Background: Aminoglycoside antibiotic resistance is largely the result of the production of enzymes that covalently modify the drugs including kinases (APHs) with structural and functional similarity to protein and lipid kinases. One of the most important aminoglycoside resistance enzymes is AAC(6′)-APH(2″), a bifunctional enzyme with both aminoglycoside acetyltransferase and kinase activities. Knowledge of enzyme active site structure is important in deciphering the molecular mechanism of antibiotic resistance and here we explored active site labeling techniques to study AAC(6′)-APH(2″) structure and function. Results: AAC(6′)-APH(2″) was irreversibly inactivated by wortmannin, a potent phosphatidylinositol 3-kinase inhibitor, through the covalent modification of a conserved lysine in the ATP binding pocket. 5′-[p-(Fluorosulfonyl)benzoyl]adenosine, an electrophilic ATP analogue and known inactivator of other APH enzymes such as APH(3′)-IIIa, did not inactivate AAC(6′)-APH(2″), and reciprocally, wortmannin did not inactivate APH(3′)-IIIa. Conclusions: These distinct active site label sensitivities point to important differences in aminoglycoside kinase active site structures and suggest that design of broad range, ATP binding site-directed inhibitors against APHs will be difficult. Nonetheless, given the sensitivity of APH enzymes to both protein and lipid kinase inhibitors, potent lead inhibitors of this important resistance enzyme are likely to be found among the libraries of compounds directed against other pharmacologically important kinases.
All Science Journal Classification (ASJC) codes
- Molecular Medicine
- Molecular Biology
- Drug Discovery
- Clinical Biochemistry