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Purification and characterization of the first archaeal aconitase from the thermoacidophilic Sulfolobus acidocaldarius.
Eur J Biochem 2001; 268(6):1760-71EJ

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 approximately 55 micromol x min(-1) x 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 degrees C, slightly under the growth optimum of S. acidocaldarius around 80 degrees C. Thermal inactivation studies of the aconitase revealed the enzymatic activity to be uninfluenced after one hour incubation at 80 degrees C. Even at 95 degrees C, a half-life of approximately 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 microM, 2.9 mM and 370 microM, respectively. The aconitase reaction was inhibited by the typical inhibitors fluorocitrate, trans-aconitate and tricarballylate. Amino-acid 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 [3Fe--4S] cluster.

Authors+Show Affiliations

Institute for Biochemistry, Medical University of Lübeck, Germany.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

11248696

Citation

Uhrigshardt, H, et al. "Purification and Characterization of the First Archaeal Aconitase From the Thermoacidophilic Sulfolobus Acidocaldarius." European Journal of Biochemistry, vol. 268, no. 6, 2001, pp. 1760-71.
Uhrigshardt H, Walden M, John H, et al. Purification and characterization of the first archaeal aconitase from the thermoacidophilic Sulfolobus acidocaldarius. Eur J Biochem. 2001;268(6):1760-71.
Uhrigshardt, H., Walden, M., John, H., & Anemüller, S. (2001). Purification and characterization of the first archaeal aconitase from the thermoacidophilic Sulfolobus acidocaldarius. European Journal of Biochemistry, 268(6), pp. 1760-71.
Uhrigshardt H, et al. Purification and Characterization of the First Archaeal Aconitase From the Thermoacidophilic Sulfolobus Acidocaldarius. Eur J Biochem. 2001;268(6):1760-71. PubMed PMID: 11248696.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Purification and characterization of the first archaeal aconitase from the thermoacidophilic Sulfolobus acidocaldarius. AU - Uhrigshardt,H, AU - Walden,M, AU - John,H, AU - Anemüller,S, PY - 2001/3/15/pubmed PY - 2001/5/5/medline PY - 2001/3/15/entrez SP - 1760 EP - 71 JF - European journal of biochemistry JO - Eur. J. Biochem. VL - 268 IS - 6 N2 - 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 approximately 55 micromol x min(-1) x 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 degrees C, slightly under the growth optimum of S. acidocaldarius around 80 degrees C. Thermal inactivation studies of the aconitase revealed the enzymatic activity to be uninfluenced after one hour incubation at 80 degrees C. Even at 95 degrees C, a half-life of approximately 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 microM, 2.9 mM and 370 microM, respectively. The aconitase reaction was inhibited by the typical inhibitors fluorocitrate, trans-aconitate and tricarballylate. Amino-acid 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 [3Fe--4S] cluster. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/11248696/Purification_and_characterization_of_the_first_archaeal_aconitase_from_the_thermoacidophilic_Sulfolobus_acidocaldarius_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0014-2956&date=2001&volume=268&issue=6&spage=1760 DB - PRIME DP - Unbound Medicine ER -