A heme-dependent enzyme forms the hydrazine in the antibiotic negamycin

• Published in: Nature chemical biology Vol. 21; no. 7; pp. 1012 - 1020
• Main Authors: Wang, Menghua, Wei, Zi-Wang, Ryan, Katherine S.
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.07.2025; Nature Publishing Group
• Subjects: 631/45/321; 631/92/60; 639/638/92/349; Anti-Bacterial Agents - biosynthesis; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - metabolism; Antibiotics; Bacteria; Biochemical Engineering; Biochemistry; Bioinformatics; Bioorganic Chemistry; Biosynthesis; Cell Biology; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Chromatography; Codons; Complementation; Dipeptides - biosynthesis; Dipeptides - chemistry; Dipeptides - metabolism; Drug resistance; Duchenne's muscular dystrophy; Dystrophy; Enzymes; Ferredoxin; Gene deletion; Genes; Genomes; Genomics; Glycine; Gram-negative bacteria; Heme; Heme - metabolism; Hereditary diseases; Hydrazine; Hydrazines; Hydrazines - chemistry; Hydrazines - metabolism; Lysine; Lysine - chemistry; Lysine - metabolism; Mass spectrometry; Multidrug resistance; Muscular dystrophy; Natural products; Nitrogen; Pathogens; Pharmacology; Proteins; Scientific imaging; Stable isotopes; Streptomyces - enzymology; Streptomyces - genetics; Streptomyces - metabolism
• ISSN: 1552-4450; 1552-4469; 1552-4469
• DOI: 10.1038/s41589-025-01898-0

Negamycin, a hydrazine-containing dipeptide-like antibiotic, was first isolated in 1970 from three strains of Streptomyces purpeofuscus . Its pronounced antibacterial properties render it an appealing candidate for combating multi-drug-resistant Gram-negative bacteria. Additionally, the unique readthrough-promoting activity makes it a subject for research as a potential therapeutic agent for Duchenne muscular dystrophy and other hereditary diseases. Here we use the unusual ( R ) - β-lysine found in negamycin as a guide to identify the biosynthetic pathway of negamycin and then carry out gene deletion and chemical complementation, stable isotope feeding and enzyme assays to elucidate the key precursors for negamycin assembly. Our work identified NegB as a lysine-2,3-aminomutase that converts lysine into ( R ) - β-lysine and NegJ as a heme-dependent, N–N bond-forming enzyme. We show that NegJ, together with a ferredoxin encoded outside of the negamycin gene cluster, directly forms hydrazinoacetic acid from glycine and nitrite. NegJ is a novel biocatalyst for N–N bond formation, and our work highlights its potential for genome mining of N–N bond-containing natural products. Negamycin is a decades-old antibiotic that can promote readthrough of premature stop codons and possesses a structure that contains an unusual N–N bond. Now, the long-mysterious negamycin gene cluster has been identified and characterized to reveal a heme-dependent enzyme that directly couples glycine and nitrite to form the N–N linkage.