Purification, characterization, and investigation of the mechanism of aminoglycoside 3'-phosphotransferase type Ia

Biochemistry. 1995 Oct 3;34(39):12681-8. doi: 10.1021/bi00039a026.


Aminoglycoside 3'-phosphotransferases [APH(3')s] are the most common cause of bacterial high-level resistance to aminoglycoside antibiotics in clinical isolates. A one-step affinity chromatography was used to purify APH(3') type Ia. The kinetic parameters for turnover of seven aminoglycosides and the corresponding minimum inhibitory concentrations for a strain of Escherichia coli harboring APH-(3')-Ia were determined. The enzyme phosphorylates its substrates with kcat/Km values of 10(6)-10(8) M-1 s-1, including substrates such as amikacin and butirosin A which traditionally have been considered poor substrates for this enzyme. The optimal pH for the phosphotransferase activity was observed to be 7.0-7.5. The purified enzyme was found to be prone to dimerization in the absence of a reducing agent. Treatment of the enzyme with trypsin excised a 4 kDa fragment from the N-terminus which contained the amino acid residue Cys-10. The 27 kDa proteolyzed APH(3')-Ia did not dimerize, suggesting that Cys-10 was involved in dimerization via a disulfide bond. The phosphorylated kanamycin A was isolated, and the phosphorylation was confirmed to occur at the 3'-hydroxyl. Furthermore, both APH(3')-Ia and APH(3')-IIa were shown to phosphorylate water ("ATP hydrolase" activity) at a rate of ca. 10(4)-10(6)-fold slower (effect on kcat/Km) than that for the phosphoryl transfer to a typical aminoglycoside. The results of product-inhibition and alternative substrate diagnostics indicate an equilibrium-random mechanism for phosphorylation of aminoglycosides by APH(3')-Ia.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Diphosphate / pharmacology
  • Anti-Bacterial Agents / pharmacology
  • Catalysis
  • Chromatography, Affinity
  • Chromatography, Gel
  • Drug Resistance, Microbial
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / drug effects
  • Escherichia coli / enzymology
  • Hydrogen-Ion Concentration
  • Kanamycin / pharmacology
  • Kanamycin Kinase
  • Kinetics
  • Phosphorylation
  • Phosphotransferases (Alcohol Group Acceptor) / antagonists & inhibitors
  • Phosphotransferases (Alcohol Group Acceptor) / chemistry
  • Phosphotransferases (Alcohol Group Acceptor) / isolation & purification*
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism
  • Substrate Specificity


  • Anti-Bacterial Agents
  • Kanamycin
  • Adenosine Diphosphate
  • Phosphotransferases (Alcohol Group Acceptor)
  • Kanamycin Kinase