Engineering a C─N Lyase for Enantioselective Hydroaminations With Secondary Amines

C─N lyases from the aspartase/fumarase superfamily are attractive biocatalysts for the production of noncanonical amino acids via selective hydroamination of α , β ‐unsaturated carboxylic acids. However, the substrate scope of these enzymes remains largely restricted to primary amines, as secondary amines are usually poorly accepted. Here, we report the engineering of ethylenediamine‐ N , N’ ‐disuccinic acid (EDDS) lyase for enantioselective hydroaminations with secondary amines as non‐native substrates. This engineered C─N lyase variant (Y26A/Q159V/R294E) not only displays a 20‐fold increase in activity for dimethylamine addition to fumarate, but also processes secondary amines that show no reactivity with the wild‐type enzyme. Expanding the substrate scope of this C─N lyase to include secondary amines establishes a viable enzymatic platform for the enantioselective synthesis of structurally diverse tertiary amines.