Structural and Mechanistic Insight into the Enantioselectivity of (R)‐Selective Styrene Monooxygenases: A Tug‐of‐War between Proximal and Distal Residues

Group E flavoprotein monooxygenases (GEMs) are well‐known for catalyzing enantioselective epoxidation reactions. However, engineering their enantioselectivity remains a significant challenge, largely due to a limited understanding of the underlying mechanisms. Among these enzymes, (R)‐selective styrene monooxygenases ((R)‐SMOs) stand out due to their unusual enantio‐switch behavior when catalyzing p‐substituted styrenes. This unique property provides an exceptional opportunity to investigate the enantiocontrol mechanisms within GEMs. In this study, we resolved the first crystal structure of an (R)‐SMO, SeStyA, derived from Streptomyces. By integrating this structural information with molecular docking and molecular dynamics (MD) simulations, we identified four key residues critical to enantiodivergency: two distal residues (S178 and A219) and two proximal residues (A59 and A312). Strikingly, a "tug‐of‐war" mechanism was revealed through saturation mutagenesis, wherein the side‐chain sizes of proximal and distal residues exerted opposing influences on enantioselectivity at the C=C bond. Leveraging this mechanistic insight, we successfully engineered SMOs with excellent (R)‐ or (S)‐enantioselectivity.