Intracellular catalase-peroxidase from the phytopathogenic fungus Magnaporthe grisea: expression analysis and biochemical characterization of the recombinant protein
Résumé
Phytopathogenic fungi like Magnaporthe grisea are unique in having two catalase-peroxidase (KatG) paralogs 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 Magnaporthe grisea KatG1 is expressed constitutively in rice blast fungus. It is the first eukaryotic catalase-peroxidase to be expressed heterologously in E. coli in high amount and purity with almost 100% haem occupancy. Recombinant MagKatG1 is an acidic, mainly homodimeric oxidoreductase with a predominant five-coordinated high-spin haem b. At 25 °C and pH 7.0, the reduction potential of the Fe(III)/Fe(II) couple is found to be (-186 ± 10) mV. It binds cyanide monophasically with an apparent bimolecular rate constant of (9.0 ± 0.4) x 105 M-1 s-1 (pH 7.0 and 25 °C) and a KD value of 1.5 µM. Its predominantly catalase activity is characterized by a pH optimum at pH 6.0 and kcat and KM values of 7010 s-1 and 4.8 mM, respectively. In addition it acts as versatile peroxidase with pH optimum in the range pH 5.0 - 5.5 using both one- (ABTS, o-dianisidine, pyrogallol, guaiacol) and two-electron donors (Br-, I-, ethanol). 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 typical representative for catalase-peroxidase of both phytopathogenic and saprotrophic fungi.
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