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

Yixin Cen, Warispreet Singh, Mamatjan Arkin, Thomas S Moody, Meilan Huang, Jiahai Zhou, Qi Wu, Manfred T Reetz

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)
15 Downloads (Pure)

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 languageEnglish
Article number3198
JournalNature Communications
Volume10
Issue number1
Early online date19 Jul 2019
DOIs
Publication statusPublished - Dec 2019
Externally publishedYes

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