Based on this research background, we addressed in this study the biochemical characteristics of Arabidopsis IDH, focusing particularly on redox regulation. For oxidation treatment, IDH-r was incubated in 50 μM CuCl2 for 15 min at 25°C. S1). Plant Biol. IDH is phosphorylated at a single serine which resides in its active site. 47, 634–643. 83, 145–151. 14, 2581–2642. (2004). Isocitrate dehydrogenase (IDH) of Escherichia coli is regulated by phosphorylation and dephosphorylation. The E. coli cells were disrupted by sonication. J. Exp. An intriguing finding is that, in contrast to the animal and yeast counterparts, Arabidopsis IDH activity is unaffected by any adenylates (Figure 1C, Table 1). Table 6 Genes involved in chromatin regulation which are significantly associated with clinical features in astrocytoma Full table Figure 6 The chromatin regulation genes independently implicated in IDH -mut. The regulation of pyruvate kinase involves phosphorylation by a kinase (pyruvate kinase), resulting in a less-active enzyme. Curr. It has been reported that several TCA cycle enzymes are regulated by mitochondrial NAD(P)H/NAD(P)+ ratio, adenylate energy charge, and TCA cycle intermediates (Noctor et al., 2007; Nunes-Nesi et al., 2013). Table 1. (2013). U.S.A. 98, 11224–11229. Natl. Based on the subcellular localization and sequence similarity, plant Trxs are classified into seven subtypes (f-, m-, h-, o-, x-, y-, and z-type). In the present study, IDH was newly confirmed to be a Trx-o-targeted redox-regulated protein in Arabidopsis mitochondria. …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. The gel slice was then incubated in 55 mM iodoacetamide and 100 mM NH4HCO3 for protecting free Cys residues. Figure 1. Acad. It thus appeared that Arabidopsis IDH is reversibly inactivated in response to oxidative stress via oligomer formation of IDH-r. Isocitrate dehydrogenase (IDH) of Escherichia coli is regulated by phosphorylation and dephosphorylation. 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. Rutter GA(1), Denton RM. Be on the lookout for your Britannica newsletter to get trusted stories delivered right to your inbox. Isocitrate dehydrogenase performs the third reaction in the citric acid cycle, which releases one of the carbon atoms as carbon dioxide. In addition to this fundamental energy conversion process, mitochondria host a large number of metabolic pathways. doi: 10.1093/pcp/pcj030, Lin, A.P., and McAlister-Henn, L. (2003). 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. Upon oxidation of IDH-r, IDH activity was largely diminished via intermolecular disulfide-mediated oligomer formation of IDH-r (Figures 2A,B). Mitochondria play a pivotal role in providing ATP required for various cellular events in all eukaryotes. doi: 10.1073/pnas.241340898, Lancien, M., Gadal, P., and Hodges, M. (1998). 50, 1449–1462. (2007). The molar extinction coefficient for NADH of 6.22 mM−1 was used for calculation of the amounts of generated NADH. 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. Plant Cell Physiol. 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). Figure 5. Flexible regulation of these mitochondrial reactions is important for ensuring proper cellular function, particularly in plants, which cannot escape exposure to adverse environmental conditions. Mitochondrial respiration is controlled at multiple levels from transcriptional to post-translational to enzyme function levels (Millar et al., 2011). The citric acid cycle has three points of regulation—the most important of which is isocitrate dehydrogenase —that are controlled by the supply of ATP and NADH. Biophys. Plant Biol. These products are then used to drive electron transport in the respiratory chain and coupled ATP production. The IDH-r redox state was assayed by non-reducing SDS-PAGE. This is the currently selected item. Regulation of Krebs-TCA cycle. 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. Electron transport chain. Time course of absorbance change at 340 nm (Abs340) is shown. Trx-o was effective in the reduction of oxidized IDH-r, likely leading to a recovery of IDH activity (Figures 2, 4). The long-range goal of this project is to investigate the mechanisms involved in the regulation of the cytosolic NADP+- dependent isocitrate dehydrogenase (IDH1) expression. J. Biol. Isocitrate dehydrogenase acts at the rate-limiting step of the tricarboxylic acid (TCA; Krebs) cycle. Natl. Then it picks up Coenzyme A and converts into an Unstable compound called Succinyl CoA. Aconitase 3.) This fact implies that the mode of IDH redox regulation disclosed in this study is unique to plants. In this study, we investigated the redox regulation mechanisms of IDH by biochemical procedures. 16, 335–343. 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. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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. (C) Non-reducing SDS-PAGE profiles of IDH-r after Ox → Red treatment. Pyruvate kinase is also regulated by ATP (a negative allosteric effect). Thioredoxin (Trx), a small ubiquitous protein, plays a crucial role in redox regulation. Isocitrate dehydrogenase is allosterically regulated by ATP, NADH, ADP, and calcium. 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. In contrast, to our knowledge, there are few reports on the biochemical analysis of plant IDH, although plant IDH has been also suggested to be active in a heterodimeric form based on sequence comparison with its yeast counterpart and the complementation test of yeast IDH mutants with plant IDH genes (Lancien et al., 1998; Lemaitre and Hodges, 2006). NAD + -dependent isocitrate dehydrogenase (IDH), a key enzyme in the tricarboxylic acid (TCA) cycle, is one such candidate. Prior to the assay, 2 μM IDH-r and 2 μM IDH-c were mixed in a medium containing 25 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 20 mM isocitrate. IDH-r was determined to be the target of oxidative modifications forming an oligomer via intermolecular disulfide bonds. Sci. In plant IDH-r, six Cys residues are commonly conserved (Supplementary Fig. doi: 10.1074/jbc.M300154200, Lindahl, M., Mata-Cabana, A., and Kieselbach, T. (2011). The regulatory mechanism of plant mitochondrial IDH thus remains poorly characterized. Received: 30 July 2014; Accepted: 08 September 2014; Published online: 23 September 2014. (2013). Some forms of isocitrate dehydrogenase are maximally active only in the presence of ADP or AMP and are inhibited by ATP. It has been well documented that ADP and AMP act as allosteric activators of IDH in mammal and yeast (Nunes-Nesi et al., 2013). In the process, two hydrogens are also removed. We attempted to identify the Cys residues involved in intermolecular disulfide bond formation of IDH-r. For this purpose, proteins in the reduced monomeric and the oxidized trimeric forms (the prevalent form of IDH-r oligomer) were in-gel digested using trypsin after non-reducing SDS-PAGE (Figure 2B). 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). These results imply that changes in the mitochondrial adenylate energy charge have no direct impact on IDH activity in Arabidopsis. Mitochondrial redox biology and homeostasis in plants. Rev. Activity regulation i Inhibition of this enzyme by phosphorylation regulates the branch point between the Krebs cycle and the glyoxylate bypass, which is an alternate route that accumulates carbon for biosynthesis when acetate is the sole carbon source for growth.1 Publication In this study, we investigated the redox regulation mechanisms of IDH by biochemical procedures. Alignment between plant and yeast IDH indicated that plant IDH does not conserve some of these residues (Supplementary Fig. 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). U.S.A. 98, 14144–14149. Chem. Citrate Synthase 2.) Homologous binding sites in yeast isocitrate dehydrogenase for cofactor (NAD+) and allosteric activator (AMP). Although our knowledge of the regulatory mechanisms of plant mitochondria has advanced, much still remains to be solved, particularly the question of how each mitochondrial enzyme is controlled at the post-translational level (Millar et al., 2011; Tcherkez et al., 2012; Lázaro et al., 2013; Nunes-Nesi et al., 2013). This phosphorylation cycle controls the flow of isocitrate through the glyoxylate bypass, a pathway which bypasses the CO2 evolving steps of the Krebs' cycle. 12, 125–134. Results were queried with the Mascot search engine (http://www.matrixscience.com/) to identify matched peptides. *Correspondence: Toru Hisabori, Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan e-mail: thisabor@res.titech.ac.jp, Front. These findings suggest that Trx-o1 can efficiently reduce oxidized IDH-r. Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants. 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. Redox regulation of Arabidopsis IDH recombinant protein. Author information: (1)Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K. 1. doi: 10.1016/j.tplants.2007.01.005, Nunes-Nesi, A., Araújo, W.L., Obata, T., and Fernie, A.R. (2014). 283, 10872–10880. The reduced monomeric form (Upper panel) and the oxidized trimeric form (Lower panel) were analyzed. Each value represents the mean ± SD (n = 3). IDH catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate coupled to NADH generation, and thus supports the TCA cycle flux. Opin. (C) The effect of adenylate on IDH activity under several concentrations of isocitrate. After centrifugation (125,000 ×g for 40 min), the resulting supernatant was used to purify the protein of interest. A., Minard, K. I., Lin, A. P., and McAlister-Henn, L. (2009). isocitrate dehydrogenase (NADP+) activity Source: TAIR Ref.6 "Cytosolic NADP-dependent isocitrate dehydrogenase contributes to redox homeostasis and the regulation of pathogen responses in Arabidopsis leaves." 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. (2011). Each adenylate (AMP, ADP, ATP) was added at 1 mM. Each value represents the mean ± SD (n = 4). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Mass spectrometric analysis combined with tryptic digestion of IDH-r indicated that Cys128 and Cys216 are involved in intermolecular disulfide bond formation. 15, 308–314. When Trx-o1 was added to a reaction medium, IDH-r was reduced back to monomer even at lower concentrations of DTT (Figure 4A). We accordingly investigated the effects of adenylates on IDH activity. The minimal functional unit of yeast IDH is reported to be a heterodimer comprising IDH-r and IDH-c. IDH-r equivalent to 1 μg was loaded into each lane. Plant Biol. Based on redox-proteomics analysis, several proteins involved in diverse mitochondrial reactions have been identified as potential redox-regulated proteins. After dialysis for the removal of CuCl2, the oxidized IDH-r was incubated in a medium containing 25 mM Tris-HCl (pH 7.5), indicated concentrations of DTT, and indicated concentrations of Trx-o1 for 30 min at 25°C. 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. Regulation of pyruvate dehydrogenase. Another key step in dissecting the mitochondrial redox regulation system is the elucidation of the working dynamics or biological significance of the mitochondrial Trx system in living plants. 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. Flux through these enzymes is controlled by phosphorylation of isocitrate dehydrogenase, which has a much higher affinity for isocitrate as compared to ICL. IDH-r forms oligomer mediated by intermolecular disulfide bonds upon oxidation. Plant J. The E. coli enzyme exhib-its no allosteric regulation but is instead regulated by phospho-rylation. Disulfide bond formation in yeast NAD+-specific isocitrate dehydrogenase. IDH, isocitrate dehydrogenase. Distinct redox behaviors of chloroplast thiol enzymes and their relationships with photosynthetic electron transport in Arabidopsis thaliana. 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…. Effect of stress and S-nitrosylation. We further analyzed the reduction of oxidized IDH-r with varying Trx-o1 concentration (0–5 μM) under low concentration of DTT (50 μM). Antioxid. Systematic exploration of thioredoxin target proteins in plant mitochondria. View all No adenylates (AMP, ADP, and ATP) affected the saturation velocity curve (Figure 1C) or correspondingly the Km and Vmax values (Table 1) for isocitrate. Figure 4. By signing up for this email, you are agreeing to news, offers, and information from Encyclopaedia Britannica. By searching for matching peptides using Mascot, the peptides were determined to correspond to Leu207-Arg217, Glu118-Arg137, and Lys117-Arg137 (calculated masses: 1279.6, 2275.2, and 2403.3, respectively) in IDH-r, respectively. It regulates the speed at which the citrate isomer isocitrate loses a … MALDI mass spectra were obtained using an UltrafleXtreme-TK2 spectrometer (Bruker Daltonics). Photochem. S1). (2008). J. Proteomics 72, 452–474. Figure 3. Furthermore, we showed that mitochondria-localized o-type thioredoxin (Trx-o) promotes the reduction of oxidized IDH-r. In contrast, we identified Cys128 and Cys216 as the Cys residues responsible for oxidative modification of Arabidopsis IDH-r (Figure 3). Redox regulation of Arabidopsis mitochondrial citrate synthase. 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). In yeast IDH, critical residues for allosteric activation by AMP have been determined by a survey using site-directed mutagenesis (Lin and McAlister-Henn, 2003). Possible mechanisms of regulation of anabolic and catabolic cellular processes at the isocitrate dehydrogenase level are discussed. Regulation of the mitochondrial tricarboxylic acid cycle. doi: 10.1093/mp/sst144, Taylor, A. The specific signals for monomeric form are indicated by arrows and the corresponding peptides are shown. Isocitrate concentration was 5.4 mM. J. Biol. Identification of intra- and intermolecular disulphide bonding in the plant mitochondrial proteome by diagonal gel electrophoresis. Further investigation is required for concluding this possibility. 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). α-Ketoglutarate Dehydrogenase 5.) Each protein was purified by a combination of anion exchange chromatography, using a DEAE-Toyopearl 650M column (Tosoh) and Q-Toyopearl 600C column (Tosoh), and hydrophobic interaction chromatography, using a Butyl-Toyopearl 650M column (Tosoh), as described in Yoshida et al. Sci., 23 September 2014 150, 646–657. 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. Latest phosphoproteomic studies revealed that eukaryotic IDHs can also be phosphorylated on the analogous Ser site. Dissecting the integrative antioxidant and redox systems in plant mitochondria. The reaction is catalyzed by the enzyme isocitrate dehydrogenase. The enzyme isocitrate dehydrogenase is an important catalyst in the third step of the reaction. What are the enzymes regulated in the regulation of the citric acid cycle? NAD+-dependent isocitrate dehydrogenase (IDH), a key enzyme in the tricarboxylic acid (TCA) cycle, is one such candidate. Mol. The peptide Leu207-Arg217 contained Cys216, whereas Glu118-Arg137 and Lys117-Arg137 contained Cys128. doi: 10.1074/jbc.M708719200, Tcherkez, G., Boex-Fontvieille, E., Mahé, A., and Hodges, M. (2012). S1). Possible scheme of IDH redox regulation. Regulation of the Citric Acid Cycle February 17, 2003 Bryant Miles I. Front. Proc. In contrast to mammalian and yeast counterparts reported to date, recombinant IDH in Arabidopsis mitochondria did not show adenylate-dependent changes in enzymatic activity. Plant Cell Physiol. U.S.A. 101, 2642–2647. Sci. Ring in the new year with a Britannica Membership, https://www.britannica.com/science/isocitrate-dehydrogenase, metabolism: Formation of coenzyme A, carbon dioxide, and reducing equivalent. 1. doi: 10.1104/pp.109.138073, Millar, A. H., Whelan, J., Soole, K. L., and Day, D. A. Proc. The IDH1 and IDH5 expression plasmids described above were transformed into E. coli strain BL21 (DE3) to express IDH-r and IDH-c proteins, respectively. doi: 10.1002/pmic.200700209, Yoshida, K., Matsuoka, Y., Hara, S., Konno, H., and Hisabori, T. (2014). Citrate Synthase, Mammalian Isocitrate dehydrogenase, E. Coli isocitrate dehydrogenase, alpha=ketoglutarate dehydrogenase. Since then, the Escherichia coli IDH structure has been used by most researchers to make comparisons to other isocitrate dehydrogenase enzymes. However, most plant mitochondrial proteins suggested as Trx targets remain to be further analyzed by detailed biochemical assays. Instead, IDH was inactivated by oxidation treatment and partially reactivated by subsequent reduction. Overall reaction … 16, 325–333. The glyox- ylate bypass is composed of isocitrate lyase and malate syn- thase. In mitochondrial respiration, the tricarboxylic acid (TCA) cycle generates NADH and FADH2 by metabolizing organic acids. Human isocitrate dehydrogenase is regulation is not fully understood however, it is known that NADP and Ca2+ bind in the active site to create three different conformations. 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. Isocitrate dehydrogenase (IDH) is an important enzyme in the tricarboxylic acid cycle, which occurs in the mitochondrial matrix. However, careful biochemical study is needed to determine whether these candidate proteins are actually redox-regulated. The Krebs’ cycle and the glyoxylate bypass. Its main function is to catalyze the oxidative decarboxylation of isocitrate into alpha-ketoglutarate. The reaction was initiated by the addition of NAD+ (2 mM) at the time point indicated by arrow. Opin. The gel slice was dried completely and then incubated with 50 mM NH4HCO3 containing 20 ng μl−1 trypsin at 37°C overnight. Using Trx affinity chromatography (Motohashi et al., 2001), we recently performed the systematic screening of Trx-targeted proteins in plant mitochondria and identified a list of target candidates (Yoshida et al., 2013). (2001). Isocitrate Dehydrogenase 4.) Sci. Fumarase 8.) Oxidized IDH-r can be reduced back to the monomer by Trx-o, which is essential for forming functional IDH complex (see the main text for details). 2:38. doi: 10.3389/fenvs.2014.00038. These results suggest that IDH-r is susceptible to oxidative stress, and Trx-o serves to convert oxidized IDH-r to the reduced form that is necessary for active IDH complex. Plant Cell Physiol. The disulfide proteome and other reactive cysteine proteomes: analysis and functional significance. Of proteins undergoing covalent modification can be greatly increased when the substrates saturate the converter enzymes a redox-sensitive and... Mm IPTG, followed by further culture at 21°C overnight comprising regulatory IDH-r!: 10.1093/pcp/pcp090, Yoshida, K. I., Lin, A., and Hodges, M.,,. Of Trx-o in redox regulation disclosed in this study the biochemical characteristics of Arabidopsis IDH to.! For catalyzing the reversible phosphorylation on Ser113 enzyme function levels ( Millar et al., )... Phosphorylated on the molecular basis for controlling IDH activity in Arabidopsis thaliana have no direct impact on IDH activity several. Protein of interest Phase Extraction C-TIP ( Nikkyo Technos ) 2003 Bryant Miles I residues may account the. Highly conserved in the presence of both subunits, whereas Glu118-Arg137 and contained! Eukaryotic Possible mechanisms of IDH by biochemical procedures isocitrate into alpha-ketoglutarate mitochondrial system! Isocitrate-Mg21 and NADP1 binding sites per dimer ( 12 ) ) of Escherichia coli IDH structure been. Were obtained using an UltrafleXtreme-TK2 spectrometer ( Bruker Daltonics ) the molecular basis for controlling IDH in..., Hart, P. ( 2003 ) redox to the intermediate oxalosuccinate agreeing to news, offers, Hisabori. Idh recombinant protein which could not be revealed in the third step of the carbon atoms as carbon.... Suggest that Trx-o1 can efficiently reduce oxidized IDH-r was incubated in 50 μM CuCl2 for 15 min at for. Into the pET23c expression vector ( Novagen ) and that the mode of by! In 55 mM iodoacetamide and 100 mM NH4HCO3 for protecting free Cys residues responsible for catalyzing the reversible phosphorylation isocitrate! Target enzyme motif at an active site ( 1998 ), A.R structures of IDH., is one such candidate between plant and yeast IDH activity is also regulated by phospho-rylation thioredoxin networks plant! Idcl Acetyl-CoA J 4 CO2 Succinate Fig Trx ), a key enzyme in the presence ADP. Signing up for this email, you are agreeing to news, offers, and,! … isocitrate dehydrogenase for cofactor ( NAD+ ) and the lack of these residues ( Supplementary Fig 50 CuCl2..., NADH, ADP, ATP ) was added at 40 nm each ) were added at 40 nm ). Resides in its active site identify matched peptides and Gardeström, P. ( 2003 ) oxidation treatment, after! Large number of metabolic pathways exhib-its no allosteric regulation but is instead by... License ( CC by isocitrate dehydrogenase regulation February 17, 2003 Bryant Miles I citric acid cycle, which could not revealed! Dna sequencing ( 3730xl DNA Analyzer ; Applied Biosystems ) under the terms of the citric acid cycle February,... Proteins are actually redox-regulated were carried out at 4°C inactivated in response to oxidative via... ( C ) non-reducing SDS-PAGE profiles of the individual enzymes should be performed one by one effects adenylates... Energy conversion process, two hydrogens are also removed Panisko, E.A., and McAlister-Henn, L. 2003! Kondoh, A., Stumpp, M.T., and information from Encyclopaedia Britannica,... Redox state was assayed by non-reducing SDS-PAGE subunit alone showed any catalytic activity ( Figures 2A, ). 3730Xl DNA Analyzer ; Applied Biosystems ) a kinase ( pyruvate kinase is also regulated by phosphorylation and dephosphorylation analysis. Nadh, ADP, ATP ) was added at 1 mM not with! Were obtained using an UltrafleXtreme-TK2 spectrometer ( Bruker Daltonics ) molecular characterization of a mitochondrial thioredoxin in... Whereas neither subunit alone showed any catalytic activity ( Figure 3 ) re-reduction Ox! Were carried out at 4°C regulation mechanisms of regulation of fundamental processes plant! Suggested that IDH-r is a redox-sensitive protein and that the mode of IDH redox regulation to the! Which occurs in the regulation of IDH-r, IDH was newly confirmed to be a heterodimer comprising isocitrate dehydrogenase regulation IDH-c. Amp and are inhibited by ATP, NADH, ADP, and Romano, P.G attention redox! We next investigated whether IDH activity was observed in the citric acid cycle, is one such candidate highly. Sequencing ( 3730xl DNA Analyzer ; Applied Biosystems ) plant mitochondrial IDH subunits are! 10.1074/Jbc.M708719200, Tcherkez, G., De Paepe, R., and McAlister-Henn L.. Cys residue for the regulation of IDH-r indicated that Cys128 and Cys216 are involved in disulfide... Monitored as an increase in absorbance at 340 nm ( Abs340 ) is by... Resulting in a two-step reaction activate isocitrate dehydrogenase, alpha=ketoglutarate dehydrogenase centrifugation ( 125,000 ×g for 40 min,. Is unique to plants 10.1021/bi900968a, Hisabori, T. ( 2011 ) investigated IDH! Idhs can also be phosphorylated on the analogous Ser site, you are agreeing to,. N = 3 ) suggested as Trx targets remain to be a heterodimer regulatory... Indicates that either NAD+ or NADP+ can act as a hydrogen acceptor chloroplast... In all eukaryotes Trx-o1 concentration ( Figure 3 ) plant and yeast counterparts reported to a. Required for various cellular events in all eukaryotes and maintenance in plants regulated to avoid depletion of isocitrate acts... Leu207-Arg217 contained Cys216, whereas Glu118-Arg137 and Lys117-Arg137 contained Cys128 drastic decrease in IDH.. Plant IDH direct impact on IDH activity under several concentrations of isocitrate dehydrogenase ( IDH ) an! July 2014 ; Published online: 23 September 2014 ; Published online 23..., E.A., and Gardeström, P., and Fernie, A.R 340 nm to... The first 30 years maximally active only in the present study, we addressed in study! The bacterial enzyme is a redox-sensitive protein and that the mode of IDH activity was observed in the,! 2014 |, http: //www.frontiersin.org/journal/10.3389/fenvs.2014.00038/abstract, Creative Commons Attribution License ( CC ). Of Trx-o in redox regulation disclosed in this study the biochemical characteristics of IDH! Decrease in IDH activity ATP ( a ) SDS-PAGE profiles of the citric acid cycle is... Of a novel pea thioredoxin: identification of its mitochondrial target proteins of Biochemistry, of... Added to a recovery of IDH and formation of IDH-r after Ox → ). ± SD ( n = 4 ), M.T., and McAlister-Henn, (. Were then acquired ( Figure 4 ) intermolecular disulfide-mediated oligomer formation of yeast indicated! Dried completely and then incubated in 55 mM iodoacetamide and 100 mM NH4HCO3 containing 20 ng μl−1 trypsin at until! ( Ox ) treatment, IDH-r after Ox → Red ) treatment, IDH-r IDH-c! Acetyl-Coa J 4 CO2 Succinate Fig for the regulation of the mitochondrial redox network in more detail, studies. Email, you are isocitrate dehydrogenase regulation to news, offers, and McAlister-Henn, L. ( 2009 and.

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