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 Magnaporthe grisea are unique in having two catalase/peroxidase (KatG) paralogues 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 KatG1 is expressed constitutively in M. grisea. It is the first eukaryotic catalase/peroxidase to be expressed heterologously in Escherichia coli in high amounts, with high purity and with almost 100% haem occupancy. Recombinant MagKatG1 is an acidic, mainly homodimeric, oxidoreductase with a predominant five-co-ordinated high-spin haem b. At 25 degrees C and pH 7.0, the E(0)' (standard reduction potential) of the Fe(III)/Fe(II) couple was found to be -186+/-10 mV. It bound cyanide monophasically with an apparent bimolecular rate constant of (9.0+/-0.4)x10(5) M(-1).s(-1) at pH 7.0 and at 25 degrees C and with a K(d) value of 1.5 muM. Its predominantly catalase activity was characterized by a pH optimum at 6.0 and k(cat) and K(m) values of 7010 s(-1) and 4.8 mM respectively. In addition, it acts as a versatile peroxidase with a pH optimum in the range 5.0-5.5 using both one-electron [2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) o-dianisidine, pyrogallol or guaiacol] and two-electron (Br(-), I(-) or ethanol) donors. 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 a typical representative for catalase/peroxidase of both phytopathogenic and saprotrophic fungi.
Zamocky
Marcel
M
Metalloprotein Research Group, Division of Biochemistry, Department of Chemistry, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria. marcel.zamocky@boku.ac.at
Furtmüller
Paul G
PG
Bellei
Marzia
M
Battistuzzi
Gianantonio
G
Stadlmann
Johannes
J
Vlasits
Jutta
J
Obinger
Christian
C
D002374:Catalase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D003001:Cloning, Molecular; D003486:Cyanides / Q000378:metabolism; D004795:Enzyme Stability; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000201:enzymology; D020082:Magnaporthe / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D008958:Models, Molecular; D012275:Oryza / Q000469:parasitology*; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D003486:Cyanides; D011994:Recombinant Proteins; D010544:Peroxidases; D002374:Catalase