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

• Published in: Nature Communications
• Main Authors: Cen, Y., Singh, W., Arkin, M., Moody, T., Huang, M., Zhou, J., Wu, Q., Reetz, M.
• Format: Article in Journal/Newspaper
• Language: English
• Published: 2019
• Subjects: Antarc*; Antarctica
• Online Access: http://hdl.handle.net/21.11116/0000-0004-65AE-7

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.