English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

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

Cen, Y., Singh, W., Arkin, M., Moody, T. S., Huang, M., Zhou, J., et al. (2019). Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution. Nature Communications, 10: 3198. doi:10.1038/s41467-019-11155-3.

Item is

Basic

show hide
Genre: Journal Article

Files

show Files

Locators

show

Creators

show
hide
 Creators:
Cen, Yixin1, 2, Author
Singh, Warispreet3, 4, Author
Arkin, Mamatjan1, Author
Moody, Thomas S.4, Author
Huang, Meilan3, Author
Zhou, Jiahai2, Author
Wu, Qi1, Author
Reetz, Manfred T.5, 6, Author           
Affiliations:
1Department of Chemistry, Zhejiang University, 310027, Hangzhou, China, ou_persistent22              
2State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 200032, Shanghai, China, ou_persistent22              
3School of Chemistry and Chemical Engineering, Queen’s University, Belfast, Northern Ireland, BT9 5AG, UK, ou_persistent22              
4Department of Biocatalysis and Isotope Chemistry, Almac Sciences, Craigavon, Northern Ireland, BT63 5QD, UK, ou_persistent22              
5Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445588              
6Chemistry Department, Philipps-University, 35032, Marburg, Germany, ou_persistent22              

Content

show
hide
Free keywords: Biocatalysis; Enzyme mechanisms; Protein design
 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

show
hide
Language(s): eng - English
 Dates: 2018-12-182019-06-242019-07-19
 Publication Status: Published online
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1038/s41467-019-11155-3
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: Nature Communications
  Abbreviation : Nat. Commun.
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: London : Nature Publishing Group
Pages: - Volume / Issue: 10 Sequence Number: 3198 Start / End Page: - Identifier: ISSN: 2041-1723
CoNE: https://pure.mpg.de/cone/journals/resource/2041-1723