Aminoglycoside 2′′-Phosphotransferase IIIa (APH(2′′)-IIIa) Prefers GTP over ATP

• Published in: The Journal of biological chemistry 2012-04, Vol.287 (16), p.12893-12903
• Main Authors: Smith, Clyde A., Toth, Marta, Frase, Hilary, Byrnes, Laura J., Vakulenko, Sergei B.
• Format: Article
• Language: English
• Subjects: Aminoglycoside Kinase; Antibiotic Resistance; Crystal Structure; Drug Design; GTP-dependent; GTPase; Protein Kinases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.341206

Contrary to the accepted dogma that ATP is the canonical phosphate donor in aminoglycoside kinases and protein kinases, it was recently demonstrated that all members of the bacterial aminoglycoside 2′′-phosphotransferase IIIa (APH(2′′)) aminoglycoside kinase family are unique in their ability to utilize GTP as a cofactor for antibiotic modification. Here we describe the structural determinants for GTP recognition in these enzymes. The crystal structure of the GTP-dependent APH(2′′)-IIIa shows that although this enzyme has templates for both ATP and GTP binding superimposed on a single nucleotide specificity motif, access to the ATP-binding template is blocked by a bulky tyrosine residue. Substitution of this tyrosine by a smaller amino acid opens access to the ATP template. Similar GTP binding templates are conserved in other bacterial aminoglycoside kinases, whereas in the structurally related eukaryotic protein kinases this template is less conserved. The aminoglycoside kinases are important antibiotic resistance enzymes in bacteria, whose wide dissemination severely limits available therapeutic options, and the GTP binding templates could be exploited as new, previously unexplored targets for inhibitors of these clinically important enzymes. Background: Aminoglycoside kinases (APHs), structurally related to the protein kinases, are responsible for acquired resistance to aminoglycoside antibiotics. Results: The structure of GTP-dependent APH(2′′)-IIIa shows that both GTP and ATP recognition sites are located on the same nucleotide binding motif. Conclusion: This enzyme uses exclusively GTP because the ATP binding site is blocked. Significance: This is the first structure of an exclusively GTP-dependent kinase enzyme.