Overexpression and characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii.
The gene coding for aminoglycoside 2'-N-acetyltransferase Ia [AAC(2')-Ia] from Providencia stuartii was amplified by PCR and cloned. The resulting construct, pACKF2, was transferred into Escherichia coli for overexpression of AAC(2')-Ia as a fusion protein with an N-terminal hexa-His tag. The fusion protein was isolated and purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose and gel permeation chromatography on Superdex 75. Comparison of the specific activity of this enzyme with that of its enterokinase-digested derivative lacking the His tag indicated that the presence of the extra N-terminal peptide does not affect activity. The temperature and pH optima for activity of both forms of the 2'-N-acetyltransferase were 20 degrees C and pH 6.0, respectively, while the enzymes were most stable at 15 degrees C and pH 8.1. The Michaelis-Menten kinetic parameters for AAC(2')-Ia at 20 degrees C and pH 6.0 were determined using a series of aminoglycoside antibiotics possessing a 2'-amino group and a concentration of acetyl coenzyme A fixed at 10 times its K(m) value of 8.75 microM. Under these conditions, gentamicin was determined to be the best substrate for the enzyme in terms of both K(m) and k(cat)/K(m) values, whereas neomycin was the poorest. Comparison of the kinetic parameters obtained with the different aminoglycosides indicated that their hexopyranosyl residues provided the most important binding sites for AAC(2')-Ia activity, while the enzyme exhibits greater tolerance further from these sites. No correlation was found between these kinetic parameters and MICs determined for P. stuartii PR50 expressing the 2'-N-acetyltransferase, suggesting that its true in vivo function is not as a resistance factor.
Franklin
K
K
Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Clarke
A J
AJ
D000123:Acetyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002876:Chromosomes, Bacterial / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008826:Microbial Sensitivity Tests; D011532:Providencia / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D000123:Acetyltransferases; C443113:aminoglycoside 2'-N-acetyltransferase