Cargando…
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...
Autores principales: | , , , , |
---|---|
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 |
_version_ | 1784872990025973760 |
---|---|
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. |
format | Online Article Text |
id | pubmed-9853848 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT schnettlerjdavid ultrahighthroughputdirectedevolutionofametalfreeabhydrolasewithacyshisasptriadintoanefficientphosphotriesterase AT kleinoskarjames ultrahighthroughputdirectedevolutionofametalfreeabhydrolasewithacyshisasptriadintoanefficientphosphotriesterase AT kaminskitomaszs ultrahighthroughputdirectedevolutionofametalfreeabhydrolasewithacyshisasptriadintoanefficientphosphotriesterase AT colinpierreyves ultrahighthroughputdirectedevolutionofametalfreeabhydrolasewithacyshisasptriadintoanefficientphosphotriesterase AT hollfelderflorian ultrahighthroughputdirectedevolutionofametalfreeabhydrolasewithacyshisasptriadintoanefficientphosphotriesterase |