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Here, we report the kinetic mechanism and structure of the aminoglycoside phosphotransferase 2\u2033\u2010IVa (APH(2\u2033)\u2010IVa), an enzyme responsible for resistance to aminoglycoside antibiotics in clinical enterococcal and staphylococcal isolates. The enzyme operates via a Bi\u2010Bi sequential mechanism in which the two substrates (ATP or GTP and an aminoglycoside) bind in a random manner. The APH(2\u2033)\u2010IVa enzyme phosphorylates various 4,6\u2010disubstituted aminoglycoside antibiotics with catalytic efficiencies (<jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>\/<jats:italic>K<\/jats:italic><jats:sub>m<\/jats:sub>) of 1.5 \u00d7 10<jats:sup>3<\/jats:sup> to 1.2 \u00d7 10<jats:sup>6<\/jats:sup> (M<jats:sup>\u22121<\/jats:sup>\u00a0s<jats:sup>\u22121<\/jats:sup>). 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\u2033)\u2010IVa 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\u2010utilizing phosphotransferases is crucial for the development of either novel aminoglycosides or, more importantly, GTP\u2010based enzyme inhibitors which would not be expected to interfere with crucial ATP\u2010dependent enzymes.<\/jats:p>","DOI":"10.1002\/pro.437","type":"journal-article","created":{"date-parts":[[2010,6,15]],"date-time":"2010-06-15T21:16:03Z","timestamp":1276636563000},"page":"1565-1576","source":"Crossref","is-referenced-by-count":33,"title":["Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase\u20102\u2033\u2010IVa"],"prefix":"10.1002","volume":"19","author":[{"given":"Marta","family":"Toth","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hilary","family":"Frase","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nuno 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