Abstract
Mitochondria are tightly linked to cellular nutrient sensing, and provide not only energy, but also intermediates for the de novo synthesis of cellular compounds including amino acids. Mitochondrial metabolic enzymes as generators and/or targets of signals are therefore important players in the distribution of intermediates between catabolic and anabolic pathways. The highly regulated 2-oxoglutarate dehydrogenase complex (OGDHC) participates in glucose oxidation via the tricarboxylic acid cycle. It occupies an amphibolic branch point in the cycle, where the energy-producing reaction of the 2-oxoglutarate degradation competes with glutamate (Glu) synthesis via nitrogen incorporation into 2-oxoglutarate. To characterize the specific impact of the OGDHC inhibition on amino acid metabolism in both plant and animal mitochondria, a synthetic analog of 2-oxoglutarate, namely succinyl phosphonate (SP), was applied to living systems from different kingdoms, both in situ and in vivo. Using a high-throughput mass spectrometry-based approach, we showed that organisms possessing OGDHC respond to SP by significantly changing their amino acid pools. By contrast, cyanobacteria which lack OGDHC do not show perturbations in amino acids following SP treatment. Increases in Glu, 4-aminobutyrate and alanine represent the most universal change accompanying the 2-oxoglutarate accumulation upon OGDHC inhibition. Other amino acids were affected in a species-specific manner, suggesting specific metabolic rearrangements and substrate availability mediating secondary changes. Strong perturbation in the relative abundance of amino acids due to the OGDHC inhibition was accompanied by decreased protein content. Our results provide specific evidence of a considerable role of OGDHC in amino acid metabolism.
Similar content being viewed by others
References
Araújo WL, Nunes-Nesi A, Trenkamp S, Bunik VI, Fernie AR (2008) Inhibition of 2-oxoglutarate dehydrogenase in potato tuber suggests the enzyme is limiting for respiration and confirms its importance in nitrogen assimilation. Plant Physiol 148(4):1782–1796. doi:10.1104/pp.108.126219
Araújo WL, Tohge T, Ishizaki K, Leaver CJ, Fernie AR (2011) Protein degradation—an alternative respiratory substrate for stressed plants. Trends Plant Sci 16(9):489–498. doi:10.1016/j.tplants.2011.05.008
Araújo WL, Tohge TL, Nunes-Nesi A, Daloso DM, Nimick M, Krahnert I, Bunik VI, Moorhead G, Fernie A (2012) Phosphonate analogs of 2-oxoglutarate perturb metabolism and gene expression in illuminated Arabidopsis leaves. Front Plant Sci 3:114. doi:10.3389/fpls.2012.00114
Asakura Y, Kimura E, Usuda Y, Kawahara Y, Matsui K, Osumi T, Nakamatsu T (2007) Altered metabolic flux due to deletion of odhA causes l-glutamate overproduction in Corynebacterium glutamicum. Appl Environ Microbiol 73(4):1308–1319. doi:10.1128/aem.01867-06
Bauwe H, Hagemann M, Fernie AR (2010) Photorespiration: players, partners and origin. Trends Plant Sci 15(6):330–336. doi:10.1016/j.tplants.2010.03.006
Bettendorff L, Peeters M, Jouan C, Wins P, Schoffeniels E (1991) Determination of thiamin and its phosphate esters in cultured neurons and astrocytes using an ion-pair reversed-phase high-performance liquid chromatographic method. Anal Biochem 198(1):52–59. doi:10.1016/0003-2697(91)90505-n
Bott M (2007) Offering surprises: TCA cycle regulation in Corynebacterium glutamicum. Trends Microbiol 15(9):417–425. doi:10.1016/j.tim.2007.08.004
Brauc S, De Vooght E, Claeys M, Höfte M, Angenon G (2011) Influence of over-expression of cytosolic aspartate aminotransferase on amino acid metabolism and defence responses against Botrytis cinerea infection in Arabidopsis thaliana. J Plant Physiol 168(15):1813–1819. doi:10.1016/j.jplph.2011.05.012
Brown GC (1992) Control of respiration and ATP synthesis in mammalian mitochondria and cells. Biochem J 284:1–13
Bunik VI, Fernie AR (2009) Metabolic control exerted by the 2-oxoglutarate dehydrogenase reaction: a cross-kingdom comparison of the crossroad between energy production and nitrogen assimilation. Biochem J 422(3):405–421. doi:10.1042/bj20090722
Bunik VI, Strumilo S (2009) Regulation of catalysis within cellular network: metabolic and signaling implications of the 2-oxoglutarate oxidative decarboxylation. Curr Chem Biol 3(3):279–290. doi:10.2174/187231309789054904
Bunik VI, Biryukov AI, Zhukov YN (1992) Inhibition of pigeon breast muscle alpha-ketoglutarate dehydrogenase by phosphonate analogues of alpha-ketoglutarate. FEBS Lett 303(2–3):197–201. doi:10.1016/0014-5793(92)80518-l
Bunik VI, Denton TT, Xu H, Thompson CM, Cooper AJL, Gibson GE (2005) Phosphonate analogs of α-ketoglutarate inhibit the activity of the α-ketoglutarate dehydrogenase complex isolated from brain and in cultured cells. Biochemistry 44:10552–10561
Bunik VI, Lovat M, Groznaya A, Graf A, Dunaeva T, Trofimova L, Sokolova N (2009) Succinyl phosphonate, a protector of the 2-oxoglutarate dehydrogenase complex, corrects behavioral impairments in rats exposed to hypoxia or ethanol. Alzheimers Dement 5(4, Supplement 1):P476–P477. doi:10.1016/j.jalz.2009.04.721
Butow RA, Avadhani NG (2004) Mitochondrial signaling: the retrograde response. Mol Cell 14(1):1–15
Cheshchevik V, Janssen AJM, Dremza IK, Zavodnik IB, Bunik VI (2010) The OGDHC-exerted control of mitochondrial respiration is increased under energy demand. In: Renner-Sattler K, Gnaiger E (eds) Mitochondrial physiology—the many functions of the organism in our cells. Steiger Druck GmbH, Axams, pp 76–77
Cooper AJL (2004) The role of glutamine transaminase K (GTK) in sulfur and alpha-keto acid metabolism in the brain, and in the possible bioactivation of neurotoxicants. Neurochem Int 44(8):557–577. doi:10.1016/j.neuint.2003.12.002
Douce R, Bourguignon J, Neuburger M, Rébeillé F (2001) The glycine decarboxylase system: a fascinating complex. Trends Plant Sci 6(4):167–176. doi:10.1016/s1360-1385(01)01892-1
Erban A, Schauer N, Fernie AR, Kopka J (2007) Nonsupervised construction and application of mass spectral and retention time index libraries from time-of-flight gas chromatography–mass spectrometry metabolite profiles. In: Weckwerth W (ed) Methods in molecular biology, vol 358. Humana Press, New York, pp 19–38. doi:10.1007/978-1-59745-244-1_2
Fang M, Toogood RD, Macova A, Ho K, Franzblau SG, McNeil MR, Sanders DAR, Palmer DRJ (2010) Succinylphosphonate esters are competitive inhibitors of MenD that show active-site discrimination between homologous alpha-ketoglutarate-decarboxylating enzymes. Biochemistry 49(12):2672–2679. doi:10.1021/bi901432d
Fernie AR, Roessner U, Trethewey RN, Willmitzer L (2001a) The contribution of plastidial phosphoglucomutase to the control of starch synthesis within the potato tuber. Planta 213(3):418–426. doi:10.1007/s004250100521
Fernie AR, Roscher A, Ratcliffe RG, Kruger NJ (2001b) Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells. Planta 212(2):250–263. doi:10.1007/s004250000386
Graf A, Kabysheva M, Klimuk E, Trofimova L, Dunaeva T, Zündorf G, Kahlert S, Reiser G, Storozhevykh T, Pinelis V, Sokolova N, Bunik V (2009) Role of 2-oxoglutarate dehydrogenase in brain pathologies involving glutamate neurotoxicity. J Mol Catal B Enzym 61(1–2):80–87. doi:10.1016/j.molcatb.2009.02.016
Hanigan MH, Ricketts WA (1993) Extracellular glutathione is a source of cysteine for cells that express gamma-glutamyl transpeptidase. Biochemistry 32(24):6302–6306. doi:10.1021/bi00075a026
Hou Y, Wang L, Ding B, Liu Y, Zhu H, Liu J, Li Y, Wu X, Yin Y, Wu G (2010) Dietary alpha-ketoglutarate supplementation ameliorates intestinal injury in lipopolysaccharide-challenged piglets. Amino Acids 39(2):555–564. doi:10.1007/s00726-010-0473-y
Hou Y, Wang L, Ding B, Liu Y, Zhu H, Liu J, Li Y, Kang P, Yin Y, Wu G (2011) Alpha-ketoglutarate and intestinal function. Front Biosci 16:1186–1196. doi:10.2741/3783
Iskakova MB, Szaflarski W, Dreyfus M, Remme J, Nierhaus KH (2006) Troubleshooting coupled in vitro transcription-translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins. Nucleic Acids Res 34(19). doi:e13510.1093/nar/gkl462
Kabysheva MS, Storozhevykh TP, Pinelis VG, Bunik VI (2009) Synthetic regulators of the 2-oxoglutarate oxidative decarboxylation alleviate the glutamate excitotoxicity in cerebellar granule neurons. Biochem Pharmacol 77(9):1531–1540. doi:10.1016/j.bcp.2009.02.001
Karaca M, Frigerio F, Maechler P (2011) From pancreatic islets to central nervous system, the importance of glutamate dehydrogenase for the control of energy homeostasis. Neurochem Int 59(4):510–517. doi:10.1016/j.neuint.2011.03.024
Kataoka M, Hashimoto KI, Yoshida M, Nakamatsu T, Horinouchi S, Kawasaki H (2006) Gene expression of Corynebacterium glutamicum in response to the conditions inducing glutamate overproduction. Lett Appl Microbiol 42(5):471–476. doi:10.1111/j.1472-765X.2006.01905.x
Kopka J, Schauer N, Krueger S, Birkemeyer C, Usadel B, Bergmuller E, Dormann P, Weckwerth W, Gibon Y, Stitt M, Willmitzer L, Fernie AR, Steinhauser D (2005) GMD@CSB.DB: the Golm Metabolome Database. Bioinformatics 21(8):1635–1638. doi:10.1093/bioinformatics/bti236
Krall L, Huege J, Catchpole G, Steinhauser D, Willmitzer L (2009) Assessment of sampling strategies for gas chromatography–mass spectrometry (GC-MS) based metabolomics of cyanobacteria. J Chromatogr B 877(27):2952–2960. doi:10.1016/j.jchromb.2009.07.006
Kuhara T, Inoue Y, Ohse M, Krasnikov B, Cooper A (2011) Urinary 2-hydroxy-5-oxoproline, the lactam form of α-ketoglutaramate, is markedly increased in urea cycle disorders. Anal Bioanal Chem 400(7):1843–1851. doi:10.1007/s00216-011-4688-x
Kwon H-B, Sabatini BL (2011) Glutamate induces de novo growth of functional spines in developing cortex. Nature 474(7349):100–104. doi:10.1038/nature09986
Laurent S, Chen H, Bédu S, Ziarelli F, Peng L, Zhang C-C (2005) Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120. Proc Nat Acad Sci USA 102(28):9907–9912. doi:10.1073/pnas.0502337102
Lisec J, Schauer N, Kopka J, Willmitzer L, Fernie AR (2006) Gas chromatography mass spectrometry-based metabolite profiling in plants. Nat Protoc 1(1):387–396. doi:10.1038/nprot.2006.59
Luedemann A, Strassburg K, Erban A, Kopka J (2008) TagFinder for the quantitative analysis of gas chromatography–mass spectrometry (GC–MS)-based metabolite profiling experiments. Bioinformatics 24(5):732–737. doi:10.1093/bioinformatics/btn023
Lytovchenko A, Beleggia R, Schauer N, Isaacson T, Leuendorf JE, Hellmann H, Rose JKC, Fernie AR (2009) Application of GC–MS for the detection of lipophilic compounds in diverse plant tissues. Plant Methods 5: Article number 4. doi:10.1186/1746-4811-5-4
McCandless DW (1982) Energy metabolism in the lateral vestibular nucleus in pyrithiamin-induced thiamin deficiency. Ann N Y Acad Sci 378:355–364. doi:10.1111/j.1749-6632.1982.tb31210.x
Mkrtchyan G, Merkushina K, Kudryavtsev P, Trofimova L, Graf N, Bunik V (2011) Brain thiamine status as an indicator of the brain functional state and response to acute hypoxia. In: Nikitina TV (ed) Warum Deutschland? Perspektiven Internationalen Zusammenarbeit Im Bereich Wissenschaft, Ausbildung, Kultur, Wirtschaft Und Politik. Elibrary Finec, Sankt-Peterburg, pp 197–202. http://elibrary.finec.ru/materials_files/360077028.pdf#page=197
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497. doi:10.1111/j.1399-3054.1962.tb08052.x
Niebisch A, Kabus A, Schultz C, Weil B, Bott M (2006) Corynebacterial protein kinase G controls 2-oxoglutarate dehydrogenase activity via the phosphorylation status of the OdhI protein. J Biol Chem 281(18):12300–12307. doi:10.1074/jbc.M512515200
Nilsen LH, Shi Q, Gibson GE, Sonnewald U (2011) Brain [U-13C]glucose metabolism in mice with decreased α-ketoglutarate dehydrogenase complex activity. J Neurosci Res 89(12):1997–2007. doi:10.1002/jnr.22606
O’Brien TA, Kluger R, Pike DC, Gennis RB (1980) Phosponate analogs of pyruvate-probes of substrate binding to pyruvate oxidase and other thiamin pyrophosphate-dependent decarboxylases. Biochim Biophys Acta 613(1):10–17. doi:10.1016/0005-2744(80)90186-2
Orlowski M, Meister A (1970) The γ-glutamyl cycle: a possible transport system for amino acids. Proc Nat Acad Sci USA 67(3):1248–1255. http://www.pnas.org/content/67/3/1248.abstract
Rakhmanova T, Popova T (2006) Regulation of 2-oxoglutarate metabolism in rat liver by NADP-isocitrate dehydrogenase and aspartate aminotransferase. Biochemistry (Moscow) 71(2):211–217. doi:10.1134/s0006297906020143
Reissner KJ, Kalivas PW (2010) Using glutamate homeostasis as a target for treating addictive disorders. Behav Pharmacol 21(5–6):514–522. doi:10.1097/FBP.0b013e32833d41b2
Rocha M, Licausi F, Araújo WL, Nunes-Nesi A, Sodek L, Fernie AR, van Dongen JT (2010) Glycolysis and the tricarboxylic acid cycle are linked by alanine aminotransferase during hypoxia induced by waterlogging of Lotus japonicus. Plant Physiol 152(3):1501–1513. doi:10.1104/pp.109.150045
Rolfe DFS, Brown GC (1997) Cellular energy utilization and molecular origin of standard metabolic rate in mammals. Physiol Rev 77(3):731–758
Runquist M, Kruger NJ (1999) Control of gluconeogenesis by isocitrate lyase in endosperm of germinating castor bean seedlings. Plant J 19(4):423–431. doi:10.1046/j.1365-313X.1999.00533.x
Sá Santos S, Gibson GE, Cooper AJL, Denton TT, Thompson CM, Bunik VI, Alves PM, Sonnewald U (2006) Inhibitors of the α-ketoglutarate dehydrogenase complex alter [1-13C]glucose and [U-13C]glutamate metabolism in cerebellar granule neuron. J Neurosci Res 83(3):450–458. doi:10.1002/jnr.20749
Schauer N, Steinhauser D, Strelkov S, Schomburg D, Allison G, Moritz T, Lundgren K, Roessner-Tunali U, Forbes MG, Willmitzer L, Fernie AR, Kopka J (2005) GC–MS libraries for the rapid identification of metabolites in complex biological samples. FEBS Lett 579(6):1332–1337. doi:10.1016/j.febslet.2005.01.029
Schultz C, Niebisch A, Gebel L, Bott M (2007) Glutamate production by Corynebacterium glutamicum: dependence on the oxoglutarate dehydrogenase inhibitor protein OdhI and protein kinase PknG. Appl Microbiol Biotechnol 76(3):691–700. doi:10.1007/s00253-007-0933-9
Schultz C, Niebisch A, Schwaiger A, Viets U, Metzger S, Bramkamp M, Bott M (2009) Genetic and biochemical analysis of the serine/threonine protein kinases PknA, PknB, PknG and PknL of Corynebacterium glutamicum: evidence for non-essentiality and for phosphorylation of OdhI and FtsZ by multiple kinases. Mol Microbiol 74(3):724–741. doi:10.1111/j.1365-2958.2009.06897.x
Shi Q, Risa Ø, Sonnewald U, Gibson GE (2009) Mild reduction in the activity of the alpha-ketoglutarate dehydrogenase complex elevates GABA shunt and glycolysis. J Neurochem 109:214–221. doi:10.1111/j.1471-4159.2009.05955.x
Shiio I, Ujigawa-Takeda K (1980) Presence and regulation of α-ketoglutarate dehydrogenase complex in a glutamate-producing bacterium, Brevibacterium flavum. Agricult Biol Chem 44(8):1897–1904. doi:10.1271/bbb1961.44.1897
Smith AC, Robinson AJ (2011) A metabolic model of the mitochondrion and its use in modelling diseases of the tricarboxylic acid cycle. BMC Syst Biol 5:102. doi:10.1186/1752-0509-5-102
Swartz J (2006) Developing cell-free biology for industrial applications. J Ind Microbiol Biotechnol 33(7):476–485. doi:10.1007/s10295-006-0127-y
Sweetlove LJ, Taylor NL, Leaver CJ (2007) Isolation of intact, functional mitochondria from the model plant Arabidopsis thaliana. Methods Mol Biol 372(1):125–136. doi:10.1007/978-1-59745-365-3_9
Tatara M, Brodzki A, Krupski W, Sliwa E, Silmanowicz P, Majcher P, Pierzynowski S, Studzinski T (2005) Effects of alpha-ketoglutarate on bone homeostasis and plasma amino acids in turkeys. Poult Sci 84(10):1604–1609
Trofimova L, Lovat M, Groznaya A, Efimova E, Dunaeva T, Maslova M, Graf A, Bunik V (2010) Behavioral impact of the regulation of the brain 2-oxoglutarate dehydrogenase complex by synthetic phosphonate analog of 2-oxoglutarate: implications into the role of the complex in neurodegenerative diseases. Int J Alzheimers Dis. doi:10.4061/2010/749061 (Article ID 749061)
Viña JR, Palacin M, Puertes IR, Hernandez R, Vina J (1989) Role of the gamma-glutamyl cycle in the regulation of amino acid translocation. Am J Physiol 257(6):E916–E922
Wu G (2009) Amino acids: metabolism, functions, and nutrition. Amino Acids 37(1):1–17. doi:10.1007/s00726-009-0269-0
Zeiger SLH, McKenzie JR, Stankowski JN, Martin JA, Cliffel DE (1802) McLaughlin B (2010) Neuron specific metabolic adaptations following multi-day exposures to oxygen glucose deprivation. Biochim Biophys Acta 11:1095–1104. doi:10.1016/j.bbadis.2010.07.013
Zhang S, Bryant DA (2011) The tricarboxylic acid cycle in Cyanobacteria. Science 334(6062):1551–1553. doi:10.1126/science.1210858
Acknowledgments
This work was supported by funding from the Russian Foundation of Basic Research (grants 10-04-90007, 11-04-91154 and 12-04-01541 to V.B.) and the Max Planck Society (WLA and ARF).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Araújo, W.L., Trofimova, L., Mkrtchyan, G. et al. On the role of the mitochondrial 2-oxoglutarate dehydrogenase complex in amino acid metabolism. Amino Acids 44, 683–700 (2013). https://doi.org/10.1007/s00726-012-1392-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00726-012-1392-x