Designing Michaelases: exploration of novel protein scaffolds for iminium biocatalysis†

Abstract

Biocatalysis is becoming a powerful and sustainable alternative for asymmetric catalysis. However, enzymes are often restricted to metabolic and less complex reactivities. This can be addressed by protein engineering, such as incorporating new-to-nature functional groups into proteins through the so-called expansion of the genetic code to produce artificial enzymes. Selecting a suitable protein scaffold is a challenging task that plays a key role in designing artificial enzymes. In this work, we explored different protein scaffolds for an abiological model of iminium-ion catalysis, Michael addition of nitromethane into E-cinnamaldehyde. We studied scaffolds looking for open hydrophobic pockets and enzymes with described binding sites for the targeted substrate. The proteins were expressed and variants harboring functional amine groups – lysine, p-aminophenylalanine, or N⁶-(D-prolyl)-L-lysine – were analyzed for the model reaction. Among the newly identified scaffolds, a thermophilic ene-reductase from Thermoanaerobacter pseudethanolicus was shown to be the most promising biomolecular scaffold for this reaction.