title: The second aconitase (AcnB) of Escherichia coli.
abstract: The second aconitase (AcnB) of Escherichia coli was partially purified from an acnA::kanR 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 center dot 85 min (131 center dot 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 lambdaacnB phages from the Kohara lambda-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 > or = 98% homogeneity from a genetically enriched source (JRG3171) and shown to be a monomeric protein of Mr 100 000 (SDS-PAGE) and 105 000 (gel filtration analysis) compared with Mr 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).
authors.affiliation: The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2UH, UK.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Bradbury
authors.affiliation: The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2UH, UK.
authors.forename: Megan J
authors.initials: MJ
authors.lastname: Gruer
authors.affiliation: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Rudd
authors.affiliation: The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2UH, UK.
authors.forename: John R
authors.initials: JR
authors.lastname: Guest
mesh_terms: D000154:Aconitate Hydratase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005784:Gene Amplification; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D015394:Molecular Structure; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D000154:Aconitate Hydratase