Overexpression and mechanistic analysis of chromosomally encoded aminoglycoside 2 '-N-acetyltransferase (AAC(2 ')-Ic) from Mycobacterium tuberculosis

The chromosomally encoded aminoglycoside N-acetyltransferase, AAC(2')-Ic, o f 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, includin g 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 cap acity of the enzyme to perform both N-acetyl and O-acetyl transfer. The enz yme exhibited "substrate activation" with many aminoglycoside substrates wh ile exhibiting Michaelis-Menten kinetics with others. Kinetic studies suppo rted a random kinetic mechanism for AAC(2')-Ic. Comparison of the kinetic p arameters of different aminoglycosides suggested that their hexopyranosyl r esidues and, to a lesser extent, the central aminocyclitol residue carry th e major determinants of substrate affinity.