Thiol-Mediated Hydrazide Installation from Diazo in the Biosynthesis of Spinamycin

Natural products featuring nitrogen-nitrogen (N-N) bonds often possess potent bioactivities, and their architectures frequently reveal novel enzymatic mechanisms. Current understanding for acylhydrazide installation is limited to a "hydrazine-first" strategy, wherein an activated acyl donor is nucleophilically attacked by a preassembled hydrazine intermediate. Spinamycin, an antifungal hydrazide-containing aryl polyene from Streptomyces albospinus, challenges this convention. Its biosynthetic machinery utilizes SpiA7, an ATP-dependent, nitrite-utilizing N-nitrosylase, to synthesize a highly transient diazo intermediate rather than a hydrazine core. How this unstable diazo compound is converted into a stable hydrazide has remained a long-standing biosynthetic mystery. Herein, we report the in vitro reconstitution of the activity of SpiA7 in conversion of the amine substrate into its mature succinylhydrazide form. Mechanistic characterization revealed that the electrophilic diazo intermediate is initially captured by thiol reagents to generate a covalent azo-sulfide intermediate (R-N═N-S-R'). This species likely undergoes a noncanonical four-electron reduction driven cooperatively by disulfide bond formation and the oxidative decarboxylation of α-ketoglutarate (αKG), which serves as the succinyl donor. Furthermore, site-directed mutagenesis identifies Cys453 of SpiA7 as a likely active-site thiol donor orchestrating this transformation. Collectively, our findings unveil an unprecedented chemical strategy for hydrazide assembly that completely bypasses a free hydrazine intermediate, expanding nature's known catalytic repertoire for building complex N-N linkages.