All phytopathogenic fungi have two catalaseeperoxidase 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 heterologouslywith almost 100% heme occupancy and comprehensively investigated by using a broad set of methods including UVeVis, 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 cyanidewas 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.

Eukaryotic Extracellular Catalase-Peroxidase from Magnaporthe grisea – Biophysical/Chemical Characterization of the First Representative from a Novel Phytopathogenic KatG Group / M., Zámocký; E., Droghetti; Bellei, Marzia; B., Gasselhuber; M., Pabst; P. G., Furtmüller; Battistuzzi, Gianantonio; G., Smulevich; C., Obinger. - In: BIOCHIMIE. - ISSN 0300-9084. - STAMPA. - 94(2012), pp. 673-683. [10.1016/j.biochi.2011.09.020]

Eukaryotic Extracellular Catalase-Peroxidase from Magnaporthe grisea – Biophysical/Chemical Characterization of the First Representative from a Novel Phytopathogenic KatG Group

BELLEI, Marzia;BATTISTUZZI, Gianantonio;
2012

Abstract

All phytopathogenic fungi have two catalaseeperoxidase 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 heterologouslywith almost 100% heme occupancy and comprehensively investigated by using a broad set of methods including UVeVis, 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 cyanidewas 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.
94
673
683
Eukaryotic Extracellular Catalase-Peroxidase from Magnaporthe grisea – Biophysical/Chemical Characterization of the First Representative from a Novel Phytopathogenic KatG Group / M., Zámocký; E., Droghetti; Bellei, Marzia; B., Gasselhuber; M., Pabst; P. G., Furtmüller; Battistuzzi, Gianantonio; G., Smulevich; C., Obinger. - In: BIOCHIMIE. - ISSN 0300-9084. - STAMPA. - 94(2012), pp. 673-683. [10.1016/j.biochi.2011.09.020]
M., Zámocký; E., Droghetti; Bellei, Marzia; B., Gasselhuber; M., Pabst; P. G., Furtmüller; Battistuzzi, Gianantonio; G., Smulevich; C., Obinger
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