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

Author

Listed:
  • Yixin Cen

    (Zhejiang University
    Chinese Academy of Sciences)

  • Warispreet Singh

    (Queen’s University
    Almac Sciences)

  • Mamatjan Arkin

    (Zhejiang University)

  • Thomas S. Moody

    (Almac Sciences)

  • Meilan Huang

    (Queen’s University)

  • Jiahai Zhou

    (Chinese Academy of Sciences)

  • Qi Wu

    (Zhejiang University)

  • Manfred T. Reetz

    (Max-Planck-Institut für Kohlenforschung
    Philipps-University)

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.

Suggested Citation

  • Yixin Cen & Warispreet Singh & Mamatjan Arkin & Thomas S. Moody & Meilan Huang & Jiahai Zhou & Qi Wu & Manfred T. Reetz, 2019. "Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution," Nature Communications, Nature, vol. 10(1), pages 1-10, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-11155-3
    DOI: 10.1038/s41467-019-11155-3
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