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Mutant APH(2'')-IIa enzymes with increased activity against amikacin and isepamicin

Marta Toth1, Hilary Frase, Joseph W Chow

  • 1Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.

Antimicrobial Agents and Chemotherapy
|February 11, 2010
PubMed

Insights

Directed evolution created mutant aminoglycoside 2''-IIa phosphotransferase enzymes. These mutants show increased resistance to specific aminoglycoside antibiotics like amikacin and isepamicin.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzyme Engineering

Background:

  • Aminoglycoside 2''-IIa phosphotransferase confers resistance to aminoglycoside antibiotics.
  • Directed evolution is a powerful tool for enzyme engineering.

Purpose of the Study:

  • To generate and characterize mutant aminoglycoside 2''-IIa phosphotransferase enzymes with altered antibiotic resistance profiles.
  • To investigate the kinetic mechanisms underlying enhanced resistance to specific aminoglycosides.

Main Methods:

  • Directed evolution using random PCR mutagenesis of the aminoglycoside 2''-IIa phosphotransferase gene.
  • Construction and characterization of specific mutant enzymes (R92H/D268N, N196D/D268N).
  • Determination of kinetic parameters (K(m), turnover) and minimum inhibitory concentrations (MICs) for various aminoglycosides.

Main Results:

  • Mutant enzymes R92H/D268N and N196D/D268N exhibited elevated resistance to amikacin and isepamicin.
  • Increased activity against isepamicin was due to decreased K(m) values.
  • Improved catalytic efficiency for amikacin resulted from both decreased K(m) and increased turnover.
  • Single amino acid substitutions (R92H, D268N) did not confer elevated MICs.

Conclusions:

  • Specific mutations in aminoglycoside 2''-IIa phosphotransferase can enhance resistance to certain aminoglycosides.
  • The kinetic basis for enhanced antibiotic resistance involves alterations in substrate binding and catalytic turnover.
  • Directed evolution is effective in generating enzymes with tailored antibiotic resistance properties.

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