Purification of two forms of kanamycin acetyltransferase from Escherichia coli.
Kanamycin acetyltransferase acylates aminoglycoside antibiotics using acetyl-CoA, and thereby conveys bacterial resistance to several clinically important antibiotics, notably amikacin. The enzyme was quantitatively and reproducibly released from Escherichia coli W677 harboring plasmid pMH67 by a modified osmotic shock procedure (bacterial cells are incubated overnight in sucrose and again without sucrose before onset of osmotic shock). The enzyme was purified by dye-ligand chromatography on Affi-Gel Blue in addition to antibiotic affinity chromatography on neomycin-Sepharose-4B. The activity did not increase with subsequent chromatography on ion-exchange, hydrophobic, or molecular-exclusion gels. However, both dye-ligand and molecular-exclusion chromatography, as well as disc-gel electrophoresis, separated the purified enzyme equally into two active protein fractions. Based on the more active of the two forms, the purification was 112-fold with a specific activity of 1.9 IU/mg. The less-active form has an unusual absorbance spectrum, with a maximum near 255 nm, which cannot be explained by the amino acid composition. Chromatography of this form alone regenerated both forms, suggesting that the enzyme is noncovalently conjugated to an uncharged chromophore, such as a lipid. The purified enzyme has a very sharp pH optimum at 5.5 with a plateau on the alkaline side, but is most stable between pH 8.5 and 9.5. Data from electrophoresis in the presence of sodium dodecyl sulfate and gel-filtration on Ultrogel AcA 44 are consistent with a tetrameric protein of 60-70,000 Da.
D000123:Acetyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D013056:Spectrophotometry, Ultraviolet
D007527:Isoenzymes; D000123:Acetyltransferases; C021115:kanamycin 6'-acetyltransferase