Mechanism of aminoglycoside 3'-phosphotransferase type IIIa: His188 is not a phosphate-accepting residue.

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Thompson PR, Hughes DW, Wright GD

Mechanism of aminoglycoside 3'-phosphotransferase type IIIa: His188 is not a phosphate-accepting residue.

Chem Biol. 1996 Sep;3(9):747-55.

PubMed ID

8939691 [ View in PubMed

]

Abstract

BACKGROUND: The enzyme aminoglycoside 3'-phosphotransferase Type IIIa (APH(3')-IIIa), confers resistance to many aminoglycoside antibiotics by regiospecific phosphorylation of their hydroxyl groups. The chemical mechanism of phosphoryl transfer is unknown. Based on sequence homology, it has been suggested that a conserved His residue, His188, could be phosphorylated by ATP, and this phospho-His would transfer the phosphate to the incoming aminoglycoside. We have used chemical modification, site-directed mutagenesis and positional isotope exchange methods to probe the mechanism of phosphoryl transfer by APH(3')-IIIa. RESULTS: Chemical modification by diethylpyrocarbonate implicated His in aminoglycoside phosphorylation by APH(3')-IIIa. We prepared His --> Ala mutants of all four His residues in APH(3')-IIIa and found minimal effects of the mutations on the steady-state phosphorylation of several aminoglycosides. One of these mutants, His188Ala, was largely insoluble when compared to the wild-type enzyme. Positional isotope exchange experiments using gamma-[18O]-ATP did not support a double-displacement mechanism. CONCLUSIONS: His residues are not required for aminoglycoside phosphorylation by APH(3')-IIIa. The conserved His 188 is thus not a phosphate accepting residue but does seem to be important for proper enzyme folding. Positional isotope exchange experiments are consistent with direct attack of the aminoglycoside hydroxyl group on the gamma-phosphate of ATP.

DrugBank Data that Cites this Article

Drug Enzymes

Drug	Enzyme	Kind	Organism	Pharmacological Action	Actions
Kanamycin	Aminoglycoside 3'-phosphotransferase	Protein	Escherichia coli	Unknown	Substrate	Details