title: Eukaryotic extracellular catalase-peroxidase from Magnaporthe grisea - Biophysical/chemical characterization of the first representative from a novel phytopathogenic KatG group.
abstract: 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.
authors.affiliation: Division of Biochemistry, Department of Chemistry, Vienna Institute of Biotechnology at BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria. marcel.zamocky@boku.ac.at
authors.forename: Marcel
authors.initials: M
authors.lastname: Zámocký
authors.forename: Enrica
authors.initials: E
authors.lastname: Droghetti
authors.forename: Marzia
authors.initials: M
authors.lastname: Bellei
authors.forename: Bernhard
authors.initials: B
authors.lastname: Gasselhuber
authors.forename: Martin
authors.initials: M
authors.lastname: Pabst
authors.forename: Paul G
authors.initials: PG
authors.lastname: Furtmüller
authors.forename: Gianantonio
authors.initials: G
authors.lastname: Battistuzzi
authors.forename: Giulietta
authors.initials: G
authors.lastname: Smulevich
authors.forename: Christian
authors.initials: C
authors.lastname: Obinger
mesh_terms: D002374:Catalase / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D020082:Magnaporthe / Q000201:enzymology* / Q000378:metabolism; D018384:Oxidative Stress; D010544:Peroxidases / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D010802:Phylogeny; D013059:Spectrum Analysis, Raman
chemical_list: D005656:Fungal Proteins; D010544:Peroxidases; D002374:Catalase