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

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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.

Original languageEnglish (US)
Pages (from-to)45876-45881
Number of pages6
JournalJournal of Biological Chemistry
Volume276
Issue number49
DOIs
StatePublished - Dec 7 2001

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aminoglycoside 2'-N-acetyltransferase
Aminoglycosides
Mycobacterium tuberculosis
Substrates
Enzymes
Kinetics
Acetyl Coenzyme A
Kanamycin
Amikacin
Acetyltransferases
Hydroxyl Radical
Kinetic parameters
Escherichia coli
Anti-Bacterial Agents
Genes
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "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.",
author = "Hegde, {Subray S.} and Farah Javid-Majd and Blanchard, {John S.}",
year = "2001",
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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.

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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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