Domain dissection and characterization of the aminoglycoside resistance enzyme ANT(3″)-Ii/AAC(6′)-IId from Serratia marcescens

► The activity of each domain of ANT(3″)/AAC(6′) is independent of the other domain. ► ANT(3″) plays a critical role in binding of aminoglycosides to AAC(6′). ► Many (d)NTPs are accepted equally well as cosubstrates of ANT(3″)/AAC(6′). ► CoA analogs are preferentially transferred to 4,6-disubstituted aminoglycosides.