Purification, characterization, and investigation of the mechanism of aminoglycoside 3'-phosphotransferase type Ia.
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.
Siregar
J J
JJ
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Miroshnikov
K
K
Mobashery
S
S
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002384:Catalysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004352:Drug Resistance, Microbial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007612:Kanamycin / Q000494:pharmacology; D019868:Kanamycin Kinase; D007700:Kinetics; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D007612:Kanamycin; D000244:Adenosine Diphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase