Semi-rational protein engineering of a novel ene-reductase from Galdieria sulphuraria for asymmetric reduction of (R)-carvone and ketoisophorone.

Asymmetric reduction of (R)-carvone and ketoisophorone by an engineered ene-reductase from Galdieria sulphuraria (GsOYE) combined with glucose dehydrogenase for NADPH regeneration were studied. A semi-rational protein engineering was used to enhance the activity and selectivity of GsOYE. Upon the sequence alignment and molecular docking results, two amino acid residues at positions 66 and 270 were selected as saturation mutation sites. Finally, a single substitution variant of GsOYE-N270A with complete conversion (100%) and diastereoselectivity (de >99%) for reduction of (R)-carvone and a double substitution variant GsOYE-Y66P/N270H with improved stereoselectivity for reduction of ketoisophorone were obtained.