Engineering of Saccharomyces pastorianus old yellow enzyme 1 for the synthesis of pharmacologically active (S)-profen derivatives

Abstract

• Identification of the key residues for the stereo-control of ene reductase OYE1. • Engineering of OYE1 for the synthesis of active ( S )-profen derivatives. • Up to >99 % stereoselectivity was observed for the asymmetric reduction. 2-Arylpropionic acid derivatives, such as ibuprofen, constitute an important group of non-steroidal anti-inflammatory drugs (NSAIDs). Biocatalytic asymmetric reduction of 2-arylacrylic acid derivatives by ene reductases (EREDs) is a valuable approach for synthesis of these derivatives. However, previous bioreduction by EREDs resulted solely in nonpharmacological ( R )-enantiomers. Here, we present the engineering of Saccharomyces pastorianus old yellow enzyme 1 (OYE1) for the synthesis of pharmacologically active ( S )-profen derivatives. By structural comparison of substrate recognition in related EREDs and analysis of non-covalent contacts in the pro- S model of OYE1, the key residues of OYE1 were identified. Mutagenesis screening at these positions successfully provided the ( S )-stereoselective OYE1 variants, which catalyzed stereoselective bioreduction of various profen precursors to afford pharmacologically active ( S )-derivatives with up to >99 % ee values. Moreover, the key residues obtained from OYE1 successfully guided the engineering of OYE2.6 from Pichia stipites and KnOYE1 from Kazachstania naganishii for ( S )-selective reduction of 2-arylacrylic acid derivatives.