Systematic Evaluation of Imine‐Reducing Enzymes: Common Principles in Imine Reductases, β‐Hydroxy Acid Dehydrogenases, and Short‐Chain Dehydrogenases/ Reductases

The enzymatic, asymmetric reduction of imines is catalyzed by imine reductases (IREDs), members of the short-chain dehydrogenase/reductase (SDR) family, and β-hydroxy acid dehydrogenase (βHAD) variants. Systematic evaluation of the structures and substrate-binding sites of the three enzyme families has revealed four common principles for imine reduction: structurally conserved cofactor-binding domains; tyrosine, aspartate, or glutamate as proton donor; at least four characteristic flanking residues that adapt the donor's pKa and polarize the substrate; and a negative electrostatic potential in the substrate-binding site to stabilize the transition state. As additional catalytically relevant positions, we propose alternative proton donors in IREDs and βHADs as well as proton relays in IREDs, βHADs, and SDRs. The functional role of flanking residues was experimentally confirmed by alanine scanning of the imine-reducing SDR from Zephyranthes treatiae. Mutating the gatekeeping phenylalanine at standard position 200 resulted in a tenfold increase in imine-reducing activity.