Crystallization and preliminary structural analysis of catalase A from Saccharomyces cerevisiae

Abstract

Abstract:Yeast peroxisomal catalase A, obtained at high yields by over expression of the C-terminally modified gene from a 2p-plasmid, has been crystallized in a form suitable for high resolution X-ray diffraction studies. Brownish crystals with bipyrimidal morphology and reaching ca. 0.8 mm in size were produced by the hanging drop method using ammonium sulphate as precipitant. These crystals diffract better than 2.0 8, resolution and belong to the hexagonal space group P6122 with unit cell parameters a = b = 184.3 8, and c = 305.5 A. An X-ray data set with 76% completeness at 3.2 A resolution was collected in a rotating anode generator using mirrors to improve the collimation of the beam. An initial solution was obtained by molecular replacement only when using a beef liver catalase tetramer model in which fragments with no sequence homology had been omitted, about 150 residues per subunit. In the structure found a single molecule of catalase A (a tetramer with accurate 222 molecular symmetry) is located in the asymmetric unit of the crystal with an estimated solvent content of about 61%. The preliminary analysis of the structure confirms the absence of a carboxy terminal domain as the one found in the catalase from Penicillium vitulue, the only other fungal catalase structure available. The NADPH binding site appears to be involved in crystal contacts, suggesting that heterogeneity in the occupancy of the nucleotide can be a major difficulty during crystallization.

Molecular cloning, characterization and gene expression of an antioxidant enzyme catalase (MrCat) from Macrobrachium rosenbergii

“…As reported by Li et al [34] the predicted tertiary structure of MrCat, His 71 was to be neighboring b2, Asn 145 was located on b4', and Tyr 357 was speculated to be in a10. These spatial locations of the catalytic residues were exactly consistent with that in catalase beef [66], human [30] and C. farreri [34] and catalase A from brewer's yeast Saccharomyces cerevisiae [67], and infer that MrCat probably performed its function in the same mechanism as human catalase.…”

Diversity of structures and properties among catalases

“…The previously reported seven catalase structures include the small subunit clade 3 enzymes from BLC [20,21], Micrococcus lysodeiticus (MLC) [22], PMC [23], Saccharomyces cerevisiae (CATA) [24,25], and human erythrocytes (HEC) [26] and the large subunit clade 2 enzymes from Penicillium vitale (PVC) [27,28] and E. coli (HPII) [29,30]. The two new structures include Pseudomonas syringae (CatF) [31,32], which provides the first look at a clade 1 catalase, and the Helicobacter pylori catalase (HPC) with and without formic acid bound [P. C. Loewen et al, unpublished].…”

Structure of catalase HPII fromEscherichia coli at 1.9 � resolution

“…To date, the structures of six heme-containing catalases have been determined including enzymes from bovine liver (BLC), 1, 2 the fungus Penicillium vitale (PVC), 3, 4 the yeast Saccharomyces cerevisiae (CatA), 5 …”