Aluminum Triggers Decreased Aconitase Activity via Fe-S Cluster Disruption and the Overexpression of Isocitrate Dehydrogenase and Isocitrate Lyase
A METABOLIC NETWORK MEDIATING CELLULAR SURVIVAL*
- Jeffrey Middaugh‡,
- Robert Hamel§,
- Gael Jean-Baptiste‡,
- Robin Beriault‡,
- Daniel Chenier‡ and
- Vasu D. Appanna‡¶
- ‡Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario P3E 2C6, Canada and §Northern Centre for Biotechnology and Clinical Research, Sudbury, Ontario P3E 6B4, Canada
- ¶ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Rd., Sudbury, Ontario P3C 2C6, Canada. Tel.: 705-675-1151 (ext. 2112); Fax: 705-675-4844; E-mail: vappanna{at}laurentian.ca.
Abstract
Although aluminum is known to be toxic to most organisms, its precise biochemical interactions are not fully understood. In the present study, we demonstrate that aluminum promotes the inhibition of aconitase (Acn) activity via the perturbation of the Fe-S cluster in Pseudomonas fluorescens. Despite the significant decrease in citrate isomerization activity, cellular survival is assured by the overexpression of isocitrate lyase and isocitrate dehydrogenase (IDH)-NADP+. 13C NMR spectroscopic studies, Blue Native PAGE, and Western blot analyses indicated that although the decrease in Acn activity is concomitant with the increase of aluminum in the culture, the amount of Acn expressed is not sensitive to the concentration of the trivalent metal. A 6-fold decrease in Acn activity and no discernable change in protein content in aluminum-stressed cultures were observed. The addition of Fe(NH4)2(SO4)2 in a reducing environment led to a significant recovery in Acn activity. This enzymatic activity reverted to normal levels when aluminum-stressed cells were transferred to either a control or an iron-supplemented medium. The overexpression of the two isocitrate-metabolizing enzymes isocitrate lyase and IDH-NADP+ appears to mitigate the deficit in Acn activity. The levels of these enzymes are dependent on the aluminum content of the culture and appear to be under transcriptional control. Hence, the regulation of the enzymes involved in the homeostasis of isocitrate constitutes a pivotal component of the global metabolic strategy that ensures the survival of this organism in an aluminum citrate environment.
Footnotes
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↵1 The abbreviations used are: CL, citrate-lyase; Acn, aconitase; IDH, isocitrate dehydrogenase; ICL, isocytrate lyase; DTT, dithiothreitol; CFE, cell-free extract; BN, Blue Native; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,-1-bis(hydroxymethyl)ethyl]glycine.
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↵* This work was supported by funding from Industry Canada and Human Resources Development Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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- Received October 22, 2004.
- Revision received November 10, 2004.
- The American Society for Biochemistry and Molecular Biology, Inc.
