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Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase-2''-IVa.


ABSTRACT: 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 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.

SUBMITTER: Toth M 

PROVIDER: S-EPMC2923509 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase-2''-IVa.

Toth Marta M   Frase Hilary H   Antunes Nuno Tiago NT   Smith Clyde A CA   Vakulenko Sergei B SB  

Protein science : a publication of the Protein Society 20100801 8


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  ...[more]

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