Nitric oxide modulates the activity of tobacco aconitase

Recent evidence suggests an important role for nitric oxide (NO) signaling in plant-pathogen interactions. Additional elucidation of the role of NO in plants will require identification of NO targets. Since aconitases are major NO targets in animals, we examined the effect of NO on tobacco (Nicotiana tabacum) aconitase. The tobacco aconitases, like their animal counterparts, were inhibited by NO donors. The cytosolic aconitase in animals, in addition to being a key redox and NO sensor, is converted by NO into an mRNA binding protein (IRP, or iron-regulatory protein) that regulates iron homeostasis. A tobacco cytosolic aconitase gene (NtACO1) whose deduced amino acid sequence shared 61% identity and 76% similarity with the human IRP-1 was cloned. Furthermore, residues involved in mRNA binding by IRP-1 were conserved in NtACO1. These results reveal additional similarities between the NO signaling mechanisms used by plants and animals.     

Recycling of RNA binding iron regulatory protein 1 into an aconitase after nitric oxide removal depends on mitochondrial ATP

Iron regulatory proteins (IRPs) control iron metabolism by specifically interacting with iron-responsive elements (IREs) on mRNAs. Nitric oxide (NO) converts IRP-1 from a [4Fe-4S] aconitase to a trans-regulatory protein through Fe-S cluster disassembly. Here, we have focused on the fate of IRE binding IRP1 from murine macrophages when NO flux stops. We show that virtually all IRP-1 molecules from NO-producing cells dissociated from IRE and recovered aconitase activity after re-assembling a [4Fe-4S] cluster in vitro. The reverse change in IRP-1 activities also occurred in intact cells no longer exposed to NO and did not require de novo protein synthesis. Likewise, inhibition of mitochondrial aconitase via NO-induced Fe-S cluster disassembly was also reversed independently of protein translation after NO removal. Our results provide the first evidence of Fe-S cluster repair of NO-modified aconitases in mammalian cells. Moreover, we show that reverse change in IRP-1 activities and repair of mitochondrial aconitase activity depended on energized mitochondria. Finally, we demonstrate that IRP-1 activation by NO was accompanied by both a drastic decrease in ferritin levels and an increase in transferrin receptor mRNA levels. However, although ferritin expression was recovered upon IRP-1-IRE dissociation, expression of transferrin receptor mRNA continued to rise for several hours after stopping NO flux.     

Knock-downs of iron-sulfur cluster assembly proteins IscS and IscU down-regulate the active mitochondrion of procyclic Trypanosoma brucei

Transformation of the metabolically down-regulated mitochondrion of the mammalian bloodstream stage of Trypanosoma brucei to the ATP-producing mitochondrion of the insect procyclic stage is accompanied by the de novo synthesis of citric acid cycle enzymes and components of the respiratory chain. Because these metabolic pathways contain multiple iron-sulfur (FeS) proteins, their synthesis, including the formation of FeS clusters, is required. However, nothing is known about FeS cluster biogenesis in trypanosomes, organisms that are evolutionarily distant from yeast and humans. Here we demonstrate that two mitochondrial proteins, the cysteine desulfurase TbiscS and the metallochaperone TbiscU, are functionally conserved in trypanosomes and essential for this parasite. Knock-downs of TbiscS and TbiscU in the procyclic stage by means of RNA interference resulted in reduced activity of the marker FeS enzyme aconitase in both the mitochondrion and cytosol because of the lack of FeS clusters. Moreover, down-regulation of TbiscS and TbiscU affected the metabolism of procyclic T. brucei so that their mitochondria resembled the organelle of the bloodstream stage; mitochondrial ATP production was impaired, the activity of the respiratory chain protein complex ubiquinol-cytochrome-c reductase was reduced, and the production of pyruvate as an end product of glucose metabolism was enhanced. These results indicate that mitochondrial FeS cluster assembly is indispensable for completion of the T. brucei life cycle.     

Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation

The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitro differentiation from wild type and mutant (Delta aco::NEO/Delta aco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO(2). Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.     

Cytosolic aconitase and ferritin are regulated by iron in Caenorhabditis elegans

Iron regulatory protein-1 (IRP-1) is a cytosolic RNA-binding protein that is a regulator of iron homeostasis in mammalian cells. IRP-1 binds to RNA structures, known as iron-responsive elements, located in the untranslated regions of specific mRNAs, and it regulates the translation or stability of these mRNAs. Iron regulates IRP-1 activity by converting it from an RNA-binding apoprotein into a [4Fe-4S] cluster protein exhibiting aconitase activity. IRP-1 is widely found in prokaryotes and eukaryotes. Here, we report the biochemical characterization and regulation of an IRP-1 homolog in Caenorhabditis elegans (GEI-22/ACO-1). GEI-22/ACO-1 is expressed in the cytosol of cells of the hypodermis and the intestine. Like mammalian IRP-1/aconitases, GEI-22/ACO-1 exhibits aconitase activity and is post-translationally regulated by iron. Although GEI-22/ACO-1 shares striking resemblance to mammalian IRP-1, it fails to bind RNA. This is consistent with the lack of iron-responsive elements in the C. elegans ferritin genes, ftn-1 and ftn-2. While mammalian ferritin H and L mRNAs are translationally regulated by iron, the amounts of C. elegans ftn-1 and ftn-2 mRNAs are increased by iron and decreased by iron chelation. Excess iron did not significantly alter worm development but did shorten their life span. These studies indicated that iron homeostasis in C. elegans shares some similarities with those of vertebrates.     

The aconitase of Escherichia coli. Nucleotide sequence of the aconitase gene and amino acid sequence similarity with mitochondrial aconitases, the iron-responsive-element-binding protein and isopropylmalate isomerases.

The nucleotide sequence of the aconitase gene (acn) of Escherichia coli was determined and used to deduce the primary structure of the enzyme. The coding region comprises 2670 bp (890 codons excluding the start and stop codons) which define a product having a relative molecular mass of 97,513 and an N-terminal amino acid sequence consistent with those determined previously for the purified enzyme. The acn gene is flanked by the cysB gene and a putative riboflavin biosynthesis gene resembling the ribA gene of Bacillus subtilis. The 1004-bp cysB--acn intergenic region contains several potential promoter and regulatory sequences. The amino acid sequence of the E. coli aconitase is similar to the mitochondrial aconitases (27-29% identity) and the isopropylmalate isomerases (20-21% identity) but it is most similar to the human iron-responsive-element-binding protein (53% identity). The three cysteine residues involved in ligand binding to the [4Fe-4S] centre are conserved in all of these proteins. Of the remaining 17 active-site residues assigned for porcine aconitase, 16 are conserved in both the bacterial aconitase and the iron-responsive-element-binding protein and 14 in the isopropylmalate isomerases. It is concluded that the bacterial and mitochondrial aconitases, the isopropylmalate isomerases and the iron-responsive-element-binding protein form a family of structurally related proteins, which does not include the Fe-S-containing fumarases. These relationships raise the possibility that the iron-responsive-element-binding protein may be a cytoplasmic aconitase and that the E. coli aconitase may have an iron-responsive regulatory function.     

Fumarate is an essential intermediary metabolite produced by the procyclic Trypanosoma brucei

The procyclic stage of Trypanosoma brucei, a parasitic protist responsible for sleeping sickness in humans, converts most of the consumed glucose into excreted succinate, by succinic fermentation. Succinate is produced by the glycosomal and mitochondrial NADH-dependent fumarate reductases, which are not essential for parasite viability. To further explore the role of the succinic fermentation pathways, we studied the trypanosome fumarases, the enzymes providing fumarate to fumarate reductases. The T. brucei genome contains two class I fumarase genes encoding cytosolic (FHc) and mitochondrial (FHm) enzymes, which account for total cellular fumarase activity as shown by RNA interference. The growth arrest of a double RNA interference mutant cell line showing no fumarase activity indicates that fumarases are essential for the parasite. Interestingly, addition of fumarate to the medium rescues the growth phenotype, indicating that fumarate is an essential intermediary metabolite of the insect stage trypanosomes. We propose that trypanosomes use fumarate as an essential electron acceptor, as exemplified by the fumarate dependence previously reported for an enzyme of the essential de novo pyrimidine synthesis (Takashima, E., Inaoka, D. K., Osanai, A., Nara, T., Odaka, M., Aoki, T., Inaka, K., Harada, S., and Kita, K. (2002) Mol. Biochem. Parasitol. 122, 189-200).     

Cytosolic Aconitase Participates in the Glyoxylate Cycle in Etiolated Pumpkin Cotyledons

Two different aconitases are known to be expressed after thegermination of oil-seed plants. One is a mitochondrial aconitasethat is involved in the tricarboxylic acid cycle. The otherparticipates in the glyoxylate cycle, playing a role in gluconeogenesisfrom stored oil. We isolated and characterized a cDNA for anaconitase from etiolated pumpkin cotyledons. The cDNA was 3,145bp long and capable of encoding a protein of 98 kDa. N-terminaland C-terminal amino acid sequences deduced from the cDNA didnot contain mitochondrial or glyoxysomal targeting signals.A search of protein databases suggested that the cDNA encodeda cytosolic aconitase. Immuno blotting analysis with a specificantibody against the aconitase expressed in Escherichia colirevealed that developmental changes in the amount of the aconitasewere correlated with changes in levels of other enzymes of theglyoxylate cycle during growth of seedlings. Further analysisby subcellular fractionation and immunofluorescence microscopyrevealed that aconitase was present only in the cytosol andmitochondria. No glyoxysomal aconitase was found in etiolatedcotyledons even though all the other enzymes of the glyoxylatecycle are known to be localized in glyoxysomes. Taken together,the data suggest that the cytosolic aconitase participates inthe glyoxylate cycle with four glyoxysomal enzymes. (Received December 1, 1994; Accepted March 17, 1995)     

The mitochondrion in bloodstream forms of Trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the F1F0-ATPase.

Bloodstream forms of Trypanosoma brucei were found to maintain a significant membrane potential across their mitochondrial inner membrane (delta psi m) in addition to a plasma membrane potential (delta psi p). Significantly, the delta psi m was selectively abolished by low concentrations of specific inhibitors of the F1F0-ATPase, such as oligomycin, whereas inhibition of mitochondrial respiration with salicylhydroxamic acid was without effect. Thus, the mitochondrial membrane potential is generated and maintained exclusively by the electrogenic translocation of H+, catalysed by the mitochondrial F1F0-ATPase at the expense of ATP rather than by the mitochondrial electron-transport chain present in T. brucei. Consequently, bloodstream forms of T. brucei cannot engage in oxidative phosphorylation. The mitochondrial membrane potential generated by the mitochondrial F1F0-ATPase in intact trypanosomes was calculated after solving the two-compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh3P+) and was shown to have a value of approximately 150 mV. When the value for the delta psi m is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton-motive force was calculated to be greater than 190 mV. It seems likely that this mitochondrial proton-motive force serves a role in the directional transport of ions and metabolites across the promitochondrial inner membrane during the bloodstream stage of the life cycle, as well as promoting the import of nuclear-encoded protein into the promitochondrion during the transformation of bloodstream forms into the next stage of the life cycle of T. brucei.     

Depletion of the thioredoxin homologue tryparedoxin impairs antioxidative defence in African trypanosomes

In trypanosomes, the thioredoxin-type protein TXN (tryparedoxin) is a multi-purpose oxidoreductase that is involved in the detoxification of hydroperoxides, the synthesis of DNA precursors and the replication of the kinetoplastid DNA. African trypanosomes possess two isoforms that are localized in the cytosol and in the mitochondrion of the parasites respectively. Here we report on the biological significance of the cTXN (cytosolic TXN) of Trypanosoma brucei for hydroperoxide detoxification. Depending on the growth phase, the concentration of the protein is 3-7-fold higher in the parasite form infecting mammals (50-100 microM) than in the form hosted by the tsetse fly (7-34 microM). Depletion of the mRNA in bloodstream trypanosomes by RNA interference revealed the indispensability of the protein. Proliferation and viability of cultured trypanosomes were impaired when TXN was lowered to 1 muM for more than 48 h. Although the levels of glutathione, glutathionylspermidine and trypanothione were increased 2-3.5-fold, the sensitivity against exogenously generated H2O2 was significantly enhanced. The results prove the essential role of the cTXN and its pivotal function in the parasite defence against oxidative stress.     

Monothiol glutaredoxin-1 is an essential iron-sulfur protein in the mitochondrion of African trypanosomes

African trypanosomes encode three monothiol glutaredoxins (1-C-Grx). 1-C-Grx1 occurs exclusively in the mitochondrion, and 1-C-Grx2 and -3 are predicted to be mitochondrial and cytosolic proteins, respectively. All three 1-C-Grx are expressed in both the mammalian bloodstream and the insect procyclic form of Trypanosoma brucei, with the highest levels found in stationary phase and starving parasites. In the rudimentary mitochondrion of bloodstream cells, 1-C-Grx1 reaches concentrations above 200 microm/subunit. Recombinant T. brucei 1-C-Grx1 exists as a noncovalent homodimer, whereas 1-C-Grx2 and 1-C-Grx3 are monomeric proteins. In vitro, dimeric 1-C-Grx1 coordinated an H(2)O(2)-sensitive [2Fe-2S] cluster that required GSH as an additional ligand. Both bloodstream and procyclic trypanosomes were refractory to down-regulation of 1-C-Grx1 expression by RNA interference. In procyclic parasites, the 1-c-grx1 alleles could only be deleted if an ectopic copy of the gene was expressed. A 5-10-fold overexpression of 1-C-Grx1 in both parasite forms did not yield a growth phenotype under optimal culture conditions. However, exposure of these cells to the iron chelator deferoxamine or H(2)O(2), but not to iron or menadione, impaired cell growth. Treatment of wild-type bloodstream parasites with deferoxamine and H(2)O(2) caused a 2-fold down- and up-regulation of 1-C-Grx1, respectively. The results point to an essential role of the mitochondrial 1-C-Grx1 in the iron metabolism of these parasites.     

The Phosphinomethylmalate Isomerase Gene pmi, Encoding an Aconitase-Like Enzyme, Is Involved in the Synthesis of Phosphinothricin Tripeptide in Streptomyces viridochromogenes

Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricin tripeptide (PTT). In the postulated biosynthetic pathway, one reaction, the isomerization of phosphinomethylmalate, resembles the aconitase reaction of the tricarboxylic acid (TCA) cycle. It was speculated that this reaction is carried out by the corresponding enzyme of the primary metabolism (C. J. Thompson and H. Seto, p. 197–222, in L. C. Vining and C. Stuttard, ed., Genetics and Biochemistry of Antibiotic Production, 1995). However, in addition to the TCA cycle aconitase gene, a gene encoding an aconitase-like protein (the phosphinomethylmalate isomerase gene, pmi) was identified in the PTT biosynthetic gene cluster by Southern hybridization experiments, using oligonucleotides which were derived from conserved amino acid sequences of aconitases. The deduced protein revealed high similarity to aconitases from plants, bacteria, and fungi and to iron regulatory proteins from eucaryotes. Pmi and the S. viridochromogenes TCA cycle aconitase, AcnA, have 52% identity. By gene insertion mutagenesis, a pmi mutant (Mapra1) was generated. The mutant failed to produce PTT, indicating the inability of AcnA to carry out the secondary-metabolism reaction. A His-tagged protein (Hispmi*) was heterologously produced in Streptomyces lividans. The purified protein showed no standard aconitase activity with citrate as a substrate, and the corresponding gene was not able to complement an acnA mutant. This indicates that Pmi and AcnA are highly specific for their respective enzymatic reactions.     

The phosphinomethylmalate isomerase gene pmi, encoding an aconitase-like enzyme, is involved in the synthesis of phosphinothricin tripeptide in Streptomyces viridochromogenes

Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricin tripeptide (PTT). In the postulated biosynthetic pathway, one reaction, the isomerization of phosphinomethylmalate, resembles the aconitase reaction of the tricarboxylic acid (TCA) cycle. It was speculated that this reaction is carried out by the corresponding enzyme of the primary metabolism (C. J. Thompson and H. Seto, p. 197-222, in L. C. Vining and C. Stuttard, ed., Genetics and Biochemistry of Antibiotic Production, 1995). However, in addition to the TCA cycle aconitase gene, a gene encoding an aconitase-like protein (the phosphinomethylmalate isomerase gene, pmi) was identified in the PTT biosynthetic gene cluster by Southern hybridization experiments, using oligonucleotides which were derived from conserved amino acid sequences of aconitases. The deduced protein revealed high similarity to aconitases from plants, bacteria, and fungi and to iron regulatory proteins from eucaryotes. Pmi and the S. viridochromogenes TCA cycle aconitase, AcnA, have 52% identity. By gene insertion mutagenesis, a pmi mutant (Mapra1) was generated. The mutant failed to produce PTT, indicating the inability of AcnA to carry out the secondary-metabolism reaction. A His-tagged protein (Hispmi*) was heterologously produced in Streptomyces lividans. The purified protein showed no standard aconitase activity with citrate as a substrate, and the corresponding gene was not able to complement an acnA mutant. This indicates that Pmi and AcnA are highly specific for their respective enzymatic reactions.     

Metabolic regulation of citrate and iron by aconitases: role of iron–sulfur cluster biogenesis

Iron and citrate are essential for the metabolism of most organisms, and regulation of iron and citrate biology at both the cellular and systemic levels is critical for normal physiology and survival. Mitochondrial and cytosolic aconitases catalyze the interconversion of citrate and isocitrate, and aconitase activities are affected by iron levels, oxidative stress and by the status of the Fe–S cluster biogenesis apparatus. Assembly and disassembly of Fe–S clusters is a key process not only in regulating the enzymatic activity of mitochondrial aconitase in the citric acid cycle, but also in controlling the iron sensing and RNA binding activities of cytosolic aconitase (also known as iron regulatory protein IRP1). This review discusses the central role of aconitases in intermediary metabolism and explores how iron homeostasis and Fe–S cluster biogenesis regulate the Fe–S cluster switch and modulate intracellular citrate flux.