Mechanistic Insights into Enantiocontrolling of Styrene Monooxygenase-Catalyzed Epoxidation of Olefins

Although styrene monooxygenases (SMOs) are widely used in the production of (S)- or (R)-enantiopure epoxides, the underlying enantiocontrol mechanism of SMO-catalyzed epoxidation remains elusive. Herein, we observed that the substrate-binding pose in the catalytic center, which was codetermined by the residues in the catalytic center and size of the alkyl moiety in the substrates, governed the enantioselectivity of the StyA-catalyzed epoxidation. The mutagenesis of the aromatic residue at site 73 into a nonaromatic residue or that of the nonaromatic residue at site 211 into aromatic residues resulted in the inversion of the enantioselectivity. The variants Y73V, V211F, V211Y, Y73V/V211F, and Y73V/V211Y of the (S)-selective StyA exhibited reversible enantioselectivity during the catalyzed epoxidation of 1-phenylcyclohexene derivatives, yielding the corresponding (R)-epoxides with high enantioselectivity (68–85% ee). Moreover, the (R)-selective variants catalyzed the olefins that harbored large hydrophobic groups, such as 1-phenylcyclohexene derivatives, into their corresponding (R)-epoxides with high enantioselectivity (up to >99% ee). However, these (R)-selective variants retained (S)-selectivity for the epoxidation of styrene, trans-β-methylstyrene, and trans-β-ethylstyrene. The elucidation of the enantiocontrolling mechanism of SMOs would thus be valuable for creating efficient styrene monooxygenases with different enantioselectivities.