A flexible active-site loop that is involved in shaping the substrate and product cleft as well as the active-site pocket was identified for the MDR-related ene/yne-reductase CaeEnR1 from Cyclocybe aegerita. Double mutations of this loop showed an up to 20-fold increase in conversion rates toward cinnamaldehyde-like substrates and a 2-fold increase in conversion toward an aliphatic and aromatic alkyne.

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.