Purification and properties of an aminoglycoside acetyltransferase from Pseudomonas aeruginosa

Abstract

An aminoglycoside 3-acetyltransferase [AAC(3)], possibly a new isoenzymic species of the 3-N-acetyltransferase group, was purified to apparent homogeneity from a crude extract of P. aeruginosa, a gentamicin-resistant clinical isolate. The method of purification was consecutive column chromatography, gel filtration, affinity chromatography and ion-exchange chromatography to give 2 protein peaks, one of which was coincident with activity and which indicated a purification of 600 (specific activity = 9.743 units mg-1 at pH 7.2, 34.degree. C). Polyacrylamide disc gel electrophoresis indicated a single protein band coincident with enzymic activity. The MW of the enzyme was .apprx. 39,000. AAC(3)-V (provisonal designation) was further characterized by stability, substrate, pH and kinetic studies. The Km was 0.724 .mu.M (sisomicin) and the Vmax was 0.102 .mu.mol min-1 mg-1 (sisomicin) at pH 7.2 and 34.degree. C. Substrate inhibition was exhibited by kanamycin A and tobramycin. Enzyme activity was significantly stabilized when preparations contained substrate.