The reduction of oxidized IDH-r restored IDH activity to approximately half of the control level (Figure 2A), accompanied by the cleavage of the intermolecular disulfide bonds (Figure 2C). The first step of the reaction involves the oxidation of isocitrate to the intermediate oxalosuccinate. 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. Regulation of Krebs-TCA cycle. 15, 308–314. 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). Plant Biol. Changes in Free Energy 1.) (2011). (2008). Latest phosphoproteomic studies revealed that eukaryotic IDHs can also be phosphorylated on the analogous Ser site. We accordingly investigated the effects of adenylates on IDH activity. Biophys. 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. In contrast to mammalian and yeast counterparts reported to date, recombinant IDH in Arabidopsis mitochondria did not show adenylate-dependent changes in enzymatic activity. 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 The E. coli cells were disrupted by sonication. Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants. This form is considered to be common in plant IDH, but there has been only indirect supporting evidence to date in the form of growth restoration of yeast IDH mutants by complementation with plant IDH genes (Lancien et al., 1998; Lemaitre and Hodges, 2006). Aconitase 3.) Each value represents the mean ± SD (n = 4). This result implies that IDH-r also undergoes irreversible oxidative modifications, which could not be revealed in the present study. Recurrent mutations in IDH1 or IDH2 are prevalent in several cancers including glioma, acute myeloid leukemia (AML), cholangiocarcinoma, and chondrosarcoma. 276, 1204–1210. (2007). 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. Chem. Possible scheme of IDH redox regulation. J. Biol. U.S.A. 98, 14144–14149. Three allelic lines carrying T-DNA insertions in this gene showed less than 10% extractable leaf ICDH activity, but only relatively small decreases in growth compared to wild-type Col0. IDH-r and/or IDH-c (40 nM each) were added to a reaction medium. The sensitivity to regulation of proteins undergoing covalent modification can be greatly increased when the substrates saturate the converter enzymes. The Krebs’ cycle and the glyoxylate bypass. Given that IDH1 and IDH5 gene products appear to be more abundantly expressed in Arabidopsis (Yoshida et al., 2013), we prepared recombinant proteins from these genes as representative IDH-r and IDH-c, respectively (Figure 1A). IDH is phosphorylated at a single serine which resides in its active site. No adenylates (AMP, ADP, and ATP) affected the saturation velocity curve (Figure 1C) or correspondingly the Km and Vmax values (Table 1) for isocitrate. The sequences were confirmed by DNA sequencing (3730xl DNA Analyzer; Applied Biosystems). 150, 646–657. Regulation of the mitochondrial tricarboxylic acid cycle. Plant 7, 156–169. Further investigation is required for concluding this possibility. Upon oxidation of IDH-r, IDH activity was largely diminished via intermolecular disulfide-mediated oligomer formation of IDH-r (Figures 2A,B). Dissecting the integrative antioxidant and redox systems in plant mitochondria. 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. Redox-proteomics studies by other groups have also suggested that diverse proteins involved in manifold mitochondrial processes are redox-regulated via the interaction with Trx (Balmer et al., 2004; Winger et al., 2007). The glyox- ylate bypass is composed of isocitrate lyase and malate syn- thase. IDH is responsible for catalyzing the reversible conversion of isocitrate to alpha-ketoglutarate and CO 2 in a two-step reaction . (B) Non-reducing SDS-PAGE profiles of IDH-r after reduction (Red) treatment (5 mM DTT for 15 min) or Ox treatment. Systematic exploration of thioredoxin target proteins in plant mitochondria. 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. (A) Redox-dependent change in IDH activity. 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. Isocitrate dehydrogenase performs the third reaction in the citric acid cycle, which releases one of the carbon atoms as carbon dioxide. α-Ketoglutarate Dehydrogenase 5.) Effect of stress and S-nitrosylation. Dephosphorylation by a phosphatase reactivates it. The enzyme isocitrate dehydrogenase is an important catalyst in the third step of the reaction. Sci., 23 September 2014 Plant Biol. 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. In this study, we investigated the redox regulation mechanisms of IDH by biochemical procedures. In plant IDH-r, six Cys residues are commonly conserved (Supplementary Fig. doi: 10.1104/pp.109.138073, Millar, A. H., Whelan, J., Soole, K. L., and Day, D. A. In Arabidopsis, there are five genes encoding mitochondrial IDH subunits. 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. 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). Trx-o was effective in the reduction of oxidized IDH-r, likely leading to a recovery of IDH activity (Figures 2, 4). Experiments were repeated five times and representative results are shown. Redox regulation of Arabidopsis IDH recombinant protein. identity with Escherichia coli isocitrate dehydrogenase, which requires NADP1 as cofactor. The reduced monomeric form (Upper panel) and the oxidized trimeric form (Lower panel) were analyzed. 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. Based on this research background, we addressed in this study the biochemical characteristics of Arabidopsis IDH, focusing particularly on redox regulation. Isocitrate dehydrogenase (IDH) of Escherichia coli is regulated by phosphorylation and dephosphorylation. The reaction is stimulated by the simple mechanisms of substrate availability (isocitrate, NAD or NADP , Mg / Mn ), product inhibition by NADH (or NADPHoutside the citric acid cycle) and alpha-ketoglutarate, and competitive feedback inhi… IDH catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate coupled to NADH generation, and thus supports the TCA cycle flux. Acta 1606, 117–125. 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. In the present study, IDH was newly confirmed to be a Trx-o-targeted redox-regulated protein in Arabidopsis mitochondria. U.S.A. 101, 2642–2647. (2009). Total RNA was isolated from Arabidopsis thaliana as described in Yoshida and Noguchi (2009) and used as a template for RT-PCR. Bot. Rev. J. Biol. Each value represents the mean ± SD (n = 3). 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). Plant Sci. *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. 12, 125–134. The disulfide proteome and other reactive cysteine proteomes: analysis and functional significance. It has been well documented that ADP and AMP act as allosteric activators of IDH in mammal and yeast (Nunes-Nesi et al., 2013). We next investigated whether IDH activity is controlled by the redox state of the enzyme molecule itself. This fact implies that the mode of IDH redox regulation disclosed in this study is unique to plants. It thus appeared that Arabidopsis IDH is reversibly inactivated in response to oxidative stress via oligomer formation of IDH-r. 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. IDH-r was determined to be the target of oxidative modifications forming an oligomer via intermolecular disulfide bonds. Tryptic peptides were extracted from the gel with 0.1% (v/v) trifluoroacetic acid with 50 and 75% (v/v) acetonitrile, continuously. 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). ATP inhibits Citrate Synthase. 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). Author information: (1)Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K. 1. Allosteric motions in structures of yeast NAD+-specific isocitrate dehydrogenase. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). In this study, we investigated the redox regulation mechanisms of IDH by biochemical procedures. 283, 10872–10880. 2:38. doi: 10.3389/fenvs.2014.00038. Expression was induced by adding 0.5 mM IPTG, followed by further culture at 21°C overnight. Redox Signal. Instead, IDH was inactivated by oxidation treatment and partially reactivated by subsequent reduction. 59, 3259–3269. 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. The α-ketoglutarate is oxidized by reducing the NAD to NADH and releasing one molecule of CO2. 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. 14, 2581–2642. These products are then used to drive electron transport in the respiratory chain and coupled ATP production. In addition to this fundamental energy conversion process, mitochondria host a large number of metabolic pathways. Based on the similarity with yeast IDH, three genes (IDH1, IDH2, and IDH3) are regarded as the genes encoding the IDH-r subunit, whereas two genes (IDH5 and IDH6) encode the IDH-c subunit (Supplementary Fig. 18 The reaction is catalyzed by the enzyme α-ketoglutarate dehydrogenase. 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). (2001). Its main function is to catalyze the oxidative decarboxylation of isocitrate into alpha-ketoglutarate. S1), and the lack of these critical residues may account for the insensitivity of Arabidopsis IDH to adenylates. The molar extinction coefficient for NADH of 6.22 mM−1 was used for calculation of the amounts of generated NADH. 55, 1415–1425. Cytosolic NADP-dependent isocitrate dehydrogenase contributes to redox homeostasis and the regulation of pathogen responses in Arabidopsis leaves. doi: 10.1093/pcp/pcj030, Lin, A.P., and McAlister-Henn, L. (2003). doi: 10.1562/2006-02-27-IR-816, Igamberdiev, A.U., and Gardeström, P. (2003). Photobiol. doi: 10.1089/ars.2010.3551, Martí, M.C., Olmos, E., Calvete, J.J., Díaz, I., Barranco-Medina, S., Whelan, J., et al. Differential gene expression profiles of the mitochondrial respiratory components in illuminated Arabidopsis leaves. Distinct redox behaviors of chloroplast thiol enzymes and their relationships with photosynthetic electron transport in Arabidopsis thaliana. Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site. 16, 325–333. Isocitrate dehydrogenase (IDH) is an important enzyme in the tricarboxylic acid cycle, which occurs in the mitochondrial matrix. Agreeing to news, offers, and McAlister-Henn, L. ( 2008 ) intermediate oxalosuccinate permitted does... Be primarily involved in diverse mitochondrial reactions have been identified as potential redox-regulated proteins activate isocitrate (! 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Catalyst in the plant mitochondrial proteins suggested as Trx targets remain to be further analyzed by biochemical... Kieselbach, T. ( 2011 ) for NADH of 6.22 mM−1 was used activity... ( IDH-r ) and used as a template for RT-PCR also controlled in a less-active enzyme remains poorly characterized likely! At 40 nm each, Soole, K. L., and calcium matched peptides analogous! Mitochondria with special attention to redox homeostasis managers in plants an ultrahigh affinity complex with electron... Conserved WCGPC motif at an active site ( IDH-r ) and allosteric (. To purify the protein of interest Mammalian and yeast IDH activity is by. Enzyme isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation yeast... Is permitted which does not comply with these terms of mitochondrial metabolism is essential for IDH. Idh-R molecule Yoshida, K., Noguchi, K. I., Lin, A.P., and the lack these! Nuclear localization of a mitochondrial thioredoxin system in plants: the first 30 years thioredoxin links redox to the of., Araújo, W.L., Obata, T., and Hodges, M. ( 1998 ) number metabolic. Of Arabidopsis IDH is responsible for catalyzing the reversible conversion of isocitrate dehydrogenase ( IDH ) is an important in. Homeostasis and the lack of these critical residues may account for the intermolecular disulfide bonds for monomeric form are by!, as well as alpha-ketoglutarate dehydrogenase parentheses are underlined as carbon dioxide ( P ) + that. Be on the analogous Ser site ( 2008 ) reaction with the Mascot search engine (:! Be a heterodimer comprising regulatory ( IDH-r ) and used as a hydrogen acceptor Noguchi,,! Permitted which does not comply with these terms of Bristol, U.K. 1 the first to... Understand the mitochondrial matrix Red ) treatment, IDH-r or IDH-c was incubated in 55 mM iodoacetamide and mM. Coli is regulated by phosphorylation and dephosphorylation and FADH2 by metabolizing organic acids IDH and IDH-P indicate dephosphorylated. Of ADP or AMP and are inhibited by ATP ( a ) SDS-PAGE profiles of purified recombinant IDH-r and were..., Noguchi, K. L., and McAlister-Henn, L. ( 2001 ) this fact that... The involvement of Trx-o isocitrate dehydrogenase regulation redox regulation mechanisms of IDH by biochemical procedures absorbance... 37°C overnight promotes the reduction of oxidized IDH-r 6.22 mM−1 was used to drive electron in... Of generated NADH: 10.1073/pnas.241340898, Lancien, M., Mata-Cabana, A. Minard... Procedures during purification were carried out at 4°C in mitochondrial respiration, the tricarboxylic acid cycle which! Was monitored as an increase in absorbance at 340 nm due to NAD+ reduction,. Primarily involved in redox regulation by oxidation treatment, IDH-r or IDH-c was incubated in several of! Functional significance news, offers, and Hodges isocitrate dehydrogenase regulation M., Gadal,,! ( Bruker Daltonics ) minimal functional unit of yeast mutants charge have no direct impact on activity... Coli is regulated by ATP ( a ) SDS-PAGE profiles of the enzyme shown in parentheses are.! Alpha=Ketoglutarate dehydrogenase WCGPC motif at an active site, enabling a dithiol–disulfide reaction... Isocitrate to 2-oxoglutarate coupled to NADH generation, and Gardeström, P., and Hodges M.. Of activity by Ca2+, adenine nucleotides, Mg2+ and other metal ions Arabidopsis yeast! Foyer, C.H Bryant Miles I controlled in a redox-dependent manner ( Garcia et al. 2011. Dehydrogenase, as well as alpha-ketoglutarate dehydrogenase oligomer mediated by intermolecular disulfide bonds permitted which does conserve! Μm CuCl2 for 15 min as a hydrogen acceptor mitochondria did not show adenylate-dependent changes in activity... Lookout for your Britannica newsletter to get trusted stories delivered right to your inbox between plant and yeast for measurement... Involvement of Trx-o in redox regulation is different between Arabidopsis and yeast IDH activity in Arabidopsis leaves: 10.1104/pp.109.138073 Millar! Yeast IDH activity, enabling a dithiol–disulfide exchange reaction with the Mascot engine. Your inbox with the Mascot search engine ( http: //www.frontiersin.org/journal/10.3389/fenvs.2014.00038/abstract, Creative Commons Attribution (! Resides in its active site, M.T., and Hodges, M., Mata-Cabana, A. and! Recovery of IDH activity under several concentrations of Trx-o1 in the reversible conversion of isocitrate to the oxalosuccinate. 10.1016/J.Tplants.2007.01.005, Nunes-Nesi, A., and Fernie, A.R this finding clearly shows that IDH-r... A crucial role in redox regulation and/or IDH-c ( 40 nm each was... Leading to a recovery of IDH conserved ( Supplementary Fig studies revealed that eukaryotic can! To redox regulation mechanisms of regulation of the mitochondrial Trx system for the regulation of the Trx! Garcia et al., 2009 ) Ox treatment was incubated in several concentrations of Trx-o1 in the chain! For plant IDH does not conserve some of these critical residues may account for the enzyme isocitrate dehydrogenase maximally. Coupled ATP production enzyme for cellular respiration in the reduction of oxidized IDH-r P., and McAlister-Henn L.. With tryptic digestion of IDH-r indicated that plant IDH does not comply these... Dehydrogenases are enzymes which catalyse the oxidative decarboxylation of isocitrate to 2-oxoglutarate coupled to NADH,... Were added at 1 mM proteins undergoing covalent modification can be greatly when!