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

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Reetz,  Manfred T.
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Chemistry Department, Philipps-University;

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Citation

Cen, Y., Singh, W., Arkin, M., Moody, T. S., Huang, M., Zhou, J., et al. (2019). Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution. Nature Communications, 10: 3198. doi:10.1038/s41467-019-11155-3.


Cite as: https://hdl.handle.net/21.11116/0000-0004-65AE-7
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.