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

• Published in: The Journal of biological chemistry Vol. 276; no. 49; pp. 45876 - 45881
• Main Authors: Hegde, Subray S., Javid-Majd, Farah, Blanchard, John S.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 07.12.2001
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M108810200

The chromosomally encoded aminoglycosideN-acetyltransferase, AAC(2′)-Ic, of Mycobacterium tuberculosis has a yet unidentified physiological function. Theaac(2′)-Ic gene was cloned and expressed inEscherichia 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 bothN-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.