Iron-dependent RNA-binding Activity of Mycobacterium Tuberculosis Aconitase

J Bacteriol. 2007 Jun;189(11):4046-52. doi: 10.1128/JB.00026-07. Epub 2007 Mar 23.

Abstract

Cellular iron levels are closely monitored by iron regulatory and sensor proteins of Mycobacterium tuberculosis for survival inside macrophages. One such class of proteins systematically studied in eukaryotes and reported in a few prokaryotes are the iron-responsive proteins (IRPs). These IRPs bind to iron-responsive elements (IREs) present at untranslated regions (UTRs) of mRNAs and are responsible for posttranscriptional regulation of the expression of proteins involved in iron homeostasis. Amino acid sequence analysis of M. tuberculosis aconitase (Acn), a tricarboxylic acid (TCA) cycle enzyme, showed the presence of the conserved residues of the IRP class of proteins. We demonstrate that M. tuberculosis Acn is bifunctional. It is a monomeric protein that is enzymatically active in converting isocitrate to cis-aconitate at a broad pH range of 7 to 10 (optimum, pH 8). As evident from gel retardation assays, M. tuberculosis Acn also behaves like an IRP by binding to known mammalian IRE-like sequences and to predicted IRE-like sequences present at the 3' UTR of thioredoxin (trxC) and the 5' UTR of the iron-dependent repressor and activator (ideR) of M. tuberculosis. M. tuberculosis Acn when reactivated with Fe(2+) functions as a TCA cycle enzyme, but upon iron depletion by a specific iron chelator, it behaves like an IRP, binding to the selected IREs in vitro. Since iron is required for the Acn activity and inhibits the RNA-binding activity of Acn, the two activities of M. tuberculosis Acn are mutually exclusive. Our results demonstrate the bifunctional nature of M. tuberculosis Acn, pointing to its likely role in iron homeostasis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aconitate Hydratase / chemistry
  • Aconitate Hydratase / genetics
  • Aconitate Hydratase / isolation & purification
  • Aconitate Hydratase / metabolism*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism*
  • Chromatography, Gel
  • Dimerization
  • Electrophoretic Mobility Shift Assay
  • Hydrogen-Ion Concentration
  • Iron / metabolism
  • Iron-Regulatory Proteins / genetics
  • Iron-Regulatory Proteins / isolation & purification
  • Iron-Regulatory Proteins / metabolism*
  • Mycobacterium tuberculosis / enzymology*
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / metabolism
  • Open Reading Frames / genetics
  • Polymerase Chain Reaction
  • Protein Binding
  • RNA / metabolism
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / isolation & purification
  • RNA-Binding Proteins / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Iron-Regulatory Proteins
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • RNA
  • Iron
  • Aconitate Hydratase