Archives of Microbiology

, Volume 186, Issue 5, pp 385–392 | Cite as

Oxidative inactivation of reduced NADP-generating enzymes in E. coli: iron-dependent inactivation with affinity cleavage of NADP-isocitrate dehydrogenase

  • Keiko Murakami
  • Ryoko Tsubouchi
  • Minoru Fukayama
  • Tadashi Ogawa
  • Masataka Yoshino
Original Paper

Abstract

Treatment of E. coli extract with iron/ascorbate preferentially inactivated NADP-isocitrate dehydrogenase without affecting glucose-6-phosphate dehydrogenase. NADP-Isocitrate dehydrogenase required divalent metals such as Mg2+, Mn2+ or Fe2+ ion. Iron/ascorbate-dependent inactivation of the enzyme was accompanied with the protein fragmentation as judged by SDS-PAGE. Catalase protecting the enzyme from the inactivation suggests that hydroxyl radical is responsible for the inactivation with fragmentation. TOF-MS analysis showed that molecular masses of the enzyme fragments were 36 and 12, and 33 and 14 kDa as minor components. Based on the amino acid sequence analyses of the fragments, cleavage sites of the enzyme were identified as Asp307-Tyr308 and Ala282-Asp283, which are presumed to be the metal-binding sites. Ferrous ion bound to the metal-binding sites of the E. coli NADP-isocitrate dehydrogenase may generate superoxide radical that forms hydrogen peroxide and further hydroxyl radical, causing inactivation with peptide cleavage of the enzyme. Oxidative inactivation of NADP-isocitrate dehydrogenase without affecting glucose 6-phosphate dehydrogenase shows only a little influence on the antioxidant activity supplying NADPH for glutathione regeneration, but may facilitate flux through the glyoxylate bypass as the biosynthetic pathway with the inhibition of the citric acid cycle under aerobic growth conditions of E. coli.

Keywords

NADP-isocitrate dehydrogenase Reactive oxygen species Affinity cleavage Metal binding Iron 

References

  1. Colman RF (1972) Role of metal ions in reactions catalyzed by pig heart triphosphopyridine nucleotide-dependent isocitrate dehydrogenase. II. Effect on catalytic properties and reactivity of amino acid residues. J Biol Chem 247:215–223PubMedGoogle Scholar
  2. Cozzone AJ, El-Mansi M (2005) Control of isocitrate dehydrogenase catalytic activity by protein phosphorylation in Escherichia coli. J Mol Microbiol Biotechnol 9:132–146PubMedCrossRefGoogle Scholar
  3. Deneke SM (2000) Thiol-based antioxidants. Curr Top Cell Regul 36:151–180PubMedCrossRefGoogle Scholar
  4. Duggleby RG (1981) A nonlinear regression program for small computers. Anal Biochem 110:9–18PubMedCrossRefGoogle Scholar
  5. Friguet B, Szweda LI, Stadtman ER (1994) Susceptibility of glucose-6-phosphate dehydrogenase modified by 4-hydroxy-2-nonenal and metal-catalyzed oxidation to proteolysis by the multicatalytic protease. Arch Biochem Biophys 311:168–173PubMedCrossRefGoogle Scholar
  6. Garnak M, Reeves HC (1979) Purification and properties of phosphorylated isocitrate dehydrogenase of Escherichia coli. J Biol Chem 254:7915–7920PubMedGoogle Scholar
  7. Grodsky NB, Soundar S, Colman RF (2000) Evaluation by site-directed mutagenesis of aspartic acid residues in the metal site of pig heart NADP-dependent isocitrate dehydrogenase. Biochemistry 39:2193–2200PubMedCrossRefGoogle Scholar
  8. Hanson RL, Rose C (1980) Effects of an insertion mutation in a locus affecting pyridine nucleotide transhydrogenase (pnt::Tn5 ) on the growth of Escherichia coli. J Bacteriol 141:401–404PubMedGoogle Scholar
  9. Holmes WH (1988) Control of flux through the citrate cycle and the glyoxylate bypass in Escherichia coli. Biochem Soc Symp 54:17–31Google Scholar
  10. Huang YC, Grodsky NB, Kim TK, Colman RF (2004) Ligands of the Mn2+ bound to porcine mitochondrial NADP-dependent isocitrate dehydrogenase, as assessed by mutagenesis. Biochemistry 43:2821–2828PubMedCrossRefGoogle Scholar
  11. Hurley JH, Thorsness PE, Ramalingam V, Helmers NH, Koshland DE, Stroud RM (1989) Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase. Proc Natl Acad Sci USA 86:8635–8639PubMedCrossRefGoogle Scholar
  12. Hurley JH, Dean AM, Koshland DE Jr, Stroud RM (1991) Catalytic mechanism of NADP+-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP+ complexes. Biochemistry 30:8671–8678PubMedCrossRefGoogle Scholar
  13. Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H, Mori H (2005) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 12:291–299PubMedGoogle Scholar
  14. Lee SM, Koh HJ, Huh TL, Park JW (1999) Radiation sensitivity of an Escherichia coli mutant lacking NADP+-dependent isocitrate dehydrogenase. Biochem Biophys Res Commun 254:647–650PubMedCrossRefGoogle Scholar
  15. Lee SM,Huh TL, Park JW (2001) Inactivation of NADP+-dependent isocitrate dehydrogenase by reactive oxygen species. Biochimie 83:1057–1065PubMedCrossRefGoogle Scholar
  16. Lee SM, Koh HJ, Park DC, Song BJ, Huh TL, Park JW (2002) Cytosolic NADP+-dependent isocitrate dehydrogenase status modulates oxidative damage to cells. Free Radic Biol Med 32:1185–1196PubMedCrossRefGoogle Scholar
  17. Maier KL, Hinze H, Meyer B, Lenz AG (1996) Metal-catalyzed inactivation of bovine glucose-6-phosphate dehydrogenase role of thiols. FEBS Lett 396:95–98PubMedCrossRefGoogle Scholar
  18. Marks PA (1966) Glucose 6-phosphate dehydrogenase—clinical aspects. In: Methods in enzymology. 9, 131–137, Academic Press, New YorkGoogle Scholar
  19. Minard KI, McAlister-Henn L (2001) Antioxidant function of cytosolic sources of NADPH in yeast. Free Radic Biol Med 31:832–843PubMedCrossRefGoogle Scholar
  20. Murakami K, Iwata S, Haneda M, Yoshino M (1997) Role of metal cations in the regulation of NADP-linked isocitrate dehydrogenase from porcine heart. Biometals 10:169–174PubMedCrossRefGoogle Scholar
  21. Ninfali P, Ditroilo M, Capellacci S, Biagiotti E (2001) Rabbit brain glucose-6-phosphate dehydrogenase: biochemical properties and inactivation by free radicals and 4-hydroxy-2-nonenal. Neuroreport 12:4149–4153PubMedCrossRefGoogle Scholar
  22. Peng L, Shimizu K (2003) Global metabolic regulation analysis for Escherichia coli K12 based on protein expression by 2-dimensional electrophoresis and enzyme activity measurement. Appl Microbial Biotechnol 61:163–178Google Scholar
  23. Sauer U, Canonaco F, Heri S, Perrenoud A, Fischer E (2004) The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli. J Biol Chem 279:6613–6619PubMedCrossRefGoogle Scholar
  24. Soundar S, Colman RF (1993) Identification of metal-isocitrate binding site of pig heart NADP-specific isocitrate dehydrogenase by affinity cleavage of the enzyme by Fe2+-isocitrate. J Biol Chem 268:5264–5271PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Keiko Murakami
    • 1
  • Ryoko Tsubouchi
    • 1
  • Minoru Fukayama
    • 2
  • Tadashi Ogawa
    • 1
  • Masataka Yoshino
    • 1
  1. 1.Department of BiochemistryAichi Medical University School of MedicineNagakute, AichiJapan
  2. 2.Central Research LaboratoryAichi Medical University School of MedicineNagakute, AichiJapan

Personalised recommendations