Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase‐2″‐IVa

Acquired resistance to aminoglycoside antibiotics primarily results from deactivation by three families of aminoglycoside‐modifying enzymes. Here, we report the kinetic mechanism and structure of the aminoglycoside phosphotransferase 2″‐IVa (APH(2″)‐IVa), an enzyme responsible for resistance to aminoglycoside antibiotics in clinical enterococcal and staphylococcal isolates. The enzyme operates via a Bi‐Bi sequential mechanism in which the two substrates (ATP or GTP and an aminoglycoside) bind in a random manner. The APH(2″)‐IVa enzyme phosphorylates various 4,6‐disubstituted aminoglycoside antibiotics with catalytic efficiencies ( k cat / K m ) of 1.5 × 10 3 to 1.2 × 10 6 (M −1  s −1 ). The enzyme uses both ATP and GTP as the phosphate source, an extremely rare occurrence in the phosphotransferase and protein kinase enzymes. Based on an analysis of the APH(2″)‐IVa structure, two overlapping binding templates specifically tuned for hydrogen bonding to either ATP or GTP have been identified and described. A detailed understanding of the structure and mechanism of the GTP‐utilizing phosphotransferases is crucial for the development of either novel aminoglycosides or, more importantly, GTP‐based enzyme inhibitors which would not be expected to interfere with crucial ATP‐dependent enzymes.