A two-metalloenzyme cascade constructs the azetidine-containing pharmacophore

Azetidine is a prominent pharmacophore present in dozens of drug-related molecules of both natural and synthetic origins. But how nature builds this moiety has long remained enigmatic. Here we address the full deciphering of a two-metalloenzyme cascade leading to polyoximic acid, an azetidine-containing moiety of the fungicide polyoxin. We demonstrate that the PolE enzyme functions as an Fe²⁺/pterin-dependent L-isoleucine desaturase. Moreover we illustrate that PolF is a new member of the emerging haem-oxygenase-like diiron oxidases, converting the desaturated L-isoleucine to polyoximic acid via an intramolecular C-N cyclization. Remarkably, we also establish that PolF exhibits dual functionality, orchestrating the sequential desaturation and cyclization with L-isoleucine as the initial substrate. Finally, our combined structural and quantum-mechanics/molecular-mechanics studies show that the PolF enzyme employs an extraordinary mechanism for the construction of the azetidine-containing moiety. These findings expand our knowledge on the catalysis of metalloenzymes and open the way for rational access of more azetidine-related molecules.