Substrate‐Guided Engineering of Imine Reductases: A Strategy for Synthesizing Tertiary Amine <i>N</i> ‐Heterocycles

The enzymatic synthesis of N‑heterocyclic tertiary amines remains underdeveloped, largely due to the time and resource demands of traditional enzyme engineering methods. Guided by key substrate features including substrate size and the positive charge of the imine intermediate, we employed a straightforward and accessible computational design to engineer the imine reductase variant M5. We successfully constructed the combinatorial mutant M5-W234I-M203A-I149G, which exhibits significantly enhanced catalytic activity toward N‑heterocyclic secondary amines, with improved substrate binding and kinetics. The variant maintained high activity across a range of secondary amines (30 mM), whether generated via a two‑step synthesis or direct use. Our results indicate that interactions between residues are a critical consideration in the construction of highly active combinatorial mutants. This work provides an accessible design strategy and establishes a rational design framework for imine reductases engineering.