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

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 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.

History of the streptothricin antibiotics and evidence for the neglect of the streptothricin resistome

“…The naturally occurring streptothricin analog BD-12 contains a formimidoylglycine group in place of β-lysine(s), notably lacking the β-amino group targeted by streptothricin acetyltransferases 48 . Neither in vitro reactions between BD-12 and a streptothricin acetyltransferase nor the aminoglycoside modifying enzyme AAC(6')-Ie-APH(6")-Ia resulted in an acetylated product, suggesting BD-12 may avoid inactivation by acetylating resistance enzymes 124 . Additional streptothricin derivatives with unnatural β-lysine modifications have been prepared through enzymatic modification in vitro, including replacement of β-lysine by 3-aminoproprionyl, 4-aminobutyl, or β-homolysine groups 124,125 .…”

“…Neither in vitro reactions between BD-12 and a streptothricin acetyltransferase nor the aminoglycoside modifying enzyme AAC(6')-Ie-APH(6")-Ia resulted in an acetylated product, suggesting BD-12 may avoid inactivation by acetylating resistance enzymes 124 . Additional streptothricin derivatives with unnatural β-lysine modifications have been prepared through enzymatic modification in vitro, including replacement of β-lysine by 3-aminoproprionyl, 4-aminobutyl, or β-homolysine groups 124,125 . Many of these streptothricin analogs have lower antimicrobial activity compared to the canonical streptothricins, suggesting that incorporating residues more similar to β-lysine, such as N-methylated ones, could potentially retain amineribosome electrostatic interactions necessary for high levels of antimicrobial activity (with ribose O2' groups at positions U1052 and C1054 16 ) while avoiding disruptive acetylation by resistance enzymes (Fig.…”

Unusual cysteine modifications in natural product biosynthesis

“… 26 A more recent study showed that Orf1, which is involved in the biosynthesis of BD-12, contains an N–O–S bridge that plays an important role in catalysis. 27 Orf1 is a noncanonical FAD-dependent enzyme responsible for appending a glycine-derived N -formimidoyl group to glycinothricin, producing the antibiotic BD-12. The X-ray crystallographic analysis revealed that Orf1 forms an oximinoglycine with an N–O–S–Cys281 substrate–enzyme bridge, which mediates the nucleophilic addition of glycinothricin and decarboxylation, leading to the cleavage of the N–O bond and the production of a formimidoylated final product.…”

“…The X-ray crystallographic analysis revealed that Orf1 forms an oximinoglycine with an N–O–S–Cys281 substrate–enzyme bridge, which mediates the nucleophilic addition of glycinothricin and decarboxylation, leading to the cleavage of the N–O bond and the production of a formimidoylated final product. 27 …”