Overexpression and Mechanistic Analysis of Chromosomally Encoded Aminoglycoside 2′-N-Acetyltransferase (AAC(2′)-Ic) from Mycobacterium tuberculosis

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.