Regulation of NAD- and NADP-dependent isocitrate dehydrogenases by reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves. Plant Cell Physiol. However, the molecular basis of IDH redox regulation is different between Arabidopsis and yeast. The biochemical properties of IDH, including action mechanisms and regulatory factors, have been best characterized in yeast (Panisko and McAlister-Henn, 2001; Lin and McAlister-Henn, 2003; Taylor et al., 2008; Garcia et al., 2009). These results imply that changes in the mitochondrial adenylate energy charge have no direct impact on IDH activity in Arabidopsis. Redox regulation of Arabidopsis mitochondrial citrate synthase. Isocitrate dehydrogenase acts at the rate-limiting step of the tricarboxylic acid (TCA; Krebs) cycle. The reaction was initiated by the addition of NAD+ (2 mM) at the time point indicated by arrow. These Cys residues are thus likely to be primarily involved in redox changes in the Arabidopsis IDH-r molecule. Figure 2. The efficiency of IDH-r reduction was highly dependent on Trx-o1 concentration (Figure 4B). 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. doi: 10.1073/pnas.191282098, Noctor, G., De Paepe, R., and Foyer, C.H. 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. doi: 10.1016/j.tplants.2007.01.005, Nunes-Nesi, A., Araújo, W.L., Obata, T., and Fernie, A.R. J. Biol. doi: 10.1562/2006-02-27-IR-816, Igamberdiev, A.U., and Gardeström, P. (2003). 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. 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…. (2011). Environ. 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). 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. Trends Plant Sci. IDH-r was determined to be the target of oxidative modifications forming an oligomer via intermolecular disulfide bonds. In the last decade, the progress of redox-proteomics analysis has provided hints that a wide variety of biological processes are governed by the redox state of their responsible enzymes (Hisabori et al., 2007; Montrichard et al., 2009; Lindahl et al., 2011). 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. The data on the structure and chemical modification of the active center functional groups suggest that isocitrate dehydrogenases isolated from various sources differ in their catalytic properties. Chem. 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… S1). The minimal functional unit of yeast IDH is reported to be a heterodimer comprising IDH-r and IDH-c. Chem. 1. Sci., 23 September 2014 Possible mechanisms of regulation of anabolic and catabolic cellular processes at the isocitrate dehydrogenase level are discussed. 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. For oxidation (Ox) treatment, IDH-r or IDH-c was incubated in 50 μM CuCl2 for 15 min. Distinct redox behaviors of chloroplast thiol enzymes and their relationships with photosynthetic electron transport in Arabidopsis thaliana. Rutter GA(1), Denton RM. U.S.A. 98, 14144–14149. Biochemistry 48, 8869–8878. Mitochondrial and nuclear localization of a novel pea thioredoxin: identification of its mitochondrial target proteins. 1. A., Minard, K. I., Lin, A. P., and McAlister-Henn, L. (2009). By signing up for this email, you are agreeing to news, offers, and information from Encyclopaedia Britannica. Respiratory carbon fluxes in leaves. 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. Acta 1606, 117–125. Biochim. B., Hu, G., Hart, P. J., and McAlister-Henn, L. (2008). (2007). Redox Signal. Disulfide bond formation in yeast NAD+-specific isocitrate dehydrogenase. 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). Instead, IDH was inactivated by oxidation treatment and partially reactivated by subsequent reduction. Mass spectrometry of IDH-r tryptic peptides. 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." Each of the amplified DNA was ligated into the pET23c expression vector (Novagen). U.S.A. 101, 2642–2647. It regulates the speed at which the citrate isomer isocitrate loses a … IDH-r and IDH-c were added at 40 nM each. 12, 125–134. 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. Copyright © 2014 Yoshida and Hisabori. 62, 79–104. J. Biol. Fumarase 8.) Mitochondrial redox biology and homeostasis in plants. Trx-dependent reduction of oxidized IDH-r. (A) Oxidized IDH-r was incubated in several concentrations of DTT in the absence (upper panel) or presence (lower panel) of 5 μM Trx-o1 for 30 min. Changes in Free Energy 1.) (2013). The transformed cells were grown at 37°C until A600 = 0.4–0.8. Sci. 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. 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). Subunit interactions of yeast NAD+-specific isocitrate dehydrogenase. Its main function is to catalyze the oxidative decarboxylation of isocitrate into alpha-ketoglutarate. (2008). (2009). This phosphorylation cycle controls the flow of isocitrate through the glyoxylate bypass, a pathway which bypasses the CO2 evolving steps of the Krebs' cycle. The reduced monomeric form (Upper panel) and the oxidized trimeric form (Lower panel) were analyzed. 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. Escherichia coli isocitrate dehydrogenase (IDH) is regulated by reversible phosphorylation on Ser113. 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). Dephosphorylation by a phosphatase reactivates it. The Krebs’ cycle and the glyoxylate bypass. In order to understand the mitochondrial redox network in more detail, biochemical studies of the individual enzymes should be performed one by one. ATP and NADH inhibit this enzyme, while ADP and calcium activate it. In contrast to mammalian and yeast counterparts reported to date, recombinant IDH in Arabidopsis mitochondria did not show adenylate-dependent changes in enzymatic activity. Isocitrate dehydrogenases are enzymes which catalyse the oxidative decarboxylation of isocitrate to 2-oxoglutarate (α-ketoglutarate). NAD+-dependent isocitrate dehydrogenase (IDH) is one of the proteins that were captured by Trx affinity chromatography (Yoshida et al., 2013). 55, 1415–1425. This phenomenon, termed zero-order ultrasensitivity, has been found to occur in the reversible phosphorylation of isocitrate dehydrogenase. 83, 145–151. In addition to this fundamental energy conversion process, mitochondria host a large number of metabolic pathways. Malate Dehydrogenase 9.) doi: 10.1074/jbc.M708719200, Tcherkez, G., Boex-Fontvieille, E., Mahé, A., and Hodges, M. (2012). No use, distribution or reproduction is permitted which does not comply with these terms. Isocitrate dehydrogenase (IDH) is an important enzyme in the tricarboxylic acid cycle, which occurs in the mitochondrial matrix. 15, 308–314. Recurrent mutations in IDH1 or IDH2 are prevalent in several cancers including glioma, acute myeloid leukemia (AML), cholangiocarcinoma, and chondrosarcoma. Annu. Results were queried with the Mascot search engine (http://www.matrixscience.com/) to identify matched peptides. 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. Mass spectrometric analysis combined with tryptic digestion of IDH-r indicated that Cys128 and Cys216 are involved in intermolecular disulfide bond formation. 47, 634–643. (C) The effect of adenylate on IDH activity under several concentrations of isocitrate. 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). Latest phosphoproteomic studies revealed that eukaryotic IDHs can also be phosphorylated on the analogous Ser site. After separation by non-reducing SDS-PAGE, stained protein bands of interest were excised from the gel and fully destained with 50 mM NH4HCO3 and 50% (v/v) acetonitrile. Hexameric oligomerization of mitochondrial peroxiredoxin PrxIIF and formation of an ultrahigh affinity complex with its electron donor thioredoxin Trx-o. 278, 12864–12872. The Isocitrate Dehydrogenase (IDH) enzyme structure in Escherichia coli was the first structure to be elucidated and understood. Isocitrate dehydrogenase (IDH) of Escherichia coli is regulated by phosphorylation and dephosphorylation. Some forms of isocitrate dehydrogenase are maximally active only in the presence of ADP or AMP and are inhibited by ATP. 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. These conformations form in the active site and are as follows: a loop is form in the inactive enzyme, a partially unraveled alpha helix in the semi open form, an… This result implies that IDH-r also undergoes irreversible oxidative modifications, which could not be revealed in the present study. doi: 10.1073/pnas.241340898, Lancien, M., Gadal, P., and Hodges, M. (1998). |, http://www.frontiersin.org/journal/10.3389/fenvs.2014.00038/abstract, Creative Commons Attribution License (CC BY). Mitochondria play a pivotal role in providing ATP required for various cellular events in all eukaryotes. 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. These products are then used to drive electron transport in the respiratory chain and coupled ATP production. 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). What regulates citrate synthase? Dissecting the integrative antioxidant and redox systems in plant mitochondria. 4:460. doi: 10.3389/fpls.2013.00460, Lemaitre, T., and Hodges, M. (2006). This finding clearly shows that both IDH-r and IDH-c are essential for functional IDH in Arabidopsis. Sci. This finding implies the significance of the mitochondrial Trx system for the regulation of TCA cycle performance. Articles. IDH-r forms oligomer mediated by intermolecular disulfide bonds upon oxidation. doi: 10.1016/j.jprot.2008.12.002, Motohashi, K., Kondoh, A., Stumpp, M.T., and Hisabori, T. (2001). 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