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Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a potent producer of superoxide anions via its NADH oxidase activity

Plant Molecular Biology volume 72, pages 301–310 (2010)Cite this article

Abstract

Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a key enzyme in purine degradation where it oxidizes hypoxanthine to xanthine and xanthine to uric acid. Electrons released from these substrates are either transferred to NAD+ or to molecular oxygen, thereby yielding NADH or superoxide, respectively. By an alternative activity, AtXDH1 is capable of oxidizing NADH with concomitant formation of NAD+ and superoxide. Here we demonstrate that in comparison to the specific activity with xanthine as substrate, the specific activity of recombinant AtXDH1 with NADH as substrate is about 15-times higher accompanied by a doubling in superoxide production. The observation that NAD+ inhibits NADH oxidase activity of AtXDH1 while NADH suppresses NAD+-dependent xanthine oxidation indicates that both NAD+ and NADH compete for the same binding-site and that both sub-activities are not expressed at the same time. Rather, each sub-activity is determined by specific conditions such as the availability of substrates and co-substrates, which allows regulation of superoxide production by AtXDH1. Since AtXDH1 exhibits the most pronounced NADH oxidase activity among all xanthine dehydrogenase proteins studied thus far, our results imply that in particular by its NADH oxidase activity AtXDH1 is an efficient producer of superoxide also in vivo.

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References

  • Alesandrini F, Mathis R, Van de Sype G, Hérouart D, Puppo A (2003) Possible roles for a cysteine protease and hydrogen peroxide in soybean nodule development and senescence. New Phytol 158:131–138

    Article  CAS  Google Scholar 

  • Amaya Y, Yamazaki K, Sato M, Noda K, Nishino T, Nishino T (1990) Proteolytic conversion of xanthine dehydrogenase from the NAD-dependent type to the O2-dependent type. Amino acid sequence of rat liver xanthine dehydrogenase and identification of the cleavage sites of the enzyme protein during irreversible conversion by trypsin. J Biol Chem 265:14170–14175

    CAS  PubMed  Google Scholar 

  • Avis PG, Bergel F, Bray RC (1955) Cellular constituents. The chemistry of xanthine oxidase. Part I. The preparation of a crystalline xanthine oxidase from cow’s milk. J Chem Soc 1100-–1105

  • Avis PG, Bergel F, Bray RC (1956) Cellular constituents. The chemistry of xanthine oxidase. Part III. Estimations of cofactors and the catalytic functions of enzyme fractions of cow’s milk. J Chem Soc 1219–1225

  • Benboubetra M, Baghiani A, Atmani D, Harrison R (2004) Physicochemical and kinetic properties of purified sheep’s milk xanthine oxidoreductase. J Dairy Sci 87:1580–1584

    CAS  PubMed  Article  Google Scholar 

  • Boland MJ (1981) NAD+: xanthine dehydrogenase from nodules of navy beans: partial purification and properties. Biochem Int 2:567–574

    CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brychkova G, Alikulov Z, Fluhr R, Sagi M (2008) A critical role for ureides in dark and senescence-induced purine remobilization is unmasked in the Atxdh1 Arabidopsis mutant. Plant J 54:496–509

    Article  CAS  PubMed  Google Scholar 

  • Corpas FJ, Gómez M, Hernández JA, Del Río LA (1993) Metabolism of activated oxygen in peroxisomes from two Pisum sativum L. cultivars with different sensitivity to sodium chloride. J Plant Physiol 141:160–165

    CAS  Google Scholar 

  • Corpas FJ, De La Colina C, Sanchez-Rasero F, Del Rio LA (1997) A role for leaf peroxisomes in the catabolism of purines. J Plant Physiol 151:246–250

    CAS  Google Scholar 

  • Corpas FJ, Palma JM, Sandalio LM, Valderrama R, Barroso JB, Del Río LA (2008) Peroxisomal xanthine oxidoreductase: characterization of the enzyme from pea (Pisum sativum L.) leaves. J Plant Physiol 165:1319–1330

    Article  CAS  PubMed  Google Scholar 

  • Corran HS, Dewan JG, Gordon AH, Green DE (1939) Xanthine oxidase and milk flavoprotein: with an Addendum by J St L Philpot. Biochem J 33:1694–1708

    CAS  PubMed  Google Scholar 

  • Datta DB, Triplett EW, Newcomb EH (1991) Localization of xanthine dehydrogenase in cowpea root nodules: implications for the interaction between cellular compartments during ureide biogenesis. Proc Natl Acad Sci USA 88:4700–4702

    Article  CAS  PubMed  Google Scholar 

  • Della Corte E, Stirpe F (1970) The regulation of xanthine oxidase. Inhibition by reduced nicotinamide-adenine dinucleotide of rat liver xanthine oxidase type D and of chick liver xanthine dehydrogenase. Biochem J 117:97–100

    CAS  PubMed  Google Scholar 

  • Della Corte E, Gozzetti G, Novello F, Stirpe F (1969) Properties of the xanthine oxidase from human liver. Biochim Biophys Acta 191:164–166

    CAS  PubMed  Google Scholar 

  • Enroth C, Eger BT, Okamoto K, Nishino T, Nishino T, Pai EF (2000) Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: structure-based mechanism of conversion. Proc Natl Acad Sci USA 97:10723–10728

    Article  CAS  PubMed  Google Scholar 

  • Escuredo PR, Minchin FR, Gogorcena Y, Iturbe-Ormaetxe I, Klucas RV, Becana M (1996) Involvement of activated oxygen in nitrateinduced senescence of pea root nodules. Plant Physiol 110:1187–1195

    CAS  PubMed  Google Scholar 

  • Evans PJ, Gallesi D, Mathieu C, Hernandez MJ, de Felipe MR, Halliwell B, Puppo A (1999) Oxidative stress occurs during soybean nodule senescence. Planta 208:73–79

    Article  CAS  Google Scholar 

  • Fridovich I (1989) Superoxide dismutases. An adaptation to a paramagnetic gas. J Biol Chem 264:7761–7764

    CAS  PubMed  Google Scholar 

  • Garattini E, Mendel RR, Romao MJ, Wright R, Terao M (2003) Mammalian molybdo-flavoenzymes, an expanding family of proteins: structure, genetics, regulation, function and pathophysiology. Biochem J 372:15–32

    Article  CAS  PubMed  Google Scholar 

  • Glatigny A, Hof P, Romao MJ, Huber R, Scazzocchio C (1998) Altered specificity mutations define residues essential for substrate positioning in xanthine dehydrogenase. J Mol Biol 278:431–438

    Article  CAS  PubMed  Google Scholar 

  • Grant JJ, Loake GJ (2000) Role of reactive oxygen intermediates and cognate redox signaling in disease resistance. Plant Physiol 124:21–29

    Article  CAS  PubMed  Google Scholar 

  • Harris CM, Massey V (1997) The reaction of reduced xanthine dehydrogenase with molecular oxygen. Reaction kinetics and measurement of superoxide radical. J Biol Chem 272:8370–8379

    Article  CAS  PubMed  Google Scholar 

  • Harrison R (2002) Structure and function of xanthine oxidoreductase: where are we now? Free Radic Biol Med 33:774–797

    Article  CAS  PubMed  Google Scholar 

  • Hesberg C, Haensch R, Mendel RR, Bittner F (2004) Tandem orientation of duplicated xanthine dehydrogenase genes from Arabidopsis thaliana: differential gene expression and enzyme activities. J Biol Chem 279:13547–13554

    Article  CAS  PubMed  Google Scholar 

  • Hille R (2006) Structur and function of xanthine oxidoreductase. Eur J Inorg Chem 10:1913–1926

    Article  CAS  Google Scholar 

  • Hille R, Massey V (1981) Studies on the oxidative half-reaction of xanthine oxidase. J Biol Chem 256:9090–9095

    CAS  PubMed  Google Scholar 

  • Hille R, Nishino T (1995) Flavoprotein structure and mechanism. 4. Xanthine oxidase and xanthine dehydrogenase. FASEB J 9:995–1003

    CAS  PubMed  Google Scholar 

  • Horecker BL, Heppel LA (1949) The reduction of cytochrom c by xanthine oxidase. J Biol Chem 178:683–690

    CAS  PubMed  Google Scholar 

  • Kuwabara Y, Nishino T, Okamoto K, Matsumura T, Eger BT, Pai EF, Nishino T (2003) Unique amino acids cluster for switching from the dehydrogenase to oxidase form of xanthine oxidoreductase. Proc Natl Acad Sci USA 100:8170–8175

    Article  CAS  PubMed  Google Scholar 

  • Lamb C, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 48:251–275

    Article  CAS  PubMed  Google Scholar 

  • Landon EJ, Myles M (1967) NADH oxidation by hypoxanthine dehydrogenase of avian kidney. Biochim Biophys Acta 143:429–431

    Article  CAS  PubMed  Google Scholar 

  • Levine A, Tenhaken R, Dixon R, Lamb C (1994) H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583–593

    Article  CAS  PubMed  Google Scholar 

  • Massey V (1959) The microestimation of succinate and the extinction coefficient of cytochrome c. Biochim Biophys Acta 34:255–256

    Article  CAS  PubMed  Google Scholar 

  • Massey V, Edmondson D (1970) On the mechanism of inactivation of xanthine oxidase by cyanide. J Biol Chem 244:1682–1691

    Google Scholar 

  • Massey V, Komai H, Palmer G, Elion GB (1970) On the mechanism of inactivation of xanthine oxidase by allopurinol and other pyrazolo[3, 4-d]pyrimidines. J Biol Chem 245:2837–2844

    CAS  PubMed  Google Scholar 

  • Miao Y, Laun T, Zimmermann P, Zentgraf U (2004) Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Mol Biol 55:853–867

    CAS  PubMed  Google Scholar 

  • Montalbini P (1992a) Inhibition of hypersensitive response by allopurinol applied to the host in the incompatible relationship between Phaseolus vulgaris and Uromyces phaseoli. J Phytopath 134:218–228

    Article  CAS  Google Scholar 

  • Montalbini P (1992b) Ureides and enzymes of ureide synthesis in wheat seeds and leaves and effect of allopurinol on Puccinia recondita f.sp. tritici infection. Plant Sci 87:225–231

    Article  CAS  Google Scholar 

  • Montalbini P (1998) Purification and some properties of xanthine dehydrogenase from wheat leaves. Plant Sci 134:89–102

    Article  CAS  Google Scholar 

  • Montalbini P (2000) Xanthine dehydrogenase from leaves of leguminous plants: purification, characterization and properties of the enzyme. J Plant Phys 156:3–16

    CAS  Google Scholar 

  • Nishino T, Nishino T (1989) The nicotinamide adenine dinucleotide-binding site of chicken liver xanthine dehydrogenase. Evidence for alteration of the redox potential of the flavin by NAD binding or modification of the NAD-binding site and isolation of a modified peptide. J Biol Chem 264:5468–5473

    CAS  PubMed  Google Scholar 

  • Nishino T, Nishino T (1997) The conversion from the dehydrogenase type to the oxidase type of rat liver xanthine dehydrogenase by modification of cysteine residues with fluorodinitrobenzene. J Biol Chem 272:29859–29864

    Article  CAS  PubMed  Google Scholar 

  • Nishino T, Nishino T, Schopfer LM, Massey V (1989) The reactivity of chicken liver xanthine dehydrogenase with molecular oxygen. J Biol Chem 264:2518–2527

    CAS  PubMed  Google Scholar 

  • Nishino T, Okamoto K, Kawaguchi Y, Hori H, Matsumura T, Eger BT, Pai EF, Nishino T (2005) Mechanism of the conversion of xanthine dehydrogenase to xanthine oxidase: identification of the two cysteine disulfide bonds and crystal structure of a non-convertible rat liver xanthine dehydrogenase mutant. J Biol Chem 280:24888–24894

    Article  CAS  PubMed  Google Scholar 

  • Pastori GM, Del Rio LA (1997) Natural senescence of pea leaves (An Activated Oxygen-Mediated Function for Peroxisomes). Plant Physiol 113:411–418

    CAS  PubMed  Google Scholar 

  • Perez-Vicente R, Alamillo JM, Cardenas J, Pineda M (1992) Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii. Biochem Biophys Acta 1117:159–166

    CAS  PubMed  Google Scholar 

  • Rajagopalan KV, Handler P (1967) Purification and properties of chicken liver xanthine dehydrogenase. J Biol Chem 242:4097–4107

    CAS  PubMed  Google Scholar 

  • Rubio MC, James EK, Clemente MR, Bucciarelli B, Fedorov M, Vance CP, Becana M (2004) Localization of superoxide dismutases and hydrogen peroxide in legume root nodules. Mol Plant-Microbe Interact 17:1294–1305

    Article  CAS  PubMed  Google Scholar 

  • Sanders A, Eisenthal R, Harrison R (1997) NADH oxidase activity of human xanthine oxidoreductase––generation of superoxide anion. Eur J Biochem 245:541–548

    Article  CAS  PubMed  Google Scholar 

  • Santos R, Hérouart D, Sigaud S, Touati D, Puppo A (2001) Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction. Mol Plant-Microbe Interact 14:86–89

    Article  CAS  PubMed  Google Scholar 

  • Sato A, Nishino T, Noda K, Amaya Y, Nishino T (1995) The structure of chicken liver xanthine dehydrogenase. cDNA cloning and the domain structure. J Biol Chem 270:2818–2826

    Article  CAS  PubMed  Google Scholar 

  • Sauer P, Frebortova J, Sebela M, Galuszka P, Jacobsen S, Pec P, Frebort I (2002) Xanthine dehydrogenase of pea seedlings: a member of the plant molybdenum oxidoreductase family. Plant Physiol Biochem 40:393–400

    Article  CAS  Google Scholar 

  • Stirpe F, Ravaioli M, Battelli MG, Musiani S, Grazi GL (2002) Xanthine oxidoreductase activity in human liver disease. Am J Gastroenterol 97:2079–2085

    Article  CAS  PubMed  Google Scholar 

  • Triplett EW, Blevins DG, Randall DD (1980) Allantoic acid synthesis in soybean root nodule cytosol via xanthine dehydrogenase. Plant Physiol 65:1203–1206

    Article  CAS  PubMed  Google Scholar 

  • Yesbergenova Z, Yang G, Oron E, Soffer D, Flur R, Sagi M (2005) The plant Mo-hydroxylases aldehyde oxidase and xanthine dehydrogenase have distinct reactive oxygen species signatures and are induced by drought and abscisic acid. Plant J 42:862–876

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We are grateful to Prof. Dr. Guenter Schwarz and Dr. Jose Santamaria Araujo (University of Cologne) for their support in protein purification, and we thank Ute Nielaender and Victoria Michael (TU Braunschweig) for excellent technical assistance.

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Affiliations

  1. Institut für Pflanzenbiologie, Technische Universität Braunschweig, Humboldtstrasse 1, 38106, Braunschweig, Germany

    Katrin Kaspari, Stefan Stagge, Ralf Rethmeier, Ralf R. Mendel & Florian Bittner

  2. Membrane Protein Research Group, Department of Biochemistry, University of Alberta, 474 Medical Sciences Building, Edmonton, Alberta, T6G 2H7, Canada

    Maryam Zarepour

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  1. Maryam Zarepour
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  2. Katrin Kaspari
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  3. Stefan Stagge
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  4. Ralf Rethmeier
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  5. Ralf R. Mendel
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  6. Florian Bittner
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Correspondence to Ralf R. Mendel.

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Zarepour, M., Kaspari, K., Stagge, S. et al. Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a potent producer of superoxide anions via its NADH oxidase activity. Plant Mol Biol 72, 301–310 (2010). https://doi.org/10.1007/s11103-009-9570-2

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  • DOI: https://doi.org/10.1007/s11103-009-9570-2

Keywords

  • Xanthine dehydrogenase
  • AtXDH1
  • NADH oxidation
  • Superoxide
  • Superoxide dismutase
  • Pichia pastoris