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.
Original language | English |
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Article number | 3198 |
Journal | Nature Communications |
Volume | 10 |
Issue number | 1 |
Early online date | 19 Jul 2019 |
DOIs | |
Publication status | Published - Dec 2019 |
Externally published | Yes |
Keywords
- Binding Sites
- Candida/enzymology
- Catalysis
- Catalytic Domain
- Crystallography, X-Ray
- Cysteine/chemistry
- Enzyme Activation
- Fungal Proteins/chemistry
- Hydrolysis
- Kinetics
- Lipase/chemistry
- Models, Molecular
- Mutation
- Protein Conformation
- Protein Engineering/methods
- Substrate Specificity