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Publication Abstract from PubMed

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 x 10(3) to 1.2 x 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 upon 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.

Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase-2-IVa.,Toth M, Frase H, Antunes NT, Smith CA, Vakulenko SB Protein Sci. 2010 Jun 15. PMID:20556826^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.