N-7′ methylation in apramycin: its biosynthesis and biological role

Apramycin, an aminoglycoside antibiotic widely used in veterinary medicine, contains a methyl group at N-7′ of the unusual octose moiety. Here we report a detailed investigation of the enzyme responsible for N-7′ methylation and the biological role of the methyl group in apramycin through a series of genetic and biochemical studies. We show that N-7′ methylation is catalyzed by a putative N-methyltransferase AprI, and occurs following the AprP-catalyzed N-7′ deacetylation step. Bioassay experiments indicate that N-7′-demethyl-apramycin exhibits lower antibacterial activity than apramycin, suggesting the bioactivity-improving effect of the methyl group. Intriguingly, the target binding assay shows that apramycin and N-7′-demethyl-apramycin exhibit the same level of affinity for the E. coli 16S rRNA, implying that apramycin may have an alternative target site in addition to the well-recognized 16S rRNA A-site. Therefore, this study provides insight into the assembly logic of apramycin at the pseudotrisaccharide stage and the development of apramycin derivatives with improved antibacterial activity based on the N-7′ methyl group.