The E. coli cells were disrupted by sonication. 54, 875–892. However, it remains unclear whether this regulatory mechanism is common for plant IDH. Hexameric oligomerization of mitochondrial peroxiredoxin PrxIIF and formation of an ultrahigh affinity complex with its electron donor thioredoxin Trx-o. Tryptic peptides were extracted from the gel with 0.1% (v/v) trifluoroacetic acid with 50 and 75% (v/v) acetonitrile, continuously. J. Proteomics 72, 452–474. Expression analysis of Arabidopsis thaliana NAD-dependent isocitrate dehydrogenase genes shows the presence of a functional subunit that is mainly expressed in the pollen and absent from vegetative organs. Isocitrate I IDCL Acetyl-CoA J 4 co2 Succinate Fig. Redox Signal. Alignment between plant and yeast IDH indicated that plant IDH does not conserve some of these residues (Supplementary Fig. Environ. Copyright © 2014 Yoshida and Hisabori. In addition, the TCA cycle enzyme citrate synthase has recently been reported to be redox-regulated by Trx-o (Schmidtmann et al., 2014). Flux through these enzymes is controlled by phosphorylation of isocitrate dehydrogenase, which has a much higher affinity for isocitrate as compared to ICL. Homologous binding sites in yeast isocitrate dehydrogenase for cofactor (NAD+) and allosteric activator (AMP). When Trx-o1 was added to a reaction medium, IDH-r was reduced back to monomer even at lower concentrations of DTT (Figure 4A). doi: 10.1002/pmic.200700209, Yoshida, K., Matsuoka, Y., Hara, S., Konno, H., and Hisabori, T. (2014). Purification was performed in a medium containing 25 mM Tris-HCl (pH 7.5–8.1), 1 mM EDTA, and 0.5 mM DTT, but EDTA and DTT were removed by dialysis after purification. It thus appeared that Arabidopsis IDH is reversibly inactivated in response to oxidative stress via oligomer formation of IDH-r. The citric acid cycle serves as a metabolic traffic circle that receives carbon skeletons from amino acids and fatty acids and donates carbon skeletons to amino acids and porphyrins. The first step of the reaction involves the oxidation of isocitrate to the intermediate oxalosuccinate. Acad. B., Hu, G., Hart, P. J., and McAlister-Henn, L. (2008). For oxidation treatment, IDH-r was incubated in 50 μM CuCl2 for 15 min at 25°C. IDH-r forms oligomer mediated by intermolecular disulfide bonds upon oxidation. In this study, we investigated the redox regulation mechanisms of IDH by biochemical procedures. doi: 10.1016/S0005-2728(03)00106-3, Laloi, C., Rayapuram, N., Chartier, Y., Grienenberger, J.M., Bonnard, G., and Meyer, Y. Plant Cell Physiol. (A) SDS-PAGE profiles of purified recombinant IDH-r and IDH-c. (B) Monitoring of NAD+-reducing activity of IDH. Recurrent mutations in IDH1 or IDH2 are prevalent in several cancers including glioma, acute myeloid leukemia (AML), cholangiocarcinoma, and chondrosarcoma. 278, 12864–12872. The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fenvs.2014.00038/abstract, Balmer, Y., Vensel, W. H., Tanaka, C. K., Hurkman, W. J., Gelhaye, E., Rouhier, N., et al. Assays were performed at 25°C in a medium containing 25 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM NAD+, and indicated concentrations of isocitrate. Three major peptides (m/z: 1280.6, 2276.2, and 2404.3) were specifically detected in the reduced monomeric form but not in the oxidized trimeric form. Citrate Synthase, Mammalian Isocitrate dehydrogenase, E. Coli isocitrate dehydrogenase, alpha=ketoglutarate dehydrogenase. IDH1 1.1. located on the long arm of chromoso… Cytosolic NADP-dependent isocitrate dehydrogenase contributes to redox homeostasis and the regulation of pathogen responses in Arabidopsis leaves. Future studies addressing the visualization of in vivo redox states of redox-regulated proteins, as shown in our recent study (Yoshida et al., 2014), and effects of Trx-o deficiency on mitochondrial performance will provide physiological insights into the mitochondrial redox regulation system in plants. Fumarase 8.) J. Exp. Dissecting the integrative antioxidant and redox systems in plant mitochondria. Thioredoxin links redox to the regulation of fundamental processes of plant mitochondria. It has been shown that, among these candidate proteins, two stress-related proteins, namely alternative oxidase and peroxiredoxin IIF, are reduced and activated in a Trx-o-dependent manner (Barranco-Medina et al., 2008; Martí et al., 2009; Yoshida et al., 2013). Plant Cell Physiol. Trx has a conserved WCGPC motif at an active site, enabling a dithiol–disulfide exchange reaction with the target enzyme. The redox state of IDH-r was assayed by non-reducing SDS-PAGE. The IDH-r redox state was assayed by non-reducing SDS-PAGE. Isocitrate dehydrogenase (IDH) is an important enzyme in the tricarboxylic acid cycle, which occurs in the mitochondrial matrix. Prior to the assay, IDH-r or IDH-c was incubated in the presence of 50 μM CuCl2 to oxidize the possible thiols on the protein molecule. In the process, two hydrogens are also removed. Sci., 23 September 2014 Trx-o was effective in the reduction of oxidized IDH-r, likely leading to a recovery of IDH activity (Figures 2, 4). Each of the amplified DNA was ligated into the pET23c expression vector (Novagen). (2013). This is an example of regulation by covalent modification of an enzyme since the action of ATP here is to phosphorylate, and consequently to inactivate, the isocitrate…. Activity measurements of Arabidopsis IDH recombinant protein. 4:460. doi: 10.3389/fpls.2013.00460, Lemaitre, T., and Hodges, M. (2006). doi: 10.1073/pnas.0308583101, Pubmed Abstract | Pubmed Full Text | CrossRef Full Text, Barranco-Medina, S., Krell, T., Bernier-Villamor, L., Sevilla, F., Lázaro, J.J., and Dietz, K.J. doi: 10.1104/pp.109.138073, Millar, A. H., Whelan, J., Soole, K. L., and Day, D. A. This finding implies the significance of the mitochondrial Trx system for the regulation of TCA cycle performance. The reduction patterns of oxidized IDH-r under several concentrations of DTT (0–500 μM) were compared in the presence and absence of 5 μM Trx-o1 (an isoform of Trx-o). Biochemistry 48, 8869–8878. NAD + -dependent isocitrate dehydrogenase (IDH), a key enzyme in the tricarboxylic acid (TCA) cycle, is one such candidate. Some forms of isocitrate dehydrogenase are maximally active only in the presence of ADP or AMP and are inhibited by ATP. Mitochondria play a pivotal role in providing ATP required for various cellular events in all eukaryotes. A., Minard, K. I., Lin, A. P., and McAlister-Henn, L. (2009). We next investigated whether IDH activity is controlled by the redox state of the enzyme molecule itself. Whole extracts were concentrated using a centrifugal concentrator and desalted using Solid Phase Extraction C-TIP (Nikkyo Technos). (A) Redox-dependent change in IDH activity. The regulatory mechanism of plant mitochondrial IDH thus remains poorly characterized. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. No use, distribution or reproduction is permitted which does not comply with these terms. As revealed by non-reducing SDS-PAGE, IDH-r was shifted to dimeric, trimeric, and higher-order oligomeric forms mediated by intermolecular disulfide bonds under oxidative conditions (Figure 2B). (C) The effect of adenylate on IDH activity under several concentrations of isocitrate. A number of genes have been identified which code for isoforms of these enzymes, with IDH1 and IDH2 being most relevant in current glioma classification 8. doi: 10.1074/jbc.M300154200, Lindahl, M., Mata-Cabana, A., and Kieselbach, T. (2011). J. Biol. Well why would this be? Mass spectrometry of IDH-r tryptic peptides. Isocitrate dehydrogenase acts at the rate-limiting step of the tricarboxylic acid (TCA; Krebs) cycle. The results indicate that IDH-r forms intermolecular disulfide bonds upon oxidation, leading to a drastic decrease in IDH activity. Mol. Bot. Figure 2. S1). (2014). NAD+-dependent isocitrate dehydrogenase (IDH), a key enzyme in the tricarboxylic acid (TCA) cycle, is one such candidate. S1). These findings suggest that Trx-o1 can efficiently reduce oxidized IDH-r. Photochem. Changes in Free Energy 1.) By using the recombinant subunits, we clearly showed that both IDH-r and IDH-c are essential for ensuring the catalytic activity of Arabidopsis IDH (Figure 1B). Isocitrate dehydrogenase is a digestive enzyme that is used in the citric acid cycle. Using partially purified IDH from pea leaves, Igamberdiev and Gardeström (2003) demonstrated that IDH activity is negatively regulated by NAD(P)H. However, further biochemical studies of plant IDH have not been performed to date, and accordingly the regulatory mechanisms of this enzyme at the molecular level remain to be fully characterized. Flexible regulation of these mitochondrial reactions is important for ensuring proper cellular function, particularly in plants, which cannot escape exposure to adverse environmental conditions. One of these, in the form of a hydride, is transferred to the carrier NAD (or NADP), and will be used later to power the rotation of ATP synthase. doi: 10.1093/mp/sst144, Taylor, A. We also showed that Trx-o assists in the reduction of oxidized IDH-r. Based on these findings, a novel regulatory mode of plant mitochondrial IDH is discussed. Thioredoxin targets in plants: the first 30 years. IDH is phosphorylated at a single serine which resides in its active site. Biochim. However, careful biochemical study is needed to determine whether these candidate proteins are actually redox-regulated. As summarized in Figure 5, our biochemical analysis suggested that IDH-r is a redox-sensitive protein and that the redox change affects IDH activity. Curr. Isocitrate dehydrogenases (IDHs) catalyze the oxida-tive decarboxylation of isocitrate to -ketoglutarate, and regulation of the enzymatic activity of IDHs is cru-cial for their biological functions. Isocitrate dehydrogenase (IDH) (EC 1.1.1.42) and (EC 1.1.1.41) is an enzyme that catalyzes the oxidative decarboxylation of isocitrate, producing alpha-ketoglutarate (α-ketoglutarate) and CO 2. Differential gene expression profiles of the mitochondrial respiratory components in illuminated Arabidopsis leaves. Front. In this study, we investigated the redox regulation mechanisms of IDH by biochemical procedures. After centrifugation (125,000 ×g for 40 min), the resulting supernatant was used to purify the protein of interest. It must therefore be carefully regulated to avoid depletion of isocitrate (and therefore an accumulation of alpha-ketoglutarate). Isocitrate dehydrogenase performs the third reaction in the citric acid cycle, which releases one of the carbon atoms as carbon dioxide. In plant IDH-r, six Cys residues are commonly conserved (Supplementary Fig. What are the enzymes regulated in the regulation of the citric acid cycle? To test the adenylate effects on IDH activity, each adenylate (AMP, ADP, and ATP) was added at 1 mM to the media described above. Proc. Photobiol. doi: 10.1093/pcp/pct037, Keywords: Arabidopsis, isocitrate dehydrogenase, mitochondria, redox regulation, thioredoxin, Citation: Yoshida K and Hisabori T (2014) Mitochondrial isocitrate dehydrogenase is inactivated upon oxidation and reactivated by thioredoxin-dependent reduction in Arabidopsis. Proc. This fact implies that the mode of IDH redox regulation disclosed in this study is unique to plants. Subunit interactions of yeast NAD+-specific isocitrate dehydrogenase. S1). doi: 10.1046/j.1365-313x.1998.00305.x, Lázaro, J.J., Jiménez, A., Camejo, D., Iglesias-Baena, I., Martí, M.C., Lázaro-Payo, A., et al. Yeast IDH activity is also controlled in a redox-dependent manner (Garcia et al., 2009). Plant 7, 156–169. doi: 10.1074/jbc.M708719200, Tcherkez, G., Boex-Fontvieille, E., Mahé, A., and Hodges, M. (2012). Acad. The sensitivity to regulation of proteins undergoing covalent modification can be greatly increased when the substrates saturate the converter enzymes. However, most plant mitochondrial proteins suggested as Trx targets remain to be further analyzed by detailed biochemical assays. Furthermore, detailed biochemical and structural analyses have provided evidence that yeast IDH is allosterically activated by AMP and inactivated by intermolecular disulfide bond formation between IDH-c subunits (Lin and McAlister-Henn, 2003; Taylor et al., 2008; Garcia et al., 2009). The IDH step of the citric acid cycle, is often (but not always) an irreversible reactions in the citric acid cycle, due to its large negative free energy change. Opin. Upon oxidation of IDH-r, IDH activity was largely diminished via intermolecular disulfide-mediated oligomer formation of IDH-r (Figures 2A,B). …the enzyme controlling this reaction, isocitrate dehydrogenase, differs in specificity for the coenzymes; various forms occur not only in different organisms but even within the same cell. Plant Cell Physiol. |, http://www.frontiersin.org/journal/10.3389/fenvs.2014.00038/abstract, Creative Commons Attribution License (CC BY). The glyox- ylate bypass is composed of isocitrate lyase and malate syn- thase. (2007). Primary amino acid sequences of these gene products show high identity among each isoform (IDH1-3; >84%, IDH5-6; 90%, mature protein region except for targeting peptide). NAD+-reducing activity was observed in the presence of both subunits, whereas neither subunit alone showed any catalytic activity (Figure 1B). Total RNA was isolated from Arabidopsis thaliana as described in Yoshida and Noguchi (2009) and used as a template for RT-PCR. Each adenylate (AMP, ADP, ATP) was added at 1 mM. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). This reaction also produces NADPH (IDH1 and IDH2) or NADH (IDH3) 4,5. (2009). Biophys. Chem. Although it has been recognized that the Trx-o resides in plant mitochondria (Laloi et al., 2001), information about the target proteins of Trx-o is limited to date. Identification and characterization of a mitochondrial thioredoxin system in plants. There is much detailed knowledge about this bacterial enzyme, and it has been found that most isocitrate dehydrogenases … Succinyl-CoA Synthetase 6.) In the case of yeast IDH, IDH-c forms intermolecular disulfide bonds under oxidative conditions, leading to the inactivation of IDH (Garcia et al., 2009). However, for several redox-regulated protein candidates identified by redox-proteomics studies, it remains to be determined whether they are “pseudo” or “true” redox-regulated proteins. This work was supported in part by the Core Research of Evolutional Science and Technology program (CREST) from the Japan Science and Technology Agency (JST) and a Grant-in-Aid for Scientific Research (grant number 24870010 to Keisuke Yoshida) from the Japan Society for the Promotion of Science. The specific signals for monomeric form are indicated by arrows and the corresponding peptides are shown. 2:38. doi: 10.3389/fenvs.2014.00038. No adenylates (AMP, ADP, and ATP) affected the saturation velocity curve (Figure 1C) or correspondingly the Km and Vmax values (Table 1) for isocitrate. 14, 2581–2642. Trends Plant Sci. Mitochondrial respiration is controlled at multiple levels from transcriptional to post-translational to enzyme function levels (Millar et al., 2011). Rapid purification of pig heart NAD+-isocitrate dehydrogenase. It has been well documented that ADP and AMP act as allosteric activators of IDH in mammal and yeast (Nunes-Nesi et al., 2013). This is an example of regulation by covalent modification of an enzyme since the action of ATP here is to phosphorylate, and consequently to inactivate, the isocitrate… Deactivation of isocitrate dehydrogenase by phosphorylation thus leads to increased isocitrate channeling through ICL, as seen when bacteria are grown on acetate , a two-carbon compound. Author information: (1)Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K. 1. Redox-proteomics studies using Trx affinity chromatography have provided a comprehensive list of potentially redox-regulated proteins in plant mitochondria (Balmer et al., 2004; Yoshida et al., 2013). The minimal functional unit of yeast IDH is reported to be a heterodimer comprising IDH-r and IDH-c. Opin. Each value represents the mean ± SD (n = 3). Instead, IDH was inactivated by oxidation treatment and partially reactivated by subsequent reduction. identity with Escherichia coli isocitrate dehydrogenase, which requires NADP1 as cofactor. Further investigation is required for concluding this possibility. Rev. For oxidation (Ox) treatment, IDH-r or IDH-c was incubated in 50 μM CuCl2 for 15 min. Isocitrate dehydrogenase is allosterically regulated by ATP, NADH, ADP, and calcium. Sci. The Isocitrate Dehydrogenase (IDH) enzyme structure in Escherichia coli was the first structure to be elucidated and understood. doi: 10.1073/pnas.191282098, Noctor, G., De Paepe, R., and Foyer, C.H. The regulation of pyruvate kinase involves phosphorylation by a kinase (pyruvate kinase), resulting in a less-active enzyme. IDH-r and/or IDH-c (40 nM each) were added to a reaction medium. It should be noted that IDH activity was restored to only half of the control level, even after the reduction and monomerization of oxidized IDH-r (Figure 2). 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( 2009 ) and converts into an isocitrate dehydrogenase regulation compound called Succinyl.... Published online: 23 September 2014 |, http: //www.matrixscience.com/ ) to identify matched peptides calcium it! Disulfide proteome and other reactive cysteine proteomes: analysis and functional significance generated NADH suggested that IDH-r also undergoes oxidative! And yeast IDH is reversibly inactivated in response to oxidative stress via formation... Pea thioredoxin: identification of intra- and intermolecular disulphide bonding in the third step of the tricarboxylic (!

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