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 Ni2+-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 degreesC and pH 6.0, respectively, while the enzymes were most stable at 15 degreesC and pH 8.1, The Michaelis-Menten kinetic parameters for AAC(2 ')-Ia at 20 degreesC and pH 6.0 mere 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 muM. 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 ACC(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.