Dissecting the cosubstrate structure requirements of the Staphylococcus aureus aminoglycoside resistance enzyme ANT(4′)

► Deoxy-ribonucleotide triphosphates (dNTPs) are better cosubstrates than ribonucleotide triphosphates (NTPs) for ANT(4′). ► The position of the triphosphate group (5′ and not 3′) on the ribose/deoxyribose ring of the cosubstrate is important for recognition by ANT(4′). ► NTPs with large substituents at the 3′-position of the ribose ring are not cosubstrates for ANT(4′). ► Nucleotidylation of aminoglycosides results in completely biologically inactive compounds.