Non‐Heme Iron Enzymes Catalyze Heterobicyclic and Spirocyclic Isoquinolone Core Formation in Piperazine Alkaloid Biosynthesis

We report the discovery and biosynthesis of new piperazine alkaloids‐arizonamides, and their derived compounds‐arizolidines, featuring heterobicyclic and spirocyclic isoquinolone skeletons, respectively. Their biosynthetic pathway involves two crucial non‐heme iron enzymes, ParF and ParG, for core skeleton construction. ParF has a dual function facilitating 2,3‐alkene formation of helvamide, as a substrate for ParG, and oxidative cleavage of piperazine. Notably, ParG exhibits catalytic versatility in multiple oxidative reactions, including cyclization and ring reconstruction. A key amino acid residue Phe67 was characterized to control the formation of the constrained arizonamide B backbone by ParG. The biosynthesis of newly identified alkaloids featuring unique heterobicyclic and spirocyclic isoquinolone skeletons, arizonamides and arizolidines, has been characterized. Notably, the non‐heme iron enzyme ParG demonstrated catalytic versatility in multiple oxidative reactions, including cyclization and ring reconstruction. This study has elucidated efficient strategies used by Nature to construct complex multicyclic heterocycle scaffolds.