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Research article

Intracellular catalase/peroxidase from the phytopathogenic rice blast fungus Magnaporthe grisea: expression analysis and biochemical characterization of the recombinant protein

Marcel Zamocky, Paul G. Furtmüller, Marzia Bellei, Gianantonio Battistuzzi, Johannes Stadlmann, Jutta Vlasits, Christian Obinger
Biochemical Journal Feb 11, 2009, 418 (2) 443-451; DOI: 10.1042/BJ20081478
Marcel Zamocky
Metalloprotein Research Group, Division of Biochemistry, Department of Chemistry, University of Natural Resources and Applied Life Sciences, Muthgasse 18 A-1190 Vienna, AustriaInstitute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia
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  • For correspondence: marcel.zamocky@boku.ac.at
Paul G. Furtmüller
Metalloprotein Research Group, Division of Biochemistry, Department of Chemistry, University of Natural Resources and Applied Life Sciences, Muthgasse 18 A-1190 Vienna, Austria
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Marzia Bellei
Department of Chemistry, University of Modena and Reggio Emilia, Modena, Italy
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Gianantonio Battistuzzi
Department of Chemistry, University of Modena and Reggio Emilia, Modena, Italy
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Johannes Stadlmann
Glycobiology Research Group, Division of Biochemistry, Department of Chemistry, University of Natural Resources and Applied Life Sciences, Muthgasse 18 A-1190 Vienna, Austria
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Jutta Vlasits
Metalloprotein Research Group, Division of Biochemistry, Department of Chemistry, University of Natural Resources and Applied Life Sciences, Muthgasse 18 A-1190 Vienna, Austria
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Christian Obinger
Metalloprotein Research Group, Division of Biochemistry, Department of Chemistry, University of Natural Resources and Applied Life Sciences, Muthgasse 18 A-1190 Vienna, Austria
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Abstract

Phytopathogenic fungi such as the rice blast fungus Magnaporthe grisea are unique in having two catalase/peroxidase (KatG) paralogues located either intracellularly (KatG1) or extracellularly (KatG2). The coding genes have recently been shown to derive from a lateral gene transfer from a (proteo)bacterial genome followed by gene duplication and diversification. Here we demonstrate that KatG1 is expressed constitutively in M. grisea. It is the first eukaryotic catalase/peroxidase to be expressed heterologously in Escherichia coli in high amounts, with high purity and with almost 100% haem occupancy. Recombinant MagKatG1 is an acidic, mainly homodimeric, oxidoreductase with a predominant five-co-ordinated high-spin haem b. At 25 °C and pH 7.0, the E0′ (standard reduction potential) of the Fe(III)/Fe(II) couple was found to be −186±10 mV. It bound cyanide monophasically with an apparent bimolecular rate constant of (9.0±0.4)×105 M−1·s−1 at pH 7.0 and at 25 °C and with a Kd value of 1.5 μM. Its predominantly catalase activity was characterized by a pH optimum at 6.0 and kcat and Km values of 7010 s−1 and 4.8 mM respectively. In addition, it acts as a versatile peroxidase with a pH optimum in the range 5.0–5.5 using both one-electron [2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) o-dianisidine, pyrogallol or guaiacol] and two-electron (Br−, I− or ethanol) donors. Structure–function relationships are discussed with respect to data reported for prokaryotic KatGs, as is the physiological role of MagKatG1. Phylogenetic analysis suggests that (intracellular) MagKatG1 can be regarded as a typical representative for catalase/peroxidase of both phytopathogenic and saprotrophic fungi.

  • catalase
  • KatG
  • Magnaporthe grisea
  • oxidative stress
  • peroxidase
  • phytopathogenic fungus

Abbreviations: ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); E0′, standard reduction potential; ECD, electronic CD; His6, hexahistidine; HS, high-spin; 3D, three-dimensional; KatG, catalase/peroxidase; katG1, gene encoding MagKatG1; katG2, gene encoding MagKatG2; LGT, lateral gene transfer; LS, low-spin; MagKatG1, intracellular KatG from Magnaporthe grisea; MagKatG2, secreted KatG from M. grisea; MCAC, metal chelate affinity chromatography; NcKatG2, KatG from Neurospora crassa; ORF, open reading frame; OTTLE, optically transparent thin-layer (spectro)electrochemical; RT–PCR, reverse-transcription PCR; SynKatG, KatG from Synechocystis PCC6803

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March 2009

Volume: 418 Issue: 2

Biochemical Journal: 418 (2)
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Intracellular catalase/peroxidase from the phytopathogenic rice blast fungus Magnaporthe grisea: expression analysis and biochemical characterization of the recombinant protein
Marcel Zamocky, Paul G. Furtmüller, Marzia Bellei, Gianantonio Battistuzzi, Johannes Stadlmann, Jutta Vlasits, Christian Obinger
Biochemical Journal Mar 2009, 418 (2) 443-451; DOI: 10.1042/BJ20081478
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Intracellular catalase/peroxidase from the phytopathogenic rice blast fungus Magnaporthe grisea: expression analysis and biochemical characterization of the recombinant protein
Marcel Zamocky, Paul G. Furtmüller, Marzia Bellei, Gianantonio Battistuzzi, Johannes Stadlmann, Jutta Vlasits, Christian Obinger
Biochemical Journal Mar 2009, 418 (2) 443-451; DOI: 10.1042/BJ20081478

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Keywords

catalase
KatG
Magnaporthe grisea
oxidative stress
peroxidase
phytopathogenic fungus

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