Computational protocol: Eukaryotic extracellular catalase–peroxidase from Magnaporthe grisea – Biophysical/chemical characterization of the first representative from a novel phytopathogenic KatG group

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Protocol publication

[…] UV–visible spectra of MagKatG2 were routinely recorded with a Hitachi U-3900 or a double-beam Cary 5 (Varian, Palo Alto, CA, US) spectrophotometer in the range 800–200 nm at 25 °C. The molar absorption coefficient of the ferric, high-spin form at Soret band maximum (102 600 M−1 cm−1) was determined from the slope of the linear plot of absorbance at 404 nm versus protein concentration determined by the Bradford method. Heme occupancy was determined with the pyridine–hemochrome method . The absorption coefficient of pure MagKatG2 at 280 nm was calculated to be ɛ280nm = 147 800 M−1 cm−1 using the protein sequence (Expasy server).Low-spin cyanide complexes were formed upon mixing of 5.5 μM of purified protein with increasing concentrations of sodium cyanide in 5 mM phosphate buffer, pH 7.5. Apparent bimolecular rate constant of complex formation was determined by conventional stopped-flow spectroscopy (SX-18MV from Applied Photophysics) as described previously . Ferrous MagKatG2 was produced by the addition of freshly prepared anaerobic solution of 2 mM sodium dithionite.Electronic circular dichroism (ECD) spectra were recorded on Chirascan equipped with a thermostatically controlled cell holder (Applied Photophysics, Leatherhead, U.K.). For recording far-UV spectra (260–190 nm), the quartz cuvette had a pathlength of 1 mm. The spectral bandwidth was 5 nm, step size 1 nm, and the scan time was 120 s (scan period 25 μs × 20,000 counts). Deconvolution of ECD spectra was performed with DichroWeb software at http://dichroweb.cryst.bbk.ac.uk based on the SP175 database . For comparison, secondary structure elements were predicted with PSIPRED server and a homology model of MagKatG2 was constructed using the SWISS-MODEL workspace . […]

Pipeline specifications

Software tools DICHROWEB, PSIPRED
Databases ExPASy
Application Circular dichroism spectroscopy
Organisms Magnaporthe grisea, Magnaporthe oryzae
Chemicals Heme