MECHANISM OF AMINOGLYCOSIDE 3'-PHOSPHOTRANSFERASE TYPE IIIA - HIS188 IS NOT A PHOSPHATE-ACCEPTING RESIDUE

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 chemica l mechanism of phosphoryl transfer is unknown. Based on sequence homol ogy, it has been suggested that a conserved His residue, His188, could be phosphorylated by ATP and this phospho-His would transfer the phos phate to the incoming aminoglycoside. We have used chemical modificati on, 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 amin oglycoside phosphorylation by APH(3')-IIIa, We prepared His --> Ala mu tants of all four His residues in APH(3')-IIIa and found minimal effec ts of the mutations on the steady-state phosphorylation of several ami noglycosides. One of these mutants, His188Ala, was largely insoluble w hen compared to the wildtype enzyme. Positional isotope exchange exper iments using gamma-[O-18]-ATP did not support a double-displacement me chanism, Conclusions: His residues are not required for aminoglycoside phosphorylation by APH(3')-IIIa. The conserved His188 is thus not a p hosphate accepting residue but does seem to be important for proper en zyme folding. Positional isotope exchange experiments are consistent w ith direct attack of the aminoglycoside hydroxyl group on the gamma-ph osphate of ATP.