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Plant Molecular Biology

, Volume 72, Issue 3, pp 301–310 | Cite as

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

  • Maryam Zarepour
  • Katrin Kaspari
  • Stefan Stagge
  • Ralf Rethmeier
  • Ralf R. MendelEmail author
  • Florian Bittner
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.

Keywords

Xanthine dehydrogenase AtXDH1 NADH oxidation Superoxide Superoxide dismutase Pichia pastoris 

Notes

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.

Supplementary material

11103_2009_9570_MOESM1_ESM.ppt (48 kb)
Supplementary material 1 (PPT 48 kb)
11103_2009_9570_MOESM2_ESM.doc (38 kb)
Supplementary material 2 (DOC 37 kb)
11103_2009_9570_MOESM3_ESM.doc (33 kb)
Supplementary material 3 (DOC 33 kb)

References

  1. 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 158:131–138CrossRefGoogle Scholar
  2. 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 265:14170–14175PubMedGoogle Scholar
  3. 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-–1105Google Scholar
  4. 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 1219–1225Google Scholar
  5. Benboubetra M, Baghiani A, Atmani D, Harrison R (2004) Physicochemical and kinetic properties of purified sheep’s milk xanthine oxidoreductase. J Dairy Sci 87:1580–1584PubMedCrossRefGoogle Scholar
  6. Boland MJ (1981) NAD+: xanthine dehydrogenase from nodules of navy beans: partial purification and properties. Biochem Int 2:567–574Google Scholar
  7. 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 72:248–254CrossRefPubMedGoogle Scholar
  8. 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 54:496–509CrossRefPubMedGoogle Scholar
  9. 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 141:160–165Google Scholar
  10. 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 151:246–250Google Scholar
  11. 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 165:1319–1330CrossRefPubMedGoogle Scholar
  12. Corran HS, Dewan JG, Gordon AH, Green DE (1939) Xanthine oxidase and milk flavoprotein: with an Addendum by J St L Philpot. Biochem J 33:1694–1708PubMedGoogle Scholar
  13. 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 88:4700–4702CrossRefPubMedGoogle Scholar
  14. 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 117:97–100PubMedGoogle Scholar
  15. Della Corte E, Gozzetti G, Novello F, Stirpe F (1969) Properties of the xanthine oxidase from human liver. Biochim Biophys Acta 191:164–166PubMedGoogle Scholar
  16. 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 97:10723–10728CrossRefPubMedGoogle Scholar
  17. 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 110:1187–1195PubMedGoogle Scholar
  18. Evans PJ, Gallesi D, Mathieu C, Hernandez MJ, de Felipe MR, Halliwell B, Puppo A (1999) Oxidative stress occurs during soybean nodule senescence. Planta 208:73–79CrossRefGoogle Scholar
  19. Fridovich I (1989) Superoxide dismutases. An adaptation to a paramagnetic gas. J Biol Chem 264:7761–7764PubMedGoogle Scholar
  20. 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 372:15–32CrossRefPubMedGoogle Scholar
  21. 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 278:431–438CrossRefPubMedGoogle Scholar
  22. Grant JJ, Loake GJ (2000) Role of reactive oxygen intermediates and cognate redox signaling in disease resistance. Plant Physiol 124:21–29CrossRefPubMedGoogle Scholar
  23. Harris CM, Massey V (1997) The reaction of reduced xanthine dehydrogenase with molecular oxygen. Reaction kinetics and measurement of superoxide radical. J Biol Chem 272:8370–8379CrossRefPubMedGoogle Scholar
  24. Harrison R (2002) Structure and function of xanthine oxidoreductase: where are we now? Free Radic Biol Med 33:774–797CrossRefPubMedGoogle Scholar
  25. 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 279:13547–13554CrossRefPubMedGoogle Scholar
  26. Hille R (2006) Structur and function of xanthine oxidoreductase. Eur J Inorg Chem 10:1913–1926CrossRefGoogle Scholar
  27. Hille R, Massey V (1981) Studies on the oxidative half-reaction of xanthine oxidase. J Biol Chem 256:9090–9095PubMedGoogle Scholar
  28. Hille R, Nishino T (1995) Flavoprotein structure and mechanism. 4. Xanthine oxidase and xanthine dehydrogenase. FASEB J 9:995–1003PubMedGoogle Scholar
  29. Horecker BL, Heppel LA (1949) The reduction of cytochrom c by xanthine oxidase. J Biol Chem 178:683–690PubMedGoogle Scholar
  30. 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 100:8170–8175CrossRefPubMedGoogle Scholar
  31. Lamb C, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 48:251–275CrossRefPubMedGoogle Scholar
  32. Landon EJ, Myles M (1967) NADH oxidation by hypoxanthine dehydrogenase of avian kidney. Biochim Biophys Acta 143:429–431CrossRefPubMedGoogle Scholar
  33. Levine A, Tenhaken R, Dixon R, Lamb C (1994) H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583–593CrossRefPubMedGoogle Scholar
  34. Massey V (1959) The microestimation of succinate and the extinction coefficient of cytochrome c. Biochim Biophys Acta 34:255–256CrossRefPubMedGoogle Scholar
  35. Massey V, Edmondson D (1970) On the mechanism of inactivation of xanthine oxidase by cyanide. J Biol Chem 244:1682–1691Google Scholar
  36. 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 245:2837–2844PubMedGoogle Scholar
  37. 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 55:853–867PubMedGoogle Scholar
  38. 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 134:218–228CrossRefGoogle Scholar
  39. 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 87:225–231CrossRefGoogle Scholar
  40. Montalbini P (1998) Purification and some properties of xanthine dehydrogenase from wheat leaves. Plant Sci 134:89–102CrossRefGoogle Scholar
  41. Montalbini P (2000) Xanthine dehydrogenase from leaves of leguminous plants: purification, characterization and properties of the enzyme. J Plant Phys 156:3–16Google Scholar
  42. 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 264:5468–5473PubMedGoogle Scholar
  43. 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 272:29859–29864CrossRefPubMedGoogle Scholar
  44. Nishino T, Nishino T, Schopfer LM, Massey V (1989) The reactivity of chicken liver xanthine dehydrogenase with molecular oxygen. J Biol Chem 264:2518–2527PubMedGoogle Scholar
  45. 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 280:24888–24894CrossRefPubMedGoogle Scholar
  46. Pastori GM, Del Rio LA (1997) Natural senescence of pea leaves (An Activated Oxygen-Mediated Function for Peroxisomes). Plant Physiol 113:411–418PubMedGoogle Scholar
  47. Perez-Vicente R, Alamillo JM, Cardenas J, Pineda M (1992) Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii. Biochem Biophys Acta 1117:159–166PubMedGoogle Scholar
  48. Rajagopalan KV, Handler P (1967) Purification and properties of chicken liver xanthine dehydrogenase. J Biol Chem 242:4097–4107PubMedGoogle Scholar
  49. 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 17:1294–1305CrossRefPubMedGoogle Scholar
  50. Sanders A, Eisenthal R, Harrison R (1997) NADH oxidase activity of human xanthine oxidoreductase––generation of superoxide anion. Eur J Biochem 245:541–548CrossRefPubMedGoogle Scholar
  51. Santos R, Hérouart D, Sigaud S, Touati D, Puppo A (2001) Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction. Mol Plant-Microbe Interact 14:86–89CrossRefPubMedGoogle Scholar
  52. 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 270:2818–2826CrossRefPubMedGoogle Scholar
  53. 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 40:393–400CrossRefGoogle Scholar
  54. Stirpe F, Ravaioli M, Battelli MG, Musiani S, Grazi GL (2002) Xanthine oxidoreductase activity in human liver disease. Am J Gastroenterol 97:2079–2085CrossRefPubMedGoogle Scholar
  55. Triplett EW, Blevins DG, Randall DD (1980) Allantoic acid synthesis in soybean root nodule cytosol via xanthine dehydrogenase. Plant Physiol 65:1203–1206CrossRefPubMedGoogle Scholar
  56. 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 42:862–876CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Maryam Zarepour
    • 2
  • Katrin Kaspari
    • 1
  • Stefan Stagge
    • 1
  • Ralf Rethmeier
    • 1
  • Ralf R. Mendel
    • 1
    Email author
  • Florian Bittner
    • 1
  1. 1.Institut für PflanzenbiologieTechnische Universität BraunschweigBraunschweigGermany
  2. 2.Membrane Protein Research Group, Department of BiochemistryUniversity of AlbertaEdmontonCanada

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