Purification and properties of two gentamicin-modifying enzymes, coded by a single plasmid pPK237 originating from Pseudomonas aeruginosa.

Publisher Website
Google Scholar
Abstract
A broad host range multiresistance plasmid pPK237, originating from P. aeruginosa mediates high-level resistance to gentamicin and tobramycin; it codes for 2 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 2 enzymes were studied after purification from an Escherichia coli K12 host. The 2 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 amino acid composition than that of the gentamicin acetyltransferase AAC(3)-I purified by Williams and Northrop. The acetyltransferase after DEAE chromatography is stable for many months at -20.degree. C, while the adenylyltransferase after purification is highly unstable; it shows enzymatic activity only in the presence of Mg2+.

This publication has 0 references indexed in Scilit: