Shield Machine-like Substrate Walking Strategy-Based Pocket Engineering of F-Amine Dehydrogenase for Accessing Structurally Diverse Fused-Ring and Linked-Ring Aryl Ketones

Although amine dehydrogenases (AmDHs) are emerging as attractive biocatalysts for chiral amine synthesis, their synthetic application in structurally diverse arylamines remains challenging, given the limited substrate acceptance. Substrate walking is an effective coevolution strategy to confer targeted substrate acceptance to an enzyme through a stepwise mutagenesis landscape adaptation. Here, based on the conventional substrate walking strategy, we report a shield machine-like substrate walking strategy to quickly evolve F-BbAmDH from Bacillus badius for accessing the difficult-to-aminate fused-ring and linked-ring aryl ketones. A set of monoring aryl ketone homologues with the benzene ring located at the end of the side-chain and regularly extended carbon skeletons was rationally selected as the transition substrates. A superior mutant library with expanded target fused-ring and linked-ring aryl ketone acceptance was identified based on the activity and specificity enhancement of the transition substrates, enabling the synthesis of pharmaceuticals and bioactive compound-related arylamines with up to 94% yield and 99% ee (R) or 99:1 cis/trans. Structure-based computational results provided molecular insights into the source of the expanded substrate acceptance. Our work demonstrates a concise engineering workflow for the collective acceptance evolution of enzymes for structurally diverse substrate panels and has promising prospects in enzyme engineering.