The second aconitase (AcnB) of Escherichia coli

Alan J. Bradbury, Megan J. Gruer, Kenneth E. Rudd, John R. Guest

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27 Scopus citations

Abstract

The second aconitase (AcnB) of Escherichia coli was partially purified from an acnA::kan(R) mutant lacking AcnA, and the corresponding polypeptide identified by activity staining and weak cross-reactivity with AcnA antiserum. The acnB gene was located at 2.85 min (131.6 kb) in a region of the chromosome previously assigned to two unidentified ORFs. Aconitase specific activities were amplified up to fivefold by infection with λacnB phages from the Kohara λ-E. coli gene library, and up to 120-fold (50% of soluble protein) by inducing transformants containing a plasmid (pGS783) in which the acnB coding region is expressed from a regulated T7 promoter. The AcnB protein was purified to ≥ 98% homogeneity from a genetically enriched source (JRG3171) and shown to be a monomeric protein of M(r) 100,000 (SDS-PAGE) and 105,000 (gel filtration analysis) compared with M(r) 93,500 predicted from the nucleotide sequence. The sequence identity between AcnA and AcnB is only 17% and the domain organization of AcnA and related proteins (1-2-3-linker-4) is rearranged in AcnB (4-1-2-3).

Original languageEnglish (US)
Pages (from-to)389-400
Number of pages12
JournalMicrobiology
Volume142
Issue number2
DOIs
StatePublished - Feb 1996

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Keywords

  • Aconitase
  • Citric acid cycle
  • Escherichia coli
  • Iron-sulphur proteins
  • Protein domains

ASJC Scopus subject areas

  • Microbiology

Cite this

Bradbury, A. J., Gruer, M. J., Rudd, K. E., & Guest, J. R. (1996). The second aconitase (AcnB) of Escherichia coli. Microbiology, 142(2), 389-400. https://doi.org/10.1099/13500872-142-2-389