Reshaping the Substrate-Binding Pocket of Ene-Reductase for Enhanced and Inverted Stereoselectivity: A Concise Access to the Stereocomplementary Chiral GABA Derivatives

Abstract

Despite the availability of numerous natural and engineered ene-reductases (ERs), enantiocomplementary synthesis of the sterically hindered molecules by ERs is still limited by poor substrate acceptance, particularly due to the insufficient complementary stereoselectivity. Herein, we reshaped the substrate-binding pocket of SeER from Saccharomyces eubayanus through semirational design, enabling ERs capable of stereocomplementary hydrogenating of the challenging substituted β-cyano cinnamic esters. Compared to the wild type, the variants exhibited enhanced activity (up to 161-fold) and catalytic efficiency k_cat/K_M (up to 358-fold), displaying potential in synthesizing various chiral β-cyano esters with high stereoselectivity (up to 99% ee). Molecular dynamics simulations demonstrated that the key for the superior catalytic performance of variants is the well-tuned substrate-binding pocket, which strengthens and stabilizes substrate recognition. Furthermore, we elucidated the practicality of the SeER variants in asymmetric synthesis of the chiral GABA derivatives (e.g., Phenibut, Baclofen, and Tolibut) via chemo-enzymatic cascade reactions.