Structural and Mechanistic Insights into Oxidative Biaryl Coupling to form Arylomycin Core by an Engineered CYP450

Arylomycin, a potent antibiotic targeting bacterial signal peptidases, 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 Cytochrome P450 enzyme 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 Arylomycin core via C-C bond formation in 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) 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 an efficient C-C formation, the reaction should undergo via biradical mechanism over hydroxylation.