Engineering of halohydrin dehalogenases for the regio- and stereoselective synthesis of (S)-4-aryl-2-oxazolidinones

Abstract

Cyanate-mediated epoxide ring-opening by halohydrin dehalogenases (HHDHs) is a promising method for the synthesis of important optically pure 2-oxazolidinones. However, the regio- and stereoselectivity of the naturally occurring HHDHs are still not satisfactory for styrene oxides (SOs). We used a structure-guided semi-rational design to improve the selectivity of HHDHs from Ilumatobacter coccineus (IcHheG), followed by site-saturation and combinatorial mutagenesis of Y18, L103, I104, and N196. The double mutant I104F/N196W showed dramatically improved selectivity toward SOs with excellent regioselectivity (>99 : 1) and stereoselectivity (E > 200), producing the corresponding useful and valuable (S)-4-aryl-2-oxazolidinone and (S)-SO with 98% ee. Moreover, it accepted a wider range of styrene oxides with moderate to excellent selectivity. Analysis of the substrate binding conformations showed that the regio- and stereoselectivity improvement in IcHheG was attributed to an increase of geometric distances to the two carbons and the oxygen atom of the epoxide ring for a single stereoisomer. This study elucidated key structural motifs responsible for regulating the regio- and stereoselectivity of IcHheG, offering theoretical guidance for protein engineering of HHDHs from other organisms.