[Artificial enzyme design for the biosynthesis of chiral ketones]

Efficient asymmetric synthesis of chiral ketones is a significant challenge in green biomanufacturing. To address the scarcity of natural secondary amine enzymes and the insufficient activation capability of existing artificial enzymes towards ketone substrates, this study developed a novel artificial secondary amine enzyme, LmrR_V99C_IIDN, based on the protein scaffold of the Lactococcus lactis multidrug resistance regulator (LmrR). A covalent modification strategy using a disulfide bond was employed to introduce an isoindoline (IIDN) moiety containing an aromatic secondary amine center into the hydrophobic cavity of the protein. This engineered enzyme activates unsaturated enone substrates via an enamine catalysis mechanism, catalyzing their asymmetric Michael addition with dibenzyl malonate. Through site-specific screening and directed evolution, the optimal mutant LmrR_V99C_N19Y_IIDN was obtained. This variant achieved a yield of 73% and an enantioselectivity of 65% in the catalytic reaction with cyclohexenone. Substrate scope studies demonstrated that the enzyme exhibited good catalytic activity and stereoselectivity towards cyclic enones (such as cyclohexenone and cyclopentenone), while showing lower catalytic efficiency for linear enones. This study provides a new biocatalytic tool for the asymmetric functionalization of ketones and highlights the potential of synergistically regulating reaction selectivity through the combination of non-natural catalytic centers and protein scaffolds. It lays a foundation for expanding the application of artificial enzymes in the synthesis of complex chiral molecules.