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

Cen, Yixin, Singh, Warispreet, Arkin, Mamatjan, Moody, Thomas S., Huang, Meilan, Zhou, Jiahai, Wu, Qi and Reetz, Manfred T. (2019) Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution. Nature Communications, 10 (1). p. 3198. ISSN 2041-1723

[img]
Preview
Text
s41467-019-11155-3.pdf - Published Version
Available under License Creative Commons Attribution 4.0.

Download (2MB) | Preview
Official URL: https://doi.org/10.1038/s41467-019-11155-3

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.

Item Type: Article
Subjects: C700 Molecular Biology, Biophysics and Biochemistry
Department: Faculties > Health and Life Sciences > Applied Sciences
Depositing User: Elena Carlaw
Date Deposited: 01 Jun 2020 15:57
Last Modified: 01 Jun 2020 16:00
URI: http://nrl.northumbria.ac.uk/id/eprint/43298

Actions (login required)

View Item View Item

Downloads

Downloads per month over past year

View more statistics