Genome Mining and Characterization of a Heme-Dependent Enzyme Catalyzing Intermolecular Nitrogen–Nitrogen Bond Formation in Hydrazinosuccinic Acid Biosynthesis

Nitrogen–nitrogen (N–N) bond-forming enzymes are rare but play vital roles in both primary and secondary metabolism. Guided by a nitric oxide synthase (NOS)-based genome mining strategy, we report the discovery and characterization of a new heme-dependent enzyme system that catalyzes intermolecular N–N bond formation. Using both in vivo and in vitro reconstitution approaches, we demonstrated that a protein complex, comprising a heme enzyme and a 2[4Fe–4S] ferredoxin partner, mediates the coupling of the α-amine group of L-aspartate with inorganic nitrogen oxide species, such as nitrite or nitric oxide, to generate hydrazinosuccinic acid, a key biosynthetic precursor in several natural product pathways. Structural modeling and site-directed mutagenesis suggest a plausible catalytic mechanism involving the formation of a reactive nitrogen intermediate, potentially a heme-bound nitrene species. These findings reveal a new family of N–N bond-forming biocatalysts that leverage inorganic nitrogen sources, offering valuable tools for genome mining and the synthetic biology.