IMR Press / FBL / Volume 4 / Issue 4 / DOI: 10.2741/perlin

Frontiers in Bioscience-Landmark (FBL) is published by IMR Press from Volume 26 Issue 5 (2021). Previous articles were published by another publisher on a subscription basis, and they are hosted by IMR Press on as a courtesy and upon agreement with Frontiers in Bioscience.

Open Access Article
Developing a snapshot of the ATP binding domain(s) of aminoglycoside phosphotransferases
Show Less
1 Department of Biology, University of Louisville, Louisville, KY 40292, USA
2 Departments of Biochemistry, University of Louisville, Louisville, KY 40292, USA
Academic Editor:Marcelo Tolmasky
Front. Biosci. (Landmark Ed) 1999, 4(4), 63–71;
Published: 1 January 1999

The aminoglycoside (AG) 3'-phosphotransferases [APH(3')s] are an important class of modifying enzymes which confer high-level resistance to those AGs actively modified by the enzymes. They catalyze the transfer of the terminal phosphate from ATP to the drug, thus preventing the AG s action at the 70S ribosome. These enzymes, which utilize ATP as a co-substrate, appear from amino acid alignments to be part of a much larger superfamily of kinases and ATP-binding proteins. Structure-function analyses have been initiated in our laboratory for APH(3')-II, whose gene was derived from transposon Tn5. Site-directed mutagenesis of the cloned APH(3')-II gene was used to genetically examine the residues in two highly-conserved motifs proposed to participate in ATP binding. Several of these residues, in fact, were shown to affect the enzyme s affinity for ATP. We have also initiated studies using photoaffinity labelling of APH(3')-II with the ATP analogs, 8-azido-ATP and 2-azido-ATP. We have shown that 8-N3ATP and 2-N3ATP can be substituted for ATP in the APH(3')-II catalyzed phosphorylation of kanamycin; such findings indicate that the interaction of these photoaffinity analogs of ATP with APH(3')-II is biologically relevant. One of the best-characterized of the APH(3') enzymes is APH(3')-IIIa, the first of the group whose structure has been analyzed by x- ray crystallography. Several studies have demonstrated that this enzyme functions by a Theorell-Chance mechanism. Moreover, the architecture of the enzyme, crystallized in the presence of ADP has revealed residues in the ATP-binding pocket which are likely to play important roles in catalysis. Once the results from biochemical analyses can be correlated with those from mutagenesis studies and x-ray crystallography, a clearer picture of the active site will be provided for an important class of AG-modifying enzymes and phosphotransferases. This picture will also allow a better understanding of these enzymes within the greater context of kinases and nucleotide-binding proteins.

Drug Resistance
Gram-Negative Aerobic Bacilli
Aminoglycoside Phosphotransferases
SiteDirected Mutagenesis
Photoaffinity Labelling
Back to top