posted on 2024-01-29, 13:34authored byHui Lin, Shengyuan Fang, Hang Gao, Zhangyuan Qin, Donglin Fan, Na Li, Zhongliu Wu, Hongge Chen
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.