Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution
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 thi...
| Published in: | Nature Communications |
|---|---|
| Main Authors: | , , , , , , , |
| Format: | Text |
| Language: | English |
| Published: |
Nature Publishing Group UK
2019
|
| Subjects: | |
| Online Access: | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642262/ http://www.ncbi.nlm.nih.gov/pubmed/31324776 https://doi.org/10.1038/s41467-019-11155-3 |
| Summary: | 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. |
|---|