Intracellular catalase/peroxidase from the phytopathogenic rice blast fungus Magnaporthe grisea: expression analysis and biochemical characterization of the recombinant protein

Phytopathogenic fungi such as the rice blast fungus Magnaporthegrisea are unique in having two catalase/peroxidase (KatG)paralogues located either intracellularly (KatG1) or extracellularly(KatG2). The coding genes have recently been shownto derive from a lateral gene transfer from a (proteo)bacterialgenome followed by gene duplication and diversification. Here wedemonstrate thatKatG1 is expressed constitutively in M. grisea. Itis the first eukaryotic catalase/peroxidase to be expressed heterologouslyin Escherichia coli in high amounts, with high purity andwith almost 100% haem occupancy. Recombinant MagKatG1is an acidic, mainly homodimeric, oxidoreductase with a predominantfive-co-ordinated high-spin haem b. At 25◦C andpH 7.0, the E0 (standard reduction potential) of the Fe(III)/Fe(II)couple was found to be −186+−10 mV. It bound cyanidemonophasically with an apparent bimolecular rate constant of(9.0+−0.4)×105 M−1 · s−1 at pH 7.0 and at 25◦C and with aKd value of 1.5 μM. Its predominantly catalase activity wascharacterized by a pH optimum at 6.0 and kcat and Km valuesof 7010 s−1 and 4.8 mM respectively. In addition, it acts as aversatile peroxidase with a pH optimum in the range 5.0–5.5using both one-electron [2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) o-dianisidine, pyrogallol or guaiacol] andtwo-electron (Br−, I− or ethanol) donors. Structure–functionrelationships are discussed with respect to data reported forprokaryotic KatGs, as is the physiological role of MagKatG1.Phylogenetic analysis suggests that (intracellular) MagKatG1 canbe regarded as a typical representative for catalase/peroxidase ofboth phytopathogenic and saprotrophic fungi.