Mechanism of aminoglycoside 3ʹ-phosphotransferase type IIIa: His188 is not a phosphate-accepting residue Original Research Article

Background: The enzyme aminoglycoside 3′-phosphotransferase Type Illa (APH(3′)-Illa), 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(3ʹ)-Illa. Results: Chemical modification by diethylpyrocarbonate implicated His in aminoglycoside phosphorylation by APH(3′)-Illa. We prepared His → Ala mutants of all four His residues in APH(3′)-Illa 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 wildtype enzyme. Positional isotope exchange experiments using γ-[18O]-ATP did not support a double-displacement mechanism. Conclusions: His residues are not required for aminoglycoside phosphorylation by APH(3′)-Illa. The conserved His188 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 γ-phosphate of ATP.