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, 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.
Hegde
S S
SS
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Javid-Majd
F
F
Blanchard
J S
JS
D000123:Acetyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D017931:DNA Primers; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity
D017931:DNA Primers; D000123:Acetyltransferases; C443113:aminoglycoside 2'-N-acetyltransferase