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

Nat Commun. 2019 Jul 19;10(1):3198. doi: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Candida / enzymology
  • Catalysis
  • Catalytic Domain
  • Crystallography, X-Ray
  • Cysteine / chemistry*
  • Enzyme Activation
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Hydrolysis
  • Kinetics
  • Lipase / chemistry*
  • Lipase / genetics
  • Lipase / metabolism
  • Models, Molecular
  • Mutation
  • Protein Conformation
  • Protein Engineering / methods
  • Substrate Specificity

Substances

  • Fungal Proteins
  • Lipase
  • lipase B, Candida antarctica
  • Cysteine