TY - JOUR
T1 - Overexpression and Mechanistic Analysis of Chromosomally Encoded Aminoglycoside 2′-N-Acetyltransferase (AAC(2′)-Ic) from Mycobacterium tuberculosis
AU - Hegde, Subray S.
AU - Javid-Majd, Farah
AU - Blanchard, John S.
PY - 2001/12/7
Y1 - 2001/12/7
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M108810200
DO - 10.1074/jbc.M108810200
M3 - Article
C2 - 11590162
AN - SCOPUS:0035824623
SN - 0021-9258
VL - 276
SP - 45876
EP - 45881
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 49
ER -