AcnC of Escherichia coli is a 2-methylcitrate dehydratase (PrpD) that can use citrate and isocitrate as substrates.
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Escherichia coli possesses two well-characterized aconitases (AcnA and AcnB) and a minor activity (designated AcnC) that is retained by acnAB double mutants and represents no more than 5% of total wild-type aconitase activity. Here it is shown that a 2-methylcitrate dehydratase (PrpD) encoded by the prpD gene of the propionate catabolic operon (prpRBCDE) is identical to AcnC. Inactivation of prpD abolished the residual aconitase activity of an AcnAB-null strain, whereas inactivation of ybhJ, an unidentified acnA paralogue, had no significant effect on AcnC activity. Purified PrpD catalysed the dehydration of citrate and isocitrate but was most active with 2-methylcitrate. PrpD also catalysed the dehydration of several other hydroxy acids but failed to hydrate cis-aconitate and related substrates containing double bonds, indicating that PrpD is not a typical aconitase but a dehydratase. Purified PrpD was shown to be a monomeric iron-sulphur protein (M(r) 54000) having one unstable [2Fe-2S] cluster per monomer, which is needed for maximum catalytic activity and can be reconstituted by treatment with Fe(2+) under reducing conditions. (+info)
Structural changes associated with switching activities of human iron regulatory protein 1.
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Metazoan iron regulatory protein 1 is a dual activity protein, being either an aconitase or a regulatory factor binding to messenger RNA involved in iron homeostasis. Sequence comparisons and site-directed mutagenesis experiments have supported a structural relationship between mitochondrial aconitase and iron regulatory protein 1. The structural properties of human recombinant iron regulatory protein 1 have been probed in the present work. Although iron-free iron regulatory protein 1 displays a significantly larger radius of gyration measured by small-angle neutron scattering than calculated for mitochondrial aconitase, binding of either the [4Fe-4S] cluster needed for aconitase activity or of a RNA substrate turns iron regulatory protein 1 into a more compact molecule. These conformational changes are associated with the gain of secondary structural elements as indicated by circular dichroism studies. They likely involve alpha-helices covering the substrate binding cleft of cytosolic aconitase, and they suggest an induced fit mechanism of iron-responsive element recognition. These studies refine previously proposed models of the "iron-sulfur switch" driving the biological function of human iron regulatory protein 1, and they provide a structural framework to probe the relevance of the numerous cellular molecules proposed to affect its function. (+info)
In vitro serial passage of Staphylococcus aureus: changes in physiology, virulence factor production, and agr nucleotide sequence.
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Recently, we observed that Staphylococcus aureus strains newly isolated from patients had twofold-higher aconitase activity than a strain passaged extensively in vitro, leading us to hypothesize that aconitase specific activity decreases over time during in vitro passage. To test this hypothesis, a strain recovered from a patient with toxic shock syndrome was serially passaged for 6 weeks, and the aconitase activity was measured. Aconitase specific activity decreased 38% (P < 0.001) by the sixth week in culture. During serial passage, S. aureus existed as a heterogeneous population with two colony types that had pronounced (wild type) or negligible zones of beta-hemolytic activity. The cell density-sensing accessory gene regulatory (agr) system regulates beta-hemolytic activity. Surprisingly, the percentage of colonies with a wild-type beta-hemolytic phenotype correlated strongly with aconitase specific activity (rho = 0.96), suggesting a common cause of the decreased aconitase specific activity and the variation in percentage of beta-hemolytic colonies. The loss of the beta-hemolytic phenotype also coincided with the occurrence of mutations in the agrC coding region or the intergenic region between agrC and agrA in the derivative strains. Our results demonstrate that in vitro growth is sufficient to result in mutations within the agr operon. Additionally, our results demonstrate that S. aureus undergoes significant phenotypic and genotypic changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains. (+info)
Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer.
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The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for amino-acid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes. The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among alpha-proteobacteria, the group of eubacteria as defined under rRNA systematics from which mitochondria arose. Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from alpha-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution. Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants. The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their alpha-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not specifically alpha-proteobacterial, homologues. A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the alpha-proteobacterial ancestor of mitochondria to branch specifically with their homologues encoded in the genomes of contemporary alpha-proteobacteria. Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the alpha-proteobacterial ancestor of mitochondria should, in phylogenetic trees, branch with homologues that are no longer found in most alpha-proteobacterial genomes, and some should reside on long branches that reveal affinity to eubacterial rather than archaebacterial homologues, but no particular affinity for any specific eubacterial donor. (+info)
Transferrin receptor-dependent iron uptake is responsible for doxorubicin-mediated apoptosis in endothelial cells: role of oxidant-induced iron signaling in apoptosis.
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In the past, investigators have successfully used iron chelators to mitigate the cardiotoxicity of doxorubicin (DOX), a widely used anticancer drug that induces reactive oxygen species (ROS), oxidative damage, and apoptosis. Although intracellular iron plays a critical role in initiating DOX-induced apoptosis, the molecular mechanism(s) that link iron, ROS, and apoptosis are still unknown. In this study, we demonstrate that apoptosis results from the exposure of bovine aortic endothelial cells to DOX and that the apoptotic cell death is accompanied by a significant increase in cellular iron ((55)Fe) uptake and activation of iron regulatory protein-1. Furthermore, DOX-induced iron uptake was shown to be mediated by the transferrin receptor (TfR)-dependent mechanism. Treatment with the anti-TfR antibody (IgA class) dramatically inhibited DOX-induced apoptosis, iron uptake, and intracellular oxidant formation as measured by fluorescence using dichlorodihydrofluorescein. Treatment with cell-permeable iron chelators and ROS scavengers inhibited DOX-induced cellular (55)Fe uptake, ROS formation, and apoptosis. Based on these findings, we conclude that DOX-induced iron signaling is regulated by the cell surface TfR expression, intracellular oxidant levels, and iron regulatory proteins. The implications of TfR-dependent iron transport in oxidant-induced apoptosis in endothelial cells are discussed. (+info)
A mitochondrial-like aconitase in the bacterium Bacteroides fragilis: implications for the evolution of the mitochondrial Krebs cycle.
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Aconitase and isocitrate dehydrogenase (IDH) enzyme activities were detected in anaerobically prepared cell extracts of the obligate anaerobe Bacteroides fragilis. The aconitase gene was located upstream of the genes encoding the other two components of the oxidative branch of the Krebs cycle, IDH and citrate synthase. Mutational analysis indicates that these genes are cotranscribed. A nonpolar in-frame deletion of the acnA gene that encodes the aconitase prevented growth in glucose minimal medium unless heme or succinate was added to the medium. These results imply that B. fragilis has two pathways for alpha-ketoglutarate biosynthesis-one from isocitrate and the other from succinate. Homology searches indicated that the B. fragilis aconitase is most closely related to aconitases of two other Cytophaga-Flavobacterium-Bacteroides (CFB) group bacteria, Cytophaga hutchinsonii and Fibrobacter succinogenes. Phylogenetic analysis indicates that the CFB group aconitases are most closely related to mitochondrial aconitases. In addition, the IDH of C. hutchinsonii was found to be most closely related to the mitochondrial/cytosolic IDH-2 group of eukaryotic organisms. These data suggest a common origin for these Krebs cycle enzymes in mitochondria and CFB group bacteria. (+info)
Iron deficiency decreases mitochondrial aconitase abundance and citrate concentration without affecting tricarboxylic acid cycle capacity in rat liver.
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Mitochondrial aconitase (m-acon) is the tricarboxylic acid (TCA) cycle enzyme that converts citrate to isocitrate. m-Acon mRNA is a potential target for regulation by iron regulatory proteins (IRPs), suggesting a link between dietary iron intake, m-acon synthesis, and energy metabolism. Our previous studies indicate that m-acon is one of a limited number of proteins that is down-regulated in iron-deficient liver. Here we use isolated hepatocytes to study the relationships among decreased m-acon abundance, TCA cycle function and cellular citrate concentration in iron deficiency. Rats were fed an iron-deficient (ID) (2 mg Fe/kg diet) diet, or they were pair-fed (PF) or freely fed (C) a control diet (50 mg Fe/kg diet) for up to 21 d. Hepatocyte total IRP activity was greater by d 2 in the ID group than in the C and PF groups and by d 10, the difference was maximal. Liver IRP activity was inversely correlated with m-acon abundance (r = -0.93, P < 0.0001). However, the decrease in m-acon abundance did not affect the ability of hepatocytes to oxidize 2-[(14)C]pyruvate or 1-[(14)C]acetate, indicating that TCA cycle capacity was not affected. Interestingly, by d 21, total liver citrate concentration was 40% lower in ID than in PF rats, suggesting enhanced utilization of citrate. However, the decrease in citrate concentration was not reflected in a change in liver total lipid concentration. Taken together, our results indicate that the iron-dependent regulation of m-acon in liver does not alter TCA cycle capacity but suggest that IRP-mediated changes in m-acon expression may modulate citrate use in other aspects of intermediary or iron metabolism. (+info)
Escherichia coli aconitases and oxidative stress: post-transcriptional regulation of sodA expression.
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Escherichia coli possesses two aconitases, a stationary-phase enzyme (AcnA), which is induced by iron and oxidative stress, and a major but less stable enzyme (AcnB), synthesized during exponential growth. In addition to the catalytic activities of the holo-proteins, the apo-proteins function as post-transcriptional regulators by site-specific binding to acn mRNAs. Thus, it has been suggested that inactivation of the enzymes could mediate a rapidly reacting post-transcriptional component of the bacterial oxidative stress response. Here it is shown that E. coli acn mutants are hypersensitive to the redox-stress reagents H(2)O(2) and methyl viologen. Proteomic analyses further revealed that the level of superoxide dismutase (SodA) is enhanced in acnB and acnAB mutants, and by exposure to methyl viologen. The amounts of other proteins, including thioredoxin reductase, 2-oxoglutarate dehydrogenase, succinyl-CoA synthetase and chaperone proteins, were also affected in the acn mutants. The altered patterns of sodA expression were confirmed in studies with sodA-lacZ reporter strains. Quantitative Northern blotting indicated that AcnA enhances the stability of the sodA transcript, whereas AcnB lowers its stability. Direct evidence that the apo-proteins have positive (AcnA) and negative (AcnB) effects on SodA synthesis was obtained from in vitro transcription-translation experiments. It is suggested that the aconitase proteins of E. coli serve as a protective buffer against the basal level of oxidative stress that accompanies aerobic growth by acting as a sink for reactive oxygen species and by modulating translation of the sodA transcript. (+info)