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

Aminoglycosides (AGs) are broad-spectrum antibiotics whose constant use and presence in growth environments has led bacteria to develop resistance mechanisms to aid in their survival. A common mechanism of resistance to AGs is their chemical modification (nucleotidylation, phosphorylation, or acetylation) by AG-modifying enzymes (AMEs). Through evolution, fusion of two AME-encoding genes has resulted in bifunctional enzymes with broader spectrum of activity. Serratia marcescens, a human enteropathogen, contains such a bifunctional enzyme, ANT(3"-Ii/AAC(6')-Ild. To gain insight into the role, effect, and importance of the union of ANT(3")-Ii and AAC(6')-Ild in this bifunctional enzyme, we separated the two domains and compared their activity to that of the full-length enzyme. We performed a thorough comparison of the substrate and cosubstrate profiles as well as kinetic characterization of the bifunctional ANT(3"-Ii/AAC(6')-ild and its individually expressed components. (C) 2013 Elsevier Masson SAS. All rights reserved.