3N4U

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

(2010) Protein Sci 19: 1565-1576

• PubMed: 20556826 Search on PubMedSearch on PubMed Central
• DOI: https://doi.org/10.1002/pro.437
• Primary Citation of Related Structures:  
  3N4T, 3N4U, 3N4V


• PubMed 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.

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Organizational Affiliation: 

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.