Structural and mechanistic insights into oxidative biaryl coupling to form the arylomycin core by an engineered CYP450†

Arylomycin, a potent antibiotic targeting bacterial signal peptidase, is difficult to synthesize experimentally due to its poor to moderate yields and the formation of a mixture of compounds. A recent experimental bioengineering work shows that the core of arylomycin can be efficiently synthesized by engineering the cytochrome P450 enzyme from Streptomyces sp.; however, the mechanism of the same was not elucidated. Herein, we have thoroughly investigated the mechanism behind the evolution of the enzyme for the synthesis of the arylomycin core via C–C bond formation in the CYP450 enzyme using hybrid QM/MM calculations, MD simulations, and DFT calculations. We show that strategic mutations such as (a) G-101 → A facilitate biaryl coupling by subtly pushing the substrate and (b) the Q-306 → H mutation creates a strong pi–pi interaction with the substrate that brings the two phenol rings of the substrate closer to undergo C–C coupling. Importantly, our QM/MM calculations show that for efficient C–C formation, the reaction should proceed via the biradical mechanism rather than hydroxylation.