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.
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.
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