Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase-2''-IVa.
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.
Toth
Marta
M
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Frase
Hilary
H
Antunes
Nuno Tiago
NT
Smith
Clyde A
CA
Vakulenko
Sergei B
SB
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000617:Aminoglycosides / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D018360:Crystallography, X-Ray; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000617:Aminoglycosides; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C048564:aminoglycoside 2''-phosphotransferase