Abstract
The chromosomally encoded aminoglycoside N-acetyltransferase, AAC(2')-Ic, of Mycobacterium tuberculosis has a yet unidentified physiological function. The aac(2')-Ic gene was cloned and expressed in Escherichia coli, and AAC(2')-Ic was purified. Recombinant AAC(2')-Ic was a soluble protein of 20,000 Da and acetylated all aminoglycosides substrates tested in vitro, including therapeutically important antibiotics. Acetyl-CoA was the preferred acyl donor. The enzyme, in addition to acetylating aminoglycosides containing 2'-amino substituents, also acetylated kanamycin A and amikacin that contain a 2'-hydroxyl substituent, although with lower activity, indicating the capacity of the enzyme to perform both N-acetyl and O-acetyl transfer. The enzyme exhibited "substrate activation" with many aminoglycoside substrates while exhibiting Michaelis-Menten kinetics with others. Kinetic studies supported a random kinetic mechanism for AAC(2')-Ic. Comparison of the kinetic parameters of different aminoglycosides suggested that their hexopyranosyl residues and, to a lesser extent, the central aminocyclitol residue carry the major determinants of substrate affinity.
Highlights
Drug in bacteria due to changes in the permeability of the outer membrane or active efflux, and most commonly (iii) enzymatic detoxification of the drug (6 –12)
In the present paper we report the cloning, overexpression, purification, substrate specificity, kinetic mechanism, and solvent kinetic isotope effects of Aminoglycoside N-acetyltransferases (AACs)(2Ј)-Ic from Mycobacterium tuberculosis
Purification and Properties of AAC(2Ј)-Ic—PCR amplification of the aac(2Ј)-Ic gene using primers AS1 and AA1 and the M. tuberculosis H37Rv genomic DNA template yielded a single fragment of the expected length
Summary
Materials—All chemicals, coenzyme A derivatives, and aminoglycoside antibiotics were purchased from Sigma-Aldrich Chemical Co. The cells were collected by centrifugation at 10,000 rpm and resuspended in buffer A (25 mM triethanolamine, pH 7.8, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol) containing protease inhibitors (Roche Molecular Biochemicals), lysozyme (0.2 g/ml), and DNase I (1 g/ml) and stirred for 20 min. The active fractions were pooled, solid ammonium sulfate was added to a final concentration of 0.5 M, and the solution was rechromatographed on phenyl-Sepharose as above. The enzyme after this step was found to be Ͼ90% pure as assessed by SDS-polyacrylamide gel electrophoresis. Kanamycin A-containing fractions, eluting at 0.4 M NaCl, were detected and quantitated using a phenol-sulfuric acid method, concentrated by lyophilization, desalted on a Sephadex G-10 column (1.5 ϫ 50 cm) using Milli-Q water, and used in kinetic studies. Where, I represents the fraction of isotope, and EVK and EV are isotope effects on V/K and V Ϫ 1, respectively
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