Tailoring the S ‐Selectivity of 2‐Succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate Synthase (MenD) from Escherichia coli

The thiamine diphosphate (ThDP)‐dependent enzyme 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate synthase from Escherichia coli ( Ec MenD, E.C. 2.2.1.9) catalyzes the carboligation of α‐ketoglutarate (α‐KG) and various benzaldehyde derivatives with excellent chemo‐ as well as high R ‐selectivity (enantiomeric excess ( ee ) >93 %) to yield chiral α‐hydroxy ketones. Based on the recently developed S ‐pocket concept, we engineered S ‐selective Ec MenD variants by optimizing the steric properties and stabilization of the acceptor substrate in the S ‐pocket. Moreover, the moderate S ‐selectivity of the Ec MenD variant I474A/F475G described recently for the carboligation of α‐KG and benzaldehyde ( ee =75 %) could be improved by selective destabilization of the R ‐pathway, which resulted in the variant I474A/F475G/R395Y ( ee =85 % S ). Subsequent investigation of the acceptor substrate range of this new variant revealed high S ‐selectivity especially with meta ‐substituted benzaldehydes, which gave access to 5‐hydroxy‐4‐oxo‐5‐arylpentanoates with excellent enantioselectivities of up to 99 % ee S . Thus, opening the S ‐pocket and simultaneous destabilization of the R ‐pathway provides a potential general new strategy to enhance the S ‐selectivity of ThDP‐dependent enzymes.