Wa-Ching Hon

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)




Dr. Daniel S. C. Yang


3',5"-aminoglycoside phosphotransferase type IIIa (APH(3')-IIIa) belongs to a family of bacterial enzymes that phosphorylate the aminoglycoside antibiotics. The modified antibiotics are rendered ineffective due to a lowered affinity for their targets in the bacterial cells. APH(3')-IIIa phosphorylates regiospecifically the 3'-and/or the 5" - hydroxyl groups of nine aminoglycosides. To gain insight into the chemical mechanisms involved in phosphate transfer, the crystal structures of APH(3')-IIIa complexed with a Mg-ATP analog and Mg-ADP have been determined to 2.4 Ȧ and 2.2 Ȧ resolution, respectively. These complexes represent the first and the last step in the enzyme kinetic reaction pathway and their structures are almost identical. Half of the protein molecule shows striking structural resemblance to the catalytic cores of protein kinases, which share less than 6% amino acid sequence identity with APH(3')-IIIa. The folding of this structurally conserved region forms the nucleotide-binding pocket in which five of the seven invariant residues form an elaborate phosphate recognition network via two Mg²⁺ ions. In the Mg-AMPPNP complex, the coordination spheres surrounding the two Mg²⁺ ions are far from the preferred octahedral geometry, which is observed in the Mg-ADP complex. The geometry of the Mg²⁺ ion that binds to the carboxylate oxygens of an invariant Asp is particularly distorted. This Mg²⁺ ion may play a role in triggering or controlling the phosphoryl transfer reaction. This hypothesis would likely have extended implications in understanding the details of the catalytic mechanisms of protein kinases. A potential antibiotic-binding site on APH(3')-IIIa is also immediately obvious. A cluster of residues conserved among the APH(3') isozymes line a partially solvent exposed pocket that displays a net electronegative surface potential. This binding pocket satisfies the requirements for binding to a broad spectrum of cationic aminoglycoside substrates.

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