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Related Experiment Videos

Structure-function analyses for aminoglycoside 3'-phosphotransferase II (APH(3')-II).

S Kocabiyik1, C Mullins, C Breeding

  • 1Dept. of Biology, University of Louisville, KY 40292.

SAAS Bulletin, Biochemistry and Biotechnology
|January 1, 1992
PubMed
Summary

Site-directed mutagenesis of the aminoglycoside phosphotransferase (APH(3')-II) gene generated mutant enzymes with reduced activity and altered substrate specificity. Random mutagenesis identified new mutations conferring amikacin resistance.

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Area of Science:

  • Molecular Biology
  • Enzymology
  • Microbiology

Background:

  • Aminoglycoside phosphotransferase (APH(3 extquotesingle)-II) confers resistance to aminoglycoside antibiotics.
  • Understanding APH(3 extquotesingle)-II structure-function relationships is crucial for combating antibiotic resistance.

Purpose of the Study:

  • To investigate the impact of specific amino acid substitutions on APH(3 extquotesingle)-II enzyme activity and substrate specificity.
  • To identify novel mutations conferring amikacin resistance through random mutagenesis.

Main Methods:

  • Site-directed mutagenesis was employed to alter conserved amino acid residues within the APH(3 extquotesingle)-II gene.
  • Random mutagenesis was performed on a strain carrying the APH(3 extquotesingle)-II gene on a conjugative plasmid.

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  • Enzyme activity and substrate resistance levels were assessed for mutant strains.

    • Main Results:

    • Mutant APH(3 extquotesingle) enzymes generally exhibited reduced activity and conferred lower levels of resistance.
    • Specific substitutions, particularly at Tyr 218, altered the substrate specificity of the enzymes.
    • Random mutagenesis yielded plasmid-borne mutations conferring amikacin resistance, with two mutations potentially located in the APH(3 extquotesingle)-II structural gene.

    Conclusions:

    • Conserved amino acid residues in APH(3 extquotesingle)-II are critical for its catalytic activity and substrate resistance.
    • Targeted mutagenesis can modulate enzyme specificity, offering insights into resistance mechanisms.
    • Random mutagenesis is effective in discovering novel resistance determinants, including potential mutations within the APH(3 extquotesingle)-II gene.