Mechanism of aminoglycoside 3'-phosphotransferase type IIIa: His188 is not a phosphate-accepting residue
UMass Chan AffiliationsDepartment of Biochemistry and Molecular Pharmacology
Magnetic Resonance Spectroscopy
Molecular Sequence Data
Phosphotransferases (Alcohol Group Acceptor)
Enzymes and Coenzymes
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AbstractBACKGROUND: 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 to 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.
SourceChem Biol. 1996 Sep;3(9):747-55. doi:10.1016/S1074-5521(96)90251-3
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/50093
At the time of publication, Paul Thompson was not yet affiliated with UMass Medical School.
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