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Purification and properties of two gentamicin-modifying enzymes, coded by a single plasmid pPK237 originating from
Abstract:
A broad host range multiresistance plasmid pPK237, originating from Pseudomonas aeruginosa mediates high-level resistance to gentamicin and tobramycin. It was found to code for two gentamicin modifying enzymes, which from their substrate profile by radioenzymatic assay were characterized as aminoglycoside acetyltransferase AAC(3)-I and aminoglycoside adenylyltransferase AAD(2"). The two enzymes were studied after purification from an Escherichia coli K12 host. The two gentamicin-modifying enzymes coded by PPK237 were completely separated by DEAE chromatography. The purification (126 fold) of the acetyltransferase was achieved by (NH4)2SO4 precipitation, DEAE chromatography and affinity chromatography. The purification of the adenylyltransferase was performed by affinity chromatography directly after (NH4)2SO4 precipitation. Both purified enzyme preparations showed a single protein band on disc electrophoresis. The Km for gentamicin C1 of the acetyltransferase was 0.066 mM. The amino acid analysis of the acetyltransferase coded by pPK237 showed a different aminoacid composition than that of the gentamicin acetyltransferase AAC(3)-I purified by Williams and Northrop17). The acetyltransferase after DEAE chromatography is stable for many months at -20 degrees C, while the adenylyltransferase after purification is highly unstable; it shows enzymatic activity only in the presence of Mg++.
Insights
This study identifies two novel gentamicin-modifying enzymes, aminoglycoside acetyltransferase (AAC(3)-I) and aminoglycoside adenylyltransferase (AAD(2")), from the Pseudomonas aeruginosa plasmid pPK237. These enzymes confer high-level resistance to gentamicin and tobramycin in bacteria.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- The plasmid pPK237 from Pseudomonas aeruginosa confers resistance to multiple antibiotics, including high-level resistance to gentamicin and tobramycin.
- This resistance is mediated by specific enzymes that modify the antibiotics, preventing their action.
Purpose of the Study:
- To characterize the gentamicin-modifying enzymes encoded by the plasmid pPK237.
- To purify and analyze the biochemical properties of these enzymes.
Main Methods:
- Enzymes were purified from an Escherichia coli K12 host carrying the pPK237 plasmid.
- Purification involved DEAE chromatography and affinity chromatography.
- Enzyme activity and substrate specificity were determined using radioenzymatic assays.
- Protein purity was assessed by disc electrophoresis, and kinetic parameters (Km) were determined.
Main Results:
- Two distinct enzymes were identified: aminoglycoside acetyltransferase AAC(3)-I and aminoglycoside adenylyltransferase AAD(2").
- Purified AAC(3)-I exhibited a Km for gentamicin C1 of 0.066 mM and showed a different amino acid composition compared to previously reported AAC(3)-I.
- AAD(2") required Mg++ for activity and was found to be unstable after purification.
Conclusions:
- The plasmid pPK237 encodes two distinct aminoglycoside-modifying enzymes responsible for gentamicin and tobramycin resistance.
- The characterized AAC(3)-I enzyme possesses unique biochemical properties, suggesting it is a novel variant.
- Understanding these resistance mechanisms is crucial for combating antibiotic resistance.