Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution

Yixin Cen; Warispreet Singh; Mamatjan Arkin; Thomas S. Moody; Meilan Huang; Jiahai  Zhou; Qi Wu; Manfred T. Reetz

doi:10.1038/s41467-019-11155-3

Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution

Yixin Cen, Warispreet Singh, Mamatjan Arkin, Thomas S. Moody, Meilan Huang^*, Jiahai Zhou^*, Qi Wu^*, Manfred T. Reetz^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

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Abstract

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.

Original language	English
Article number	3198
Number of pages	10
Journal	Nature Communications
Volume	10
DOIs	https://doi.org/10.1038/s41467-019-11155-3
Publication status	Published - 19 Jul 2019

Bibliographical note

This is a interdisciplinary search in collaboration with experimentalists from Zhejiang Univ, Institute of Max Plank, Chinese Academy of Science. The work was funded by Invest NI in collaboration with Almac.

Nature Communications is a peer-reviewed journal with an Impact Factor of 14.9

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10.1038/s41467-019-11155-3Licence: CC BY

Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution
Copyright 2019 the authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
Final published version, 1.94 MBLicence: CC BY

Meilan Huang
- m.huangqub.acuk
- School of Chemistry and Chemical Engineering - Senior Lecturer
- Theoretical and Applied Catalysis
Person: Academic

Cite this

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title = "Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution",

abstract = "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.",

author = "Yixin Cen and Warispreet Singh and Mamatjan Arkin and Moody, {Thomas S.} and Meilan Huang and Jiahai Zhou and Qi Wu and Reetz, {Manfred T.}",

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AU - Cen, Yixin

AU - Singh, Warispreet

AU - Arkin, Mamatjan

AU - Moody, Thomas S.

AU - Huang, Meilan

AU - Zhou, Jiahai

AU - Wu, Qi

AU - Reetz, Manfred T.

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N2 - 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.

AB - 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.

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Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution

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