-Throughput Directed Evolution of a Metal-Free α/β-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase
title Ultrahigh-Throughput Directed Evolution of a Metal-Free α/β-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase
title_full Ultrahigh-Throughput Directed Evolution of a Metal-Free α/β-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase
title_fullStr Ultrahigh-Throughput Directed Evolution of a Metal-Free α/β-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase
title_full_unstemmed Ultrahigh-Throughput Directed Evolution of a Metal-Free α/β-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase
title_short Ultrahigh-Throughput Directed Evolution of a Metal-Free α/β-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase
title_sort ultrahigh-throughput directed evolution of a metal-free α/β-hydrolase with a cys-his-asp triad into an efficient phosphotriesterase
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9853848/
https://www.ncbi.nlm.nih.gov/pubmed/36583539
http://dx.doi.org/10.1021/jacs.2c10673
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