title: Crystallization and preliminary structural analysis of catalase A from Saccharomyces cerevisiae.
abstract: Yeast peroxisomal catalase A, obtained at high yields by over expression of the C-terminally modified gene from a 2 mu-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 A resolution and belong to the hexagonal space group P6(1)22 with unit cell parameters a = b = 184.3 A 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 vitalae, 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.
authors.affiliation: C.I.D. (CSIC), Barcelona, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Berthet
authors.forename: L M
authors.initials: LM
authors.lastname: Nykyri
authors.forename: J
authors.initials: J
authors.lastname: Bravo
authors.forename: M J
authors.initials: MJ
authors.lastname: Mate
authors.forename: C
authors.initials: C
authors.lastname: Berthet-Colominas
authors.forename: P M
authors.initials: PM
authors.lastname: Alzari
authors.forename: F
authors.initials: F
authors.lastname: Koller
authors.forename: I
authors.initials: I
authors.lastname: Fita
mesh_terms: D002374:Catalase / Q000737:chemistry*; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D002374:Catalase