Probing the Mechanism of CAL‐B‐Catalyzed aza‐Michael Addition of Aniline Compounds with Acrylates Using Mutation and Molecular Docking Simulations

• Published in: ChemistrySelect
• Main Authors: Gu, Bo, E Hu, Zu−, Yang, Zeng‐Jie, Li, Jun, Zhou, Zi‐Wen, Wang, Na, Yu, Xiao‐Qi
• Other Authors: National Natural Science Foundation of China
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 2019
• Subjects: Antarc*; Antarctica
• Online Access: http://dx.doi.org/10.1002/slct.201900112; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fslct.201900112; https://onlinelibrary.wiley.com/doi/pdf/10.1002/slct.201900112; https://onlinelibrary.wiley.com/doi/full-xml/10.1002/slct.201900112

Abstract CAL−B (Lipase B from Candida antarctica ) catalyzed aza‐Michael addition of a set of aniline compounds with acrylates under mild conditions was described. A systematic study allowed to determine the appropriate solvent, enzyme loading, reaction temperature and time. In order to speculate and verify its mechanism, the wild‐type and three mutants (S105 A, H224 A and I189 A) of CAL−B were expressed. Some control experiments demonstrated the active site was responsible for the enzymatic process, in which Ser105 and His224 played a crucial role. Besides, the mutation of Ile189 also affected its activity a lot. Based on these results, a docking experiment was performed to speculate the mechanism: the oxyanion hole (Thr40 and Gln106) of the active site activated the acrylates and stabilized the transition states. The Ile189 residues, as an important part of active cavity, could form a strong hydrophobic interaction with substrates. And the Ser105 and His224 residues were responsible for proton transfer during the catalytic process. This would help to understand the promiscuity of CAL−B, and provide ideas to design novel enzyme to improve the efficiency of its promiscuity.