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 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.
Thompson
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PR
Department of Biochemistry, McMaster University, Hamilton, ON, Canada L8N3Z5. wrighte@fhs.csu.mcmaster.ca
Hughes
D W
DW
Wright
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GD
D000255:Adenosine Triphosphate / Q000378:metabolism; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D016983:Enterococcus / Q000201:enzymology*; D006639:Histidine / Q000378:metabolism*; D019868:Kanamycin Kinase; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D016415:Sequence Alignment; D013210:Staphylococcus / Q000201:enzymology*
D009844:Oligosaccharides; D010710:Phosphates; D006639:Histidine; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D004047:Diethyl Pyrocarbonate