Purification, crystallization and phase determination of the DR1998 haembcatalase fromDeinococcus radiodurans

Abstract

The protective mechanisms of Deinococcus radiodurans against primary reactive oxygen species involve nonenzymatic scavengers and a powerful enzymatic antioxidant system including catalases, peroxidases and superoxide dismutases that prevents oxidative damage. Catalase is an enzyme that is responsible for the conversion of H 2 O 2 to O 2 and H 2 O, protecting the organism from the oxidative effect of H 2 O 2 . This study reports the purification and crystallization of the DR1998 catalase from D. radiodurans. The crystals diffracted to 2.6 Å resolution and belonged to space group C222 1 , with unit-cell parameters a = 97.33, b = 311.88, c = 145.63 Å , suggesting that they contain four molecules per asymmetric unit. The initial phases were determined by molecular replacement and the obtained solution shows the typical catalase quaternary structure. A preliminary model of the protein structure has been built and refinement is currently in progress.

Structure of the monofunctional heme catalase DR1998 from Deinococcus radiodurans

“…[36]. Structural refinement proceeded smoothly, and led to R work /R free of 0.2224/0.2436 and a final R factor of 0.2363, with all available diffraction data.…”

“…(chains A, B, C, and D) are arranged as two dimers, which, by crystallographic symmetry (chains A 0 , B 0 , C 0 , and D 0 ), form two typical catalase tetramers: tetramer 1, formed by chains A, B, A 0 , and B 0 ; and tetramer 2, formed by chains C, D, C 0 , and D 0 [36]. Superimposition of the independent molecules showed a low rmsd of~0.…”

“…The residue numbering is from DR1998; the corresponding residues for the other catalases are presented in Table 2 X-ray diffraction, crystal structure determination, and refinement Diffraction data were measured at 100 K at beamline ID29 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) by the use of X-rays with a wavelength of 0.826 A and a PILATUS 6M detector. Data collection, processing, crystallographic characterization and structure determination have been described previously [36], and are summarized in Table 1.…”

“…Protein purification and crystallization DR1998 was obtained from D. radiodurans as previously described [36]. A 300 L culture of D. radiodurans was grown at 30°C to an attenuance at 600 nm of 2.0, in M53 medium containing 0.5% (w/v) yeast extract, 0.5% (w/v) glucose, 0.5% (w/v) NaCl, and 1% (w/v) casein.…”

“…The final fraction containing DR1998 eluted at 600 mM sodium acetate. SDS/PAGE was used to confirm the purity of the protein, which was then used in crystallization trials as described in [36]. Crystals were obtained at room temperature with the hanging-drop vapor diffusion method, by Amino acids are represented as sticks, with carbon atoms in gray for DR1998, yellow for beef liver catalase, and green for Es.…”