Overexpression and Characterization of the Chromosomal Aminoglycoside 2′- N -Acetyltransferase of Providencia stuartii

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Abstract
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°C and pH 6.0, respectively, while the enzymes were most stable at 15°C and pH 8.1. The Michaelis-Menten kinetic parameters for AAC(2′)-Ia at 20°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 μM. 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.

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