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The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer. Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at approximately 2.0-A resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel. The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure. It is hypothesized that the slightly larger H(2)O(2) passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-A resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel. Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222-point group symmetry of the native enzyme. An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism. The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled.

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Haem-containing catalases are homotetrameric molecules that degrade hydrogen peroxide. Phylogenetically, the haem-containing catalases can be grouped into three main lines or clades. The crystal structures of seven catalases have been determined, all from clades II and III. In order to obtain a structure of an enzyme from clade I, which includes all plant, algae and some bacterial enzymes, two bacterial catalases, CatF from Pseudomonas syringae and Kat from Listeria seeligeri, have been crystallized by the hanging-drop vapour-diffusion technique, using PEG and ammonium sulfate as precipitants, respectively. Crystals of P. syringae CatF, with a plate-like morphology, belong to the monoclinic space group P2(1), with unit-cell parameters a = 60.6, b = 153.9, c = 109.2 A, beta = 102.8 degrees. From these crystals a diffraction data set to 1.8 A resolution with 98% completeness was collected using synchrotron radiation. Crystals of L. seeligeri Kat, with a well developed bipyramidal morphology, belong to space group I222 (or I2(1)2(1)2(1)), with unit-cell parameters a = 74.4, b = 121.3, c = 368.5 A. These crystals diffracted beyond 2.2 A resolution when using synchrotron radiation, but presented anisotropic diffraction, with the weakest direction perpendicular to the long c axis.

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BACKGROUND: The biosynthesis of key metabolic components is of major interest to biologists. Studies of de novo purine synthesis are aimed at obtaining a deeper understanding of this central pathway and the development of effective chemotherapeutic agents. Phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) synthase catalyses the seventh step out of ten in the biosynthesis of purine nucleotides. To date, only one structure of an enzyme involved in purine biosynthesis has been reported: adenylosuccinate synthetase, which catalyses the first committed step in the synthesis of AMP from IMP. RESULTS: We report the first three-dimensional structure of a SAICAR synthase, from Saccharomyces cerevisiae. It is a monomer with three domains. The first two domains consist of antiparallel beta sheets and the third is composed of two alpha helices. There is a long deep cleft made up of residues from all three domains. Comparison of SAICAR synthases by alignment of their sequences reveals a number of conserved residues, mostly located in the cleft. The presence of two sulphate ions bound in the cleft, the structure of SAICAR synthase in complex with ATP and a comparison of this structure with that of other ATP-dependent proteins point to the interdomain cleft as the location of the active site. CONCLUSIONS: The topology of the first domain of SAICAR synthase resembles that of the N-terminal domain of proteins belonging to the cyclic AMP-dependent protein kinase family. The fold of the second domain is similar to that of members of the D-alanine:D-alanine ligase family. Together these enzymes form a new superfamily of mononucleotide-binding domains. There appears to be no other enzyme, however, which is composed of the same combination of three domains, with the individual topologies found in SAICAR synthase.

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A heme d prosthetic group with the configuration of a cis-hydroxychlorin gamma-spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical. For both catalases the heme d is rotated 180 degrees about the axis defined by the alpha-gamma-meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase. Only six residues in the heme pocket, preserved in P. vitale and HPII, differ from those found in the bovine catalase. In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme. These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction.

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Crystals of phosphoribosylaminoimidazolesuccinocarboxamide synthase (EC 6.3.2.6) from the yeast Saccharomyces cerevisiae were grown by the vapor diffusion hanging-drop technique, using ammonium sulfate as the precipitant. The crystals had dimensions up to 1.2 mm. X-ray diffraction experiments indicated a space group of P2(1)2(1)2(1) and unit cell parameters of a = 62.3 A, b = 63.5 A and c = 80.9 A, with one molecule in the asymmetric unit. Native data have been collected to 2.5 A resolution.

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The three-dimensional crystal structure of catalase from Micrococcus lysodeikticus has been solved by multiple isomorphous replacement and refined at 1.5 A resolution. The subunit of the tetrameric molecule of 222 symmetry consists of a single polypeptide chain of about 500 amino acid residues and one haem group. The crystals belong to space group P4(2)2(1)2 with unit cell parameters a = b = 106.7 A, c = 106.3 A, and there is one subunit of the tetramer per asymmetric unit. The amino acid sequence has been tentatively determined by computer graphics model building and comparison with the known three-dimensional structure of beef liver catalase and sequences of several other catalases. The atomic model has been refined by Hendrickson and Konnert's least-squares minimisation against 94,315 reflections between 8 A and 1.5 A. The final model consists of 3,977 non-hydrogen atoms of the protein and haem group, 426 water molecules and one sulphate ion. The secondary and tertiary structures of the bacterial catalase have been analyzed and a comparison with the structure of beef liver catalase has been made.

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The three-dimensional structure analysis of crystalline fungal catalase from Penicillium vitale has been extended to 2.0 A resolution. The crystals belong to space group P3(1)21, with the unit cell parameters of a = b = 144.4 A and c = 133.8 A. The asymmetric unit contains half a tetrameric molecule of 222 symmetry. Each subunit is a single polypeptide chain of approximately 670 amino acid residues and binds one heme group. The amino acid sequence has been tentatively determined by computer graphics model building (using the FRODO system) and comparison with the known sequence of beef liver catalase. The atomic model has been refined by the Hendrickson & Konnert (1981) restrained least-squares program against 68,000 reflections between 5 A and 2 A resolution. The final R-factor is 0.31 after 24 refinement cycles. The secondary and tertiary structure of the catalase has been analyzed.

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The structures of Penicillium vitale and beef liver catalase have been determined to atomic resolution. Both catalases are tetrameric proteins with deeply buried heme groups. The amino acid sequence of beef liver catalase is known and contains (at least) 506 amino acid residues. Although the sequence of P. vitale catalase has not yet been determined chemically, 670 residues have been built into the 2 A resolution electron density map and have been given tentative assignments. A large portion of each catalase molecule (91% of residues in beef liver catalase and 68% of residues in P. vitale catalase) shows structural homology. The root-mean-square deviation between 458 equivalenced C alpha atoms is 1.17 A. The dissimilar parts include a small fragment of the N-terminal arm and an additional "flavodoxin-like" domain at the carboxy end of the polypeptide chain of P. vitale catalase. In contrast, beef liver catalase contains one bound NADP molecule per subunit in a position equivalent to the chain region, leading to the flavodoxin-like domain, of P. vitale catalase. The position and orientation of the buried heme group in the two catalases, relative to the mutually perpendicular molecular dyad axes, are identical within experimental error. A mostly hydrophobic channel leads to the buried heme group. The surface opening to the channel differs due to the different disposition of the amino-terminal arm and the presence of the additional flavodoxin-like domain in P. vitale catalase. Possible functional implications of these comparisons are discussed.

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