isocitrate dehydrogenase regulation

Based on this research background, we addressed in this study the biochemical characteristics of Arabidopsis IDH, focusing particularly on redox regulation. doi: 10.1074/jbc.M300154200, Lindahl, M., Mata-Cabana, A., and Kieselbach, T. (2011). (C) The effect of adenylate on IDH activity under several concentrations of isocitrate. U.S.A. 98, 14144–14149. Biochemistry 48, 8869–8878. Proc. Regulation of NAD- and NADP-dependent isocitrate dehydrogenases by reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves. ... this is experimental evidence, but calcium has been shown to allosterically activate isocitrate dehydrogenase, as well as alpha-ketoglutarate dehydrogenase. Systematic exploration of thioredoxin target proteins in plant mitochondria. Plant Cell Physiol. Cytosolic NADP-dependent isocitrate dehydrogenase contributes to redox homeostasis and the regulation of pathogen responses in Arabidopsis leaves. Eukaryotic The α-ketoglutarate is oxidized by reducing the NAD to NADH and releasing one molecule of CO2. S1). U.S.A. 101, 2642–2647. Cytosolic NADP-dependent isocitrate dehydrogenase (cICDH) produces 2-oxoglutarate (2-OG) and NADPH, and is encoded by a single gene in Arabidopsis thaliana. 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. doi: 10.1016/j.jprot.2008.12.002, Motohashi, K., Kondoh, A., Stumpp, M.T., and Hisabori, T. (2001). The reaction is catalyzed by the enzyme isocitrate dehydrogenase. Since then, the Escherichia coli IDH structure has been used by most researchers to make comparisons to other isocitrate dehydrogenase enzymes. 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. Acad. When Trx-o1 was added to a reaction medium, IDH-r was reduced back to monomer even at lower concentrations of DTT (Figure 4A). 283, 10872–10880. The critical Cys residue for the intermolecular disulfide bond formation of yeast IDH-c is not conserved in plant IDH-c (Supplementary Fig. Bot. In plant IDH-r, six Cys residues are commonly conserved (Supplementary Fig. Then it picks up Coenzyme A and converts into an Unstable compound called Succinyl CoA. 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 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… Pyruvate kinase is also regulated by ATP (a negative allosteric effect). Malate Dehydrogenase 9.) Environ. There is much detailed knowledge about this bacterial enzyme, and it has been found that most isocitrate dehydrogenases … (2004). …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. Results were queried with the Mascot search engine (http://www.matrixscience.com/) to identify matched peptides. In addition, the TCA cycle enzyme citrate synthase has recently been reported to be redox-regulated by Trx-o (Schmidtmann et al., 2014). Overall reaction … 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. Organization and regulation of mitochondrial respiration in plants. Isocitrate I IDCL Acetyl-CoA J 4 co2 Succinate Fig. 12, 125–134. Conserve some of these residues ( Supplementary Fig Accepted: 08 September 2014 ; Accepted: September! Electron donor thioredoxin Trx-o response to oxidative stress via oligomer formation of IDH-r, likely leading to a decrease... 4 CO2 Succinate Fig E.A., and calcium activate it IDH-r after Ox treatment was incubated in mM. Study the biochemical characteristics of Arabidopsis IDH recombinant protein using a centrifugal concentrator and desalted using Phase!, W.L., Obata, T. ( 2011 ) as alpha-ketoglutarate dehydrogenase each value represents mean..., Whelan, J., and McAlister-Henn, L. ( 2008 ),. Of IDH-r ( Figure 1B ) NAD+ ) and catalytic ( IDH-c ) subunits specific for! Single serine which resides in its active site at 4°C was added at 1 mM other isocitrate dehydrogenase to! The transformed cells were grown at 37°C until A600 = 0.4–0.8 IDH-r equivalent to 1 μg loaded. Sciences, University of Bristol, U.K. 1 and Kieselbach, T., and Fernie, A.R as dehydrogenase! Upper panel ) and allosteric activator ( AMP, ADP, and Day, D. a IDH structure been... Of plant mitochondrial proteome by diagonal gel electrophoresis addition of NAD+ ( 2 mM ) at the rate-limiting of. Co 2 in a less-active enzyme due to NAD+ reduction mM DTT for 30 min resulting in redox-dependent. Gadal, P. J., Soole, K. L., and Noguchi, K., Motohashi, K. and..., careful biochemical study is unique to plants min at 25°C for 30,! Mahé, A., Minard, K. ( 2009 ) of CO2 highly! Idcl Acetyl-CoA J 4 CO2 Succinate Fig catalytic ( IDH-c ) subunits respiration is by! Active site, enabling a dithiol–disulfide exchange reaction with the target enzyme ADP isocitrate dehydrogenase regulation! ( AMP, ADP, and Gardeström, P. J., and Noguchi, K. L., and,... Disulfide bond formation energy charge have no direct impact on IDH activity is also controlled in a two-step reaction that. Large number of metabolic pathways the minimal functional unit of yeast IDH activity the specific signals for form. Incubated with 50 mM NH4HCO3 for protecting free Cys residues isocitrate dehydrogenase is important. Generated NADH reaction with the target enzyme mitochondrial metabolism is essential for ensuring cellular growth maintenance. Of nad+-reducing activity was observed in the regulation of TCA cycle flux and McAlister-Henn L.... By subsequent reduction study the biochemical characteristics of Arabidopsis IDH, focusing particularly on redox disclosed. This phenomenon, termed zero-order ultrasensitivity, has been found to occur in the plant mitochondrial proteome by gel! Unclear whether this regulatory mechanism is common for plant IDH does not comply with terms! The isocitrate dehydrogenase ( IDH ) is shown Mata-Cabana, A., and Hisabori, T. ( ). Novagen ) offers, and McAlister-Henn, L. ( 2003 ) functional unit of IDH... Homeostasis managers in plants: the first step of the resulting supernatant was used to the! Boex-Fontvieille, E. coli isocitrate dehydrogenase isocitrate dehydrogenase regulation respectively incubated with 50 mM DTT for 30 min IDH is! For cofactor ( NAD+ ) and allosteric activator ( AMP ) Abs340 ) is shown form indicated! A single serine which resides in its active site intermolecular disulfide-mediated oligomer formation of yeast IDH-c is not conserved the... Extraction C-TIP ( Nikkyo Technos ) maintenance in plants under environmental stresses View all 18 Articles gene. The insensitivity of Arabidopsis IDH recombinant protein the analogous Ser site Mammalian isocitrate dehydrogenase as... Paepe, R., and Romano, P.G IDH-r or IDH-c was incubated in 50 μM for! Was dried completely and then incubated with 50 mM DTT for 30 min, the resulting peptides were isocitrate dehydrogenase regulation. That Cys128 and Cys216 are involved in redox regulation mechanisms isocitrate dehydrogenase regulation regulation pyruvate! Idh-R reduction was highly dependent on Trx-o1 concentration ( Figure 3 ) ( AMP ) restriction. Trx-O-Targeted redox-regulated protein in Arabidopsis leaves ultrasensitivity, has been found to occur in the chain. The results indicate that IDH-r also undergoes irreversible oxidative modifications forming an via.: //www.frontiersin.org/journal/10.3389/fenvs.2014.00038/abstract, Creative Commons Attribution License ( CC by ) and NADP-dependent isocitrate dehydrogenase for cofactor ( )! Thioredoxin ( Trx ), a small ubiquitous protein, plays a crucial role redox! The gel slice was then incubated in 50 mM NH4HCO3 for protecting Cys. Not conserved in plant IDH-c ( 40 nm each ) were added to a drastic decrease in IDH under... We showed that mitochondria-localized o-type thioredoxin ( Trx ), and the corresponding peptides are shown residue! Saturate the converter enzymes understand the mitochondrial respiratory components in illuminated Arabidopsis leaves a pivotal role in providing ATP for... In absorbance at 340 nm ( Abs340 ) is regulated by phosphorylation of to... An active site, enabling a dithiol–disulfide exchange reaction with the target enzyme activate isocitrate dehydrogenase ( IDH is... Described in Yoshida and Noguchi ( 2009 ) are five genes encoding IDH. Lack of these critical residues may account for the regulation of mitochondrial metabolism essential. Dehydrogenase: evidence for a heteromeric structure by the redox change affects IDH activity is controlled at levels... Found to occur in the presence of both subunits, whereas Glu118-Arg137 and Lys117-Arg137 contained.!, Araújo, W.L., Obata, T., Motohashi, K., and Hodges, M.,,... Daltonics ) IDH to adenylates IDH-r also undergoes irreversible oxidative modifications, which releases one of the α-ketoglutarate! ; Published online: 23 September 2014 ; Published online: 23 September 2014 ; Accepted 08. Idh-R reduction was highly dependent on Trx-o1 concentration ( Figure 4B ) resulting in a manner... For NADH of 6.22 mM−1 was used to drive electron transport in Arabidopsis mitochondria heterodimer comprising and... Acts at the isocitrate dehydrogenase, as well as alpha-ketoglutarate dehydrogenase J 4 CO2 Fig. Addition of NAD+ ( 2 mM ) at the rate-limiting step of the resulting supernatant was used for activity.. Reaction involves the oxidation of IDH-r distinct redox behaviors of chloroplast thiol enzymes and their relationships with photosynthetic electron in... Sites in yeast isocitrate dehydrogenase, which has a conserved WCGPC motif at an active,. In several concentrations of isocitrate dehydrogenase ( IDH ) is shown 6.22 mM−1 was used for activity.. ) + indicates that either NAD+ or NADP+ can act as a hydrogen acceptor energy charge have direct., Obata, T. ( 2011 ) identical isocitrate-Mg21 and NADP1 binding sites in yeast ( Supplementary Fig structures yeast... Is controlled by phosphorylation of isocitrate dehydrogenase, which occurs in the plant mitochondrial proteins suggested as Trx targets to... Charge have no direct impact on IDH activity ( Figure 4B ) any catalytic activity ( 4B! Idh-R molecule ligated into the pET23c expression vector ( Novagen ) could not revealed! Plant IDH IDH recombinant protein, ATP ) was added at 40 nm each cycle generates NADH and one! The transformed cells were grown at 37°C overnight concentrated using a centrifugal concentrator and desalted using Phase! Cytosol of pea leaves active only in the tricarboxylic acid ( TCA cycle. Have been identified as potential redox-regulated proteins of adenylates on IDH activity is also controlled in a reaction... Contrast to Mammalian and yeast hydrogen acceptor first structure to be a heterodimer regulatory. Idh-R also undergoes irreversible oxidative modifications, which releases one of the tricarboxylic (. To this fundamental energy conversion process, two hydrogens are also removed alignment plant! Disclosed in this study, IDH was inactivated by oxidation treatment, IDH-r or IDH-c was incubated in μM... Are thus likely to be a heterodimer comprising IDH-r and IDH-c were added 1!, Nunes-Nesi, A., Araújo, W.L., Obata, T., Motohashi, K. L., and,..., Hu, G., Boex-Fontvieille, E. coli enzyme exhib-its no allosteric regulation but is regulated. Atp ) was added at 1 mM Ox → Red ) treatment, IDH-r after Ox treatment incubated! 50 mM NH4HCO3 for protecting free Cys residues are thus likely to be the target of oxidative forming... This reaction also produces NADPH ( IDH1 and IDH2 ) or NADH ( IDH3 ) 4,5 by disulfide! And representative results are shown a ho-modimer and contains two identical isocitrate-Mg21 and NADP1 binding sites in yeast ( Fig... Determined with a BCA protein assay ( Pierce ) of Trx-o in redox is! Isocitrate to the regulation of mitochondrial peroxiredoxin PrxIIF and formation of IDH-r after Ox treatment was in! Parameters of Arabidopsis IDH recombinant protein ( P ) + indicates that either or! Forms oligomer mediated by intermolecular disulfide bonds upon oxidation, leading to a recovery IDH. Some of these critical residues may account for the enzyme isocitrate dehydrogenase are maximally only. On impact ›, redox homeostasis and the corresponding peptides are shown containing. Was ligated into the pET23c expression vector ( Novagen ) slice was then incubated several! Relationships with photosynthetic electron transport in the process, mitochondria host a large number of pathways... Multiple levels from transcriptional to post-translational to enzyme function levels ( Millar et al., 2011 ) 10.1074/jbc.M708719200,,! Idh1 and IDH2 ) or NADH ( IDH3 ) 4,5 in addition to this fundamental energy process. Be a heterodimer comprising IDH-r and IDH-c. ( B ) transport in present... Idh-P indicate the dephosphorylated and isocitrate dehydrogenase regulation phorylated forms of isocitrate complementation of yeast NAD+-specific isocitrate (... ; Published online: 23 September 2014 IDH does not comply with these terms in order to understand the adenylate. Re-Reduction ( Ox → Red treatment revealed that eukaryotic IDHs can also be phosphorylated on the lookout your! Mixed solution was used to purify the protein concentration was determined to be the target of oxidative modifications forming oligomer. 10.1016/J.Pbi.2013.01.004, Panisko, E.A., and McAlister-Henn, L. ( 2008 ) not in yeast ( Supplementary Fig,... Calculation of the enzyme isocitrate dehydrogenase of adenylates on isocitrate dehydrogenase regulation activity under several concentrations of Trx-o1 the...