Eukaryotic extracellular catalase–peroxidase from Magnaporthe grisea – Biophysical/chemical characterization of the first representative from a novel phytopathogenic KatG group

All phytopathogenic fungi have two catalase–peroxidase paralogues located either intracellularly (KatG1) or extracellularly (KatG2). Here, for the first time a secreted bifunctional, homodimeric catalase–peroxidase (KatG2 from the rice blast fungus Magnaporthe grisea) has been produced heterologousl...

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Bibliographic Details
Published in:Biochimie 2012-03, Vol.94 (3), p.673-683
Main Authors: Zámocký, Marcel, Droghetti, Enrica, Bellei, Marzia, Gasselhuber, Bernhard, Pabst, Martin, Furtmüller, Paul G., Battistuzzi, Gianantonio, Smulevich, Giulietta, Obinger, Christian
Format: Article
Language:English
Subjects:
Adducts
Catalase
Catalase - chemistry
Catalase - classification
Catalase - metabolism
Cyanide
Data processing
Extracellular catalase–peroxidase
Fungal Proteins - chemistry
Fungal Proteins - metabolism
Heme
Histidine
Host-pathogen interactions
Magnaporthe - enzymology
Magnaporthe - metabolism
Magnaporthe grisea
Mass spectroscopy
Oryza sativa
Oxidative Stress
Peroxidase
Peroxidases - chemistry
Peroxidases - classification
Peroxidases - metabolism
Peroxidases–catalase superfamily
pH effects
Phylogeny
Phytopathogen
Phytopathogenic fungi
Raman spectroscopy
Reduction potential
Research Paper
Resonance Raman spectroscopy
rice blast
Spectroscopy
Spectrum Analysis, Raman
Structure-function relationships
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Summary:All phytopathogenic fungi have two catalase–peroxidase paralogues located either intracellularly (KatG1) or extracellularly (KatG2). Here, for the first time a secreted bifunctional, homodimeric catalase–peroxidase (KatG2 from the rice blast fungus Magnaporthe grisea) has been produced heterologously with almost 100% heme occupancy and comprehensively investigated by using a broad set of methods including UV–Vis, ECD and resonance Raman spectroscopy (RR), thin-layer spectroelectrochemistry, mass spectrometry, steady-state & presteady-state spectroscopy. RR spectroscopy reveals that MagKatG2 shows a unique mixed-spin state, non-planar heme b, and a proximal histidine with pronounced imidazolate character. At pH 7.0 and 25 °C, the standard reduction potential E°′ of the Fe(III)/Fe(II) couple for the high-spin native protein was found to fall in the range typical for the KatG family. Binding of cyanide was relatively slow at pH 7.0 and 25 °C and with a Kd value significantly higher than for the intracellular counterpart. Demonstrated by mass spectrometry MagKatG2 has the typical Trp118-Tyr251-Met277 adduct that is essential for its predominantly catalase activity at the unique acidic pH optimum. In addition, MagKatG2 acts as a versatile peroxidase using both one- and two-electron donors. Based on these data, structure–function relationships of extracellular eukaryotic KatGs are discussed with respect to intracellular KatGs and possible role(s) in host–pathogen interaction. [Display omitted] ► Bifunctional KatG2s secreted by phytopathogenic fungi protect them from oxidative stress. ► MagKatG2 from the rice blast fungus shows a mixed-spin state & non-planar heme b. ► Bifunctional MagKatG2 acts as versatile peroxidase with one- and two-electron donors. ► Phytopathogenic secretory catalase–peroxidases are interesting targets for pest control.
ISSN:0300-9084
1638-6183
DOI:10.1016/j.biochi.2011.09.020