Michael H. Perlin¹, Scott A. Brown¹, and Jaydev N. Dholakia²

1Departments of Biology, and ² Biochemistry, University of Louisville, Louisville, KY 40292 USA

TABLE OF CONTENTS

1. Abstract

2. Introduction- Resistance to aminoglycoside antibiotics

3. Aminoglycoside Phosphotransferase (APH) Enzymes

3.1. The best-characterized APHs

3.1.1. APH(3')-II

3.1.1.1. Site-directed mutagenesis
3.1.1.2. Photoaffinity labelling

3.1.2. APH(3')-III

3.1.2.1. Kinetic studies
3.1.2.2. Crystal structure
3.1.2.3. Protein kinase inhibitors
3.1.2.4. NMR spectroscopy

4. Perspectives

5. Acknowledgments

6. References

1. ABSTRACT

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.