Mass spectrometric analyses for the C281-dimedone adduct and the sulfinylated peptide fragments of Orf1

ABSTRACT: 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 non-canonical 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.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Yung-Lin Wang  

LAB HEAD: Tsung-Lin Li

PROVIDER: PXD041104 | Pride | 2023-05-10

REPOSITORIES: Pride