Revisiting the Catalytic Cycle and Kinetic Mechanism of ANT(2″): A Structural and Kinetic Study

Aminoglycoside antibiotics have lost much of their effectiveness due to widespread resistance, primarily via covalent modification. One of the most ubiquitous enzymes responsible for aminoglycoside resistance is ANT(2″), which catalyzes a nucleotidylation reaction. Due to its clinical importance, much research has focused on dissecting the mechanism of action, some of it dating back more than 30 years. Here we present structural data for catalytically informative states of the enzyme, i.e. ANT(2″) in complex with AMP and tobramycin (inactive-intermediate state), and in complex with adenylyl-2″-tobramycin, pyrophosphate, and Mn2+(product-bound state). These two structures in conjunction with our previously reported structure of ANT(2″)’s substrate-bound complex, capture clinical states along ANT(2″)’s reaction coordinate. Additionally, ITC-based studies are presented that assess the order of substrate binding and product re-lease. Combined, these results outline a kinetic mechanism for ANT(2″) that contrad...