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 Pseudomonas aeruginosa, a gentamicin-resistant clinical isolate. The method of purification was consecutive column chromatography--(i) gel filtration, (ii) affinity chromatography, and (iii) ion-exchange chromatography--to give two 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 degrees C). Polyacrylamide disc gel electrophoresis indicated a single protein band coincident with enzymic activity. The molecular weight of the enzyme was about 39 000. AAC(3)-V (provisonal designation) was further characterized by stability, substrate, pH, and kinetic studies. The Km was 0.724 microM (sisomicin), and the Vmax was 0.102 mumol min-1 mg-1 (sisomicin) at pH 7.2 and 34 degrees C. Substrate inhibition was exhibited by kanamycin A and tobramycin. Studies showed that enzyme activity was significantly stabilized when preparations contained substrate.