Enzymatic Tailoring in Luzopeptin Biosynthesis Involves Cytochrome P450‐Mediated Carbon–Nitrogen Bond Desaturation for Hydrazone Formation

Luzopeptins and related decadepsipeptides are bisintercalator nonribosomal peptides featuring rare acyl‐substituted tetrahydropyridazine‐3‐carboxylic acid (Thp) subunits that are critical to their biological activities. Herein, we reconstitute the biosynthetic tailoring pathway in luzopeptin A biosynthesis through in vivo genetic and in vitro biochemical approaches. Significantly, we revealed a multitasking cytochrome P450 enzyme that catalyzes four consecutive oxidations including the highly unusual carbon–nitrogen bond desaturation, forming the hydrazone‐bearing 4‐OH‐Thp residues. Moreover, we identified a membrane‐bound acyltransferase that likely mediates the subsequent O‐acetylation extracellularly, as a potential self‐protective strategy for the producer strain. Further genome mining of novel decadepsipeptides and an associated P450 enzyme have provided mechanistic insights into the P450‐mediated carbon–nitrogen bond desaturation. Our results not only reveal the molecular basis of pharmacophore formation in bisintercalator decadepsipeptides, but also expand the catalytic versatility of P450 family enzymes. Luzopeptins are potent antitumor and antiviral agents with their biological activities controlled by rare modified tetrahydropyridazine structural subunits. Reconstitution of the tailoring pathway in luzopeptin A biosynthesis reveals the molecular basis of this key pharmacophore formation, which involves unusual carbon–nitrogen bond desaturation by multitasking cytochrome P450 and extracellular O‐acetylation by integral membrane acyltransferase.