Purification and characterization of the first archaeal aconitase from the thermoacidophilic Sulfolobus acidocaldarius

Helge Uhrigshardt, Michael Walden, Harald John, Stefan Anemüller

16 Zitate (Scopus)

Abstract

The first archaeal aconitase was isolated from the cytosol of the thermoacidophilic Sulfolobus acidocaldarius. Interestingly, the enzyme was copurified with an isocitrate lyase. This enzyme, directly converting isocitrate, the reaction product of the aconitase reaction, was also unknown in crenarchaeota, thus far. Both proteins could only be separated by SDS gel electrophoresis yielding apparent molecular masses of 96 kDa for the aconitase and 46 kDa for the isocitrate lyase. Despite of its high oxygen sensitivity, the aconitase could be enriched 27-fold to a specific activity of ≈ 55 μmol·min 1·mg 1, based on the direct aconitase assay system. Maximal enzyme activities were measured at pH 7.4 and the temperature optimum for the archaeal enzyme was recorded at 75°C, slightly under the growth optimum of S. acidocaldarius around 80°C. Thermal inactivation studies of the aconitase revealed the enzymatic activity to be uninfluenced after one hour incubation at 80°C. Even at 95°C, a half-life of ≈ 14 min was determined, clearly defining it as a thermostable protein. The apparent K m values for the three substrates cis-aconitate, citrate and isocitrate were found as 108 μM, 2.9 mM and 370 μM, respectively. The aconitase reaction was inhibited by the typical inhibitors fluorocitrate, trans-aconitate and tricarballylate. Aminoacid sequencing of three internal peptides of the S. acidocaldarius aconitase revealed the presence of highly conserved residues in the archaeal enzyme. By amino-acid sequence alignments, the S. acidocaldarius sequence was found to be highly homologous to either other putative archaeal or known eukaryal and bacterial sequences. As shown by EPR-spectroscopy, the enzyme hosts an interconvertible [3Fe24S] cluster.

OriginalspracheEnglisch
ZeitschriftEuropean Journal of Biochemistry
Jahrgang268
Ausgabenummer6
Seiten (von - bis)1760-1771
Seitenumfang12
ISSN0014-2956
DOIs
PublikationsstatusVeröffentlicht - 2001

Strategische Forschungsbereiche und Zentren

  • Forschungsschwerpunkt: Infektion und Entzündung - Zentrum für Infektions- und Entzündungsforschung Lübeck (ZIEL)

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