Shifting the Substrate Scope of an Ene/Yne‐Reductase by Loop Engineering

Abstract Medium‐chain dehydrogenase/reductase‐superfamily related (MDR) ene‐reductases are highly neglected biocatalysts that have not yet been systematically studied in terms of rational protein engineering. Therefore, we crystallized the MDR‐related CaeEnR1 in its apo‐ and binary‐complex and searched for secondary structures that might influence enzyme activity toward specific substrate classes. By comparing the apo‐ and binary‐complexes, a flexible active‐site loop was identified that is involved in shaping the substrate and product cleft as well as the active‐site pocket. Furthermore, we could show that an active‐site loop undergoes drastic re‐arrangement after cofactor/substrate binding, revealing an open–closed mechanism. Based on these results, we subjected this active‐site loop to an alanine‐scan. The identified hits were then designed toward substituted cinnamaldehyde‐like substrates and an aliphatic/aromatic alkyne. From all tested amino acids P64, Y69, and S70 were identified as the most influential amino acid residues. Particularly the double mutations P64F/Y69A and Y69F/S70A showed an up to 20‐fold increase in conversion rates toward cinnamaldehyde‐like substrates. In contrast, a 2‐fold increase in conversion toward the aliphatic and aromatic alkyne was achieved with the S70F variant. With this study we state the foundation of protein engineering of the neglected MDR‐related ERs which opens up a new pathway for tailored biocatalysts.