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 <i>Streptomyces</i> 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 <i>via</i> 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 <i>via</i> the biradical mechanism rather than hydroxylation.