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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
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.
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.
- 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.