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.

Details

Title
Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution
Author
Cen, Yixin 1 ; Singh, Warispreet 2   VIAFID ORCID Logo  ; Arkin, Mamatjan 3 ; Moody, Thomas S 4 ; Huang, Meilan 5 ; Zhou, Jiahai 6 ; Wu, Qi 3 ; Reetz, Manfred T 7 

1  Department of Chemistry, Zhejiang University, Hangzhou, China; State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China 
2  School of Chemistry and Chemical Engineering, Queen’s University, Belfast, Northern Ireland, UK; Department of Biocatalysis and Isotope Chemistry, Almac Sciences, Craigavon, Northern Ireland, UK 
3  Department of Chemistry, Zhejiang University, Hangzhou, China 
4  Department of Biocatalysis and Isotope Chemistry, Almac Sciences, Craigavon, Northern Ireland, UK 
5  School of Chemistry and Chemical Engineering, Queen’s University, Belfast, Northern Ireland, UK 
6  State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China 
7  Max-Planck-Institut für Kohlenforschung, Mülheim an der Ruhr, Germany; Chemistry Department, Philipps-University, Marburg, Germany 
Pages
1-10
Publication year
2019
Publication date
Jul 2019
Publisher
Nature Publishing Group
e-ISSN
20411723
Source type
Scholarly Journal
Language of publication
English
DOI
https://doi.org/10.1038/s41467-019-11155-3
ProQuest document ID
2260417815
Copyright
© 2019. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.