TY - JOUR
T1 - Eukaryotic extracellular catalase-peroxidase from Magnaporthe grisea - Biophysical/chemical characterization of the first representative from a novel phytopathogenic KatG group
AU - Zámocký, Marcel
AU - Droghetti, Enrica
AU - Bellei, Marzia
AU - Gasselhuber, Bernhard
AU - Pabst, Martin
AU - Furtmüller, Paul G.
AU - Battistuzzi, Gianantonio
AU - Smulevich, Giulietta
AU - Obinger, Christian
PY - 2012
Y1 - 2012
N2 - 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 K d 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.
AB - 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 K d 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.
KW - Extracellular catalase-peroxidase
KW - Oxidative stress
KW - Peroxidases-catalase superfamily
KW - Phytopathogen
KW - Reduction potential
KW - Resonance Raman spectroscopy
UR - http://www.scopus.com/inward/record.url?scp=84857060979&partnerID=8YFLogxK
U2 - 10.1016/j.biochi.2011.09.020
DO - 10.1016/j.biochi.2011.09.020
M3 - Article
C2 - 21971530
AN - SCOPUS:84857060979
SN - 0300-9084
VL - 94
SP - 673
EP - 683
JO - Biochimie
JF - Biochimie
IS - 3
ER -