Mechanistic analysis of Mycobacterium tuberculosis Rv1347c, a lysine Nε-acyltransferase involved in mycobactin biosynthesis

Brenda A. Frankel, John S. Blanchard

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17 Citations (Scopus)

Abstract

Mycobactin acylation plays a crucial role in the ability of Mycobacterium tuberculosis to acquire intracellular iron during infection. M. tuberculosis Rv1347c, the lysine Nε-acyltransferase responsible for mycobactin acylation, represents a valid target for the development of novel anti-tubercular agents. Here we investigate the substrate specificity of Rv1347c, evaluate its kinetic mechanism and probe the contributions of active-site residues to catalysis. Our results confirm that Rv1347c demonstrates a preference for longer acyl-chains and suggest that mycobactin acylation occurs subsequent to mycobactin core assembly. Steady-state bisubstrate kinetics and dead-end inhibitor studies support a random sequential kinetic mechanism. Analysis of the pH dependence of kcat/Km revealed the presence of two groups that must be deprotonated for efficient catalysis. Mutagenesis of His130 and Asp168 indicated that both residues are critical for acyltransferase activity and suggests that His130 is responsible for general base activation of the ε-amino group of lysine.

Original languageEnglish (US)
Pages (from-to)259-266
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume477
Issue number2
DOIs
StatePublished - Sep 15 2008

Fingerprint

Acyltransferases
Biosynthesis
Acylation
Lysine
Catalysis
Kinetics
Mutagenesis
Substrate Specificity
Mycobacterium tuberculosis
Catalytic Domain
Iron
Chemical activation
mycobactins
Mycobacterium tuberculosis Rv1347c
Substrates
Infection

Keywords

  • GNAT
  • Lysine N-acyltransferase
  • Mycobactin
  • Rv1347c
  • Siderophore
  • Tuberculosis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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title = "Mechanistic analysis of Mycobacterium tuberculosis Rv1347c, a lysine Nε-acyltransferase involved in mycobactin biosynthesis",
abstract = "Mycobactin acylation plays a crucial role in the ability of Mycobacterium tuberculosis to acquire intracellular iron during infection. M. tuberculosis Rv1347c, the lysine Nε-acyltransferase responsible for mycobactin acylation, represents a valid target for the development of novel anti-tubercular agents. Here we investigate the substrate specificity of Rv1347c, evaluate its kinetic mechanism and probe the contributions of active-site residues to catalysis. Our results confirm that Rv1347c demonstrates a preference for longer acyl-chains and suggest that mycobactin acylation occurs subsequent to mycobactin core assembly. Steady-state bisubstrate kinetics and dead-end inhibitor studies support a random sequential kinetic mechanism. Analysis of the pH dependence of kcat/Km revealed the presence of two groups that must be deprotonated for efficient catalysis. Mutagenesis of His130 and Asp168 indicated that both residues are critical for acyltransferase activity and suggests that His130 is responsible for general base activation of the ε-amino group of lysine.",
keywords = "GNAT, Lysine N-acyltransferase, Mycobactin, Rv1347c, Siderophore, Tuberculosis",
author = "Frankel, {Brenda A.} and Blanchard, {John S.}",
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AU - Frankel, Brenda A.

AU - Blanchard, John S.

PY - 2008/9/15

Y1 - 2008/9/15

N2 - Mycobactin acylation plays a crucial role in the ability of Mycobacterium tuberculosis to acquire intracellular iron during infection. M. tuberculosis Rv1347c, the lysine Nε-acyltransferase responsible for mycobactin acylation, represents a valid target for the development of novel anti-tubercular agents. Here we investigate the substrate specificity of Rv1347c, evaluate its kinetic mechanism and probe the contributions of active-site residues to catalysis. Our results confirm that Rv1347c demonstrates a preference for longer acyl-chains and suggest that mycobactin acylation occurs subsequent to mycobactin core assembly. Steady-state bisubstrate kinetics and dead-end inhibitor studies support a random sequential kinetic mechanism. Analysis of the pH dependence of kcat/Km revealed the presence of two groups that must be deprotonated for efficient catalysis. Mutagenesis of His130 and Asp168 indicated that both residues are critical for acyltransferase activity and suggests that His130 is responsible for general base activation of the ε-amino group of lysine.

AB - Mycobactin acylation plays a crucial role in the ability of Mycobacterium tuberculosis to acquire intracellular iron during infection. M. tuberculosis Rv1347c, the lysine Nε-acyltransferase responsible for mycobactin acylation, represents a valid target for the development of novel anti-tubercular agents. Here we investigate the substrate specificity of Rv1347c, evaluate its kinetic mechanism and probe the contributions of active-site residues to catalysis. Our results confirm that Rv1347c demonstrates a preference for longer acyl-chains and suggest that mycobactin acylation occurs subsequent to mycobactin core assembly. Steady-state bisubstrate kinetics and dead-end inhibitor studies support a random sequential kinetic mechanism. Analysis of the pH dependence of kcat/Km revealed the presence of two groups that must be deprotonated for efficient catalysis. Mutagenesis of His130 and Asp168 indicated that both residues are critical for acyltransferase activity and suggests that His130 is responsible for general base activation of the ε-amino group of lysine.

KW - GNAT

KW - Lysine N-acyltransferase

KW - Mycobactin

KW - Rv1347c

KW - Siderophore

KW - Tuberculosis

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