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 Kd 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.

High Conformational Stability of Secreted Eukaryotic Catalase-peroxidases

“…Heterologous Expression and Purification of MagKatG2-Recombinant catalase-peroxidases (wild-type MagKatG2 and single and double mutants) were expressed in Escherichia coli strain BL21 Star TM (DE3) (Invitrogen) and purified to homogeneity as described previously (15). Briefly, the inducible expression with 0.5% lactose overnight at 16°C yielded on average 30 mg of soluble KatG2 per liter of M9ZL medium supplemented with hemin (75 M final concentration).…”

“…His-tagged protein (with added protease inhibitors PMSF and leupeptin) was eluted with a linear gradient of 0 -500 mM imidazole in 50 mM sodium phosphate, pH 7.5, 500 mM NaCl. Subsequent purification by hydroxyapatite column separated the high-spin heme fractions (15), which were pooled and concentrated with a Centriprep 50K (Millipore, for 30 min, 1,500 ϫ g) and further purified by size exclusion chromatography (15). Dimeric protein fractions were concentrated up to 11 mg/ml in Centriprep 50K in 5 mM phosphate buffer, pH 7.5, and used for crystallization studies.…”

“…To date, only a small number of biochemical studies of eukaryotic fungal catalase-peroxidases have been reported, mainly focused on the intracellular KatG1 from Neurospora crassa (NcKatG1, originally named CAT-2) (22) and the rice blast fungus Magnaporthe grisea (MagKatG1) (14). Recently, the first biochemical data about the secreted KatG2 from M. grisea (MagKatG2) have been reported (15). Here we extend the characterization of MagKatG2 with the report of its crystal structure, the first of an eukaryotic catalase-peroxidase, at 1.55 Å resolution.…”

“…1a). Currently, the nonsecreted or intracellular KatG1s are found in nonpathogenic and pathogenic fungi (13,14), whereas the extracellular KatG2s or secreted KatG2s are found only in pathogenic fungi (15), suggesting that they are an important defense against the oxidative burst produced by infected plants (16 -18).…”

Redundant Catalases Detoxify Phagocyte Reactive Oxygen and Facilitate Histoplasma capsulatum Pathogenesis

“…In the fungal kingdom, nonenzymatic strategies include production of melanin to absorb ROS or reductants such as thioredoxin to mend oxidative protein damage (8)(9)(10). While most organisms express a variety of antioxidant factors to cope with metabolically derived intracellular ROS, microbial pathogens must employ additional, often extracellular, factors to defend against ROS produced by host cells and the host environment (11)(12)(13)(14)(15)(16)(17)(18). For microbial pathogens that infect ROS-producing phagocytic cells, the ability to defend against phagocyte-derived ROS becomes even more imperative.…”