Mechanism (biology)

Gene

Quantum mechanics

Catalysis

Candida antarctica

Philosophy

Protein design

Biology

Active site

Protein engineering

Biochemistry

Protein structure

Computational biology

Cysteine

Stereochemistry

Physics

Mutant

Epistemology

Enzyme

Hydrolysis

Enzyme kinetics

Chemistry

Catalytic triad

Lipase

Directed evolution

Nature Communications

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.

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