Discovery of a Heme‐Dependent Enzyme Catalyzing Nitrogen–Nitrogen Bond Formation in Kinamycin Biosynthesis

A nitrogen-nitrogen (N─N) bond is a core feature of diverse natural products with interesting structural and biological properties. Kinamycin and lomaiviticin, featuring a diazobenzo[b]fluorene core, exhibit exceptional potency as chemotherapeutic agents. However, the N─N bond forming step in their biosynthesis has remained elusive. Through extensive mutagenesis and biochemical studies, we herein report that Alp1J, belonging to a new family of heme-dependent enzymes, catalyzes the N─N bond formation in kinamycin biosynthesis. Interestingly, Alp1J forms a stable complex with its partner ferredoxin Alp1I, which can protect the cofactors and is critical for the N─N bond formation activity. With its partner ferredoxin, Alp1J catalyzes formation of the hydrazine intermediate directly from l-aspartate and nitrite by a pathway involving four-electron reduction. Our findings expand the knowledge of enzymatic N─N bond formation and show the potential for the discovery and development of novel N─N bond containing natural products through genome mining and synthetic biology.