N-Formimidoylation/-iminoacetylation requires a specialized enzyme employing FAD-dependent and ligand-protein NOS bridge dual chemistry

Abstract Oxidized cysteine residues are highly reactive and may form covalent conjugates with other residues in proteins to enable proper functionalities, for example, the lysine-cysteine NOS bridge identified recently as an allosteric redox switch. To our knowledge, the NOS-covalent species per se directly engaging in reactions has not been previously demonstrated. Here, we report a non-canonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin forming BD-12, an enzymatic process demanding a cascade of delicate chemical reactions. To illustrate how it proceeds, we pursed X-ray crystallography whereby crystal complexes display two tetrameric oligomers in an asymmetric unit. Unlike canonical FAD-dependent oxidoreductases, two substrate-binding sites that sit 13.5 Å apart were uncovered one for glycine and the other for glycinothricin/glycylthricin. An intermediate-enzyme adduct in an NOS-covalent linkage was spotted at the latter, where it acts as though a two-scissile-bond linkage rod permits nucleophilic addition and cofactor-free decarboxylation reactions to take place. The chain length of nucleophilic acceptors contends with bond cleavage sites at either N–O or O–S accounting for N-formimidoylation or -iminoacetylation. The resultant is no more sensitive to aminoglycoside-modifying enzymes, an acute strategy that antibiotic-producing species employs to reinvigorate its armory countering drug resistance of competing species.