A two-metalloenzyme cascade constructs the azetidine-containing pharmacophore

Rong Gong, Yao Qu, Jia Liu, Xuan Zhang, Liwei Zhou, Zhu Tian, Xinyue Zeng, Boyu Jin, Zhen Li, Le Yu, Ruyi Chen, Yang Zhou, Langxing Liao, Liulin Yang, Xuemin Song, You-Sheng Cai, Kun Shen, Zixin Deng, Zhengyu Zhang, Binju Wang & Wenqing Chen

Nature Chemistry 2025

https://www.nature.com/articles/s41557-025-01949-y

Abstract

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.