Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution
Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but thi...
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ftpubman:oai:pure.mpg.de:item_3150154 2023-08-27T04:05:42+02:00 Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution Cen, Y. Singh, W. Arkin, M. Moody, T. Huang, M. Zhou, J. Wu, Q. Reetz, M. 2019-07-19 http://hdl.handle.net/21.11116/0000-0004-65AE-7 eng eng info:eu-repo/semantics/altIdentifier/doi/10.1038/s41467-019-11155-3 http://hdl.handle.net/21.11116/0000-0004-65AE-7 Nature Communications info:eu-repo/semantics/article 2019 ftpubman https://doi.org/10.1038/s41467-019-11155-3 2023-08-02T00:02:19Z Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105 − /His224 + rather than a concerted process. Article in Journal/Newspaper Antarc* Antarctica Max Planck Society: MPG.PuRe Nature Communications 10 1 |
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Max Planck Society: MPG.PuRe |
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English |
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Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105 − /His224 + rather than a concerted process. |
format |
Article in Journal/Newspaper |
author |
Cen, Y. Singh, W. Arkin, M. Moody, T. Huang, M. Zhou, J. Wu, Q. Reetz, M. |
spellingShingle |
Cen, Y. Singh, W. Arkin, M. Moody, T. Huang, M. Zhou, J. Wu, Q. Reetz, M. Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution |
author_facet |
Cen, Y. Singh, W. Arkin, M. Moody, T. Huang, M. Zhou, J. Wu, Q. Reetz, M. |
author_sort |
Cen, Y. |
title |
Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution |
title_short |
Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution |
title_full |
Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution |
title_fullStr |
Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution |
title_full_unstemmed |
Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution |
title_sort |
artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution |
publishDate |
2019 |
url |
http://hdl.handle.net/21.11116/0000-0004-65AE-7 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
Nature Communications |
op_relation |
info:eu-repo/semantics/altIdentifier/doi/10.1038/s41467-019-11155-3 http://hdl.handle.net/21.11116/0000-0004-65AE-7 |
op_doi |
https://doi.org/10.1038/s41467-019-11155-3 |
container_title |
Nature Communications |
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10 |
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1 |
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1775357444148428800 |