N-Formimidoylation/-iminoacetylation modification in aminoglycosides requires FAD-dependent and ligand-protein NOS bridge dual chemistry

Yung Lin Wang, Chin Yuan Chang, Ning Shian Hsu, I. Wen Lo, Kuan Hung Lin, Chun Liang Chen, Chi Fon Chang, Zhe Chong Wang, Yasushi Ogasawara, Tohru Dairi, Chitose Maruyama, Yoshimitsu Hamano*, Tsung Lin Li*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of which the allosteric redox switch formed by the lysine-cysteine NOS bridge is an example. Here, we report a noncanonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin to form the antibiotic BD-12. X-ray crystallography was used to investigate this complex enzymatic process, which showed Orf1 has two substrate-binding sites that sit 13.5 Å apart unlike canonical FAD-dependent oxidoreductases. One site could accommodate glycine and the other glycinothricin or glycylthricin. Moreover, an intermediate-enzyme adduct with a NOS-covalent linkage was observed in the later site, where it acts as a two-scissile-bond linkage facilitating nucleophilic addition and cofactor-free decarboxylation. The chain length of nucleophilic acceptors vies with bond cleavage sites at either N–O or O–S accounting for N-formimidoylation or N-iminoacetylation. The resultant product is no longer sensitive to aminoglycoside-modifying enzymes, a strategy that antibiotic-producing species employ to counter drug resistance in competing species.

Original languageEnglish
Article number2528
JournalNature Communications
Issue number1
StatePublished - Dec 2023


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