Journal
NATURE COMMUNICATIONS
Volume 10, Issue -, Pages -Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41467-019-11155-3
Keywords
-
Categories
Funding
- Max-Planck-Society
- Arthur C. Cope Fund
- National Natural Science Foundation of China [21574113]
- Zhejiang Provincial Natural Science Foundation [LY19B020014]
- INVEST NI Research and Development Program
- Strategic Priority Research Program (B) of the Chinese Academy of Sciences [XDB20000000]
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available