Chemomimetic design and directed evolution of enzymes for iminium biocatalysis Guangcai Xu 1 , Michele Crotti 2 , Andreas Kunzendorf 3 , Gerrit J. Poelarends* 1 University of Manchester, UK, 2 University of Groningen, The Netherlands Manchester Institute of Biotechnology, University of Manchester, UKDepartment of Chemistry, University College London, UKInstitute of Biochemistry, University of Greifswald, GermanyDeveloping new enzymes is crucial for the application of biocatalysis in conquering challenging synthesis objectives. In addition to natural enzyme discovery, fruitful new-to-natural enzymes can be obtained by chemomimetic redesign of existing enzymes. Organocatalysis was originally bio-inspired and has become the third pillar of asymmetric catalysis. Herein, we transferred iminium catalysis, a fundamental branch of organocatalysis back to enzyme platforms to create novel biocatalysts. By repurposing two natural enzymes with catalytic amines: 4-oxalocrotonate tautomerase (4-OT, catalytic N-terminal Proline) and deoxyribose-phosphate aldolase (DERA, catalytic Lysine), we have created various novel biocatalysts to promote valuable transformations inspired by iminium catalysis. We used directed evolution to boost these iminium catalysis activities. Using 4-OT as a starting enzyme, we have developed: a Michaelase promoting the conjugate additions of nitromethane to α,β-unsaturated aldehydes, a non-natural cofactor-independent peroxyzyme catalysing epoxidation reactions, and a cofactor-independent enzyme for cyclopropanation reactions. We have also switched the catalytic machinery of DERA from enamine to iminium catalysis to promote Michael addition reactions. Our research on developing new activities in enzymes by iminium catalysis illustrates the power of combining chemomimetic biocatalyst design and directed evolution to generate useful new-to-nature enzymes. References 1. Angew. Chem. Int. Ed. 2022 , 61 , e202203613 Angew. Cem. Int. Ed. 2022 , 61 , e202113970 2. Chem. Eur. J. 2022 , 28 , e202201651 3. Angew. Chem. Int. Ed. 2021 , 60 , 24059-24063 4. ACS Catal. 2021 , 11 , 13236-13243 5. Angew. Chem. Int. Ed. 2020 , 59 , 10374-10378
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