Substrate Conformational Switch Governs Selective C–S Bond Formation by P450 CxnD

Sulfur-containing heterocycles represent important structural motifs in natural products and pharmaceutical compounds. P450 enzyme CxnD catalyzes the intramolecular C-S bond formation that constructs the tricyclic dihydrothiopyranoindole scaffold during chuangxinmycin biosynthesis. Here, we combine multiscale simulations to elucidate the molecular mechanism underlying this selective transformation. Our results reveal that following initial N-H abstraction by Compound I, the substrate radical undergoes a pronounced conformational rearrangement that promotes subsequent S-H abstraction and properly aligns the reactive centers for selective C-S bond formation. The conformational switch is driven by attenuation of the hydrogen-bonding interaction between the indole radical and the heme moiety, which permits displacement of the indole nitrogen away from the iron center and disruption of the original salt bridge with Lys238. This enables stabilization of a new reactive conformer through formation of a salt bridge with Lys61. Moreover, comparative simulations of the substrate enantiomer reveal that conformational flexibility of the substrate radical serves as a key determinant of reactivity in the CxnD/2S-5 system. Together, these findings illuminate how substrate conformational dynamics critically modulate the selectivity of C-S coupling in P450-catalyzed reactions.