A. I. Grebenko

Three-dimensional structure of catalase from Micrococcus lysodeikticus at 1.5 Å resolution

The three‐dimensional crystal structure of catalase from Micrococcus lysodeikticus has been solved by multiple isomorphous replacement and refined at 1.5 Å 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 P42212 with unit cell parameters a = b = 106,7 Å, c = 106,3 Å, 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 Konnerts least‐squares minimisation against 94,315 reflections between 8 Å and 1.5 Å. The final model consists or 3,977 non‐hydrogen atoms of the protein and haem group, 426 water molecules and ones sulphate ion. The secondary and tertiary sructures of the bacterial catalase have been analyzed and a comparison with the structure of beef liver catalase has been made.

Structure of the Heme d of Penicillium vitale and Escherichia coli Catalases

A heme d prosthetic group with the configuration of a cis-hydroxychlorin -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 α--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.

The structures of Micrococcus lysodeikticus catalase, its ferryl intermediate (compound II) and NADPH complex

The crystal structure of the bacterial catalase from Micrococcus lysodeikticus has been refined using the gene-derived sequence both at 0.88 A resolution using data recorded at 110 K and at 1.5 A resolution with room-temperature data. The atomic resolution structure has been refined with individual anisotropic atomic thermal parameters. This has revealed the geometry of the haem and surrounding protein, including many of the H atoms, with unprecedented accuracy and has characterized functionally important hydrogen-bond interactions in the active site. The positions of the H atoms are consistent with the enzymatic mechanism previously suggested for beef liver catalase. The structure reveals that a 25 A long channel leading to the haem is filled by partially occupied water molecules, suggesting an inherent facile access to the active site. In addition, the structures of the ferryl intermediate of the catalase, the so-called compound II, at 1.96 A resolution and the catalase complex with NADPH at 1.83 A resolution have been determined. Comparison of compound II and the resting state of the enzyme shows that the binding of the O atom to the iron (bond length 1.87 A) is associated with increased haem bending and is accompanied by a distal movement of the iron and the side chain of the proximal tyrosine. Finally, the structure of the NADPH complex shows that the cofactor is bound to the molecule in an equivalent position to that found in beef liver catalase, but that only the adenine part of NADPH is visible in the present structure.

The structure of SAICAR synthase: an enzyme in the de novo pathway of purine nucleotide biosynthesis.

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.

X-ray diffraction study of Penicillium Vitale catalase in the complex with aminotriazole

The three-dimensional structure of the enzyme catalase from Penicillium vitale in a complex with the inhibitor aminotriazole was solved and refined by protein X-ray crystallography methods. An analysis of the three-dimensional structure of the complex showed that the inhibition of the enzyme occurs as a result of the covalent binding of aminotriazole to the amino-acid residue His64 in the active site of the enzyme. An investigation of the three-dimensional structure of the complex resulted in the amino-acid residues being more precisely identified. The binding sites of saccharide residues and calcium ions in the protein molecule were found.

X-ray diffraction study of the complex of the enzyme SAICAR synthase with substrate analogues

The three-dimensional structure of the complex of the enzyme SAICAR synthase with analogues of natural substrates, namely, phosphoribosylaminoimidazolecarboxamide (AICAR) and succinic acid, was determined and refined by methods of protein crystallography. Two AICAR-binding sites were revealed in the protein molecule. One of these sites is located in the active center of the enzyme in the vicinity of the ATP-binding site, which has been found in the complex of SAICAR synthase with ATP that had been studied earlier. The second AICAR-binding site is located at the periphery of the protein molecule and coincides with the additional ATP-binding site present in the complexes studied earlier. The binding site of succinic acid was revealed in the active center of the enzyme in the vicinity of the AICAR molecule. The electron density distribution for the AICAR molecule in the active center is indicative of the possible lability of the atomic group of the adenine base.

X-ray diffraction study of the complexes of SAICAR synthase with adenosinetriphosphate

The three-dimensional structures of two enzyme-substrate complexes of SAICAR synthase from the yeast Saccharomyces cerevisiae with adenosinetriphosphate (ATP) prepared under different conditions were studied by X-ray diffraction analysis and then refined. An enzyme molecule was shown to contain two binding sites of ATP. One of these sites is located in the central cavity of the enzyme molecule and apparently binds the ATP molecule directly involved in the enzymatic reaction. In the complexes, the phosphate groups of ATP occupying this site adopt different conformations depending on the Mg2+ concentration. The functional role of the second binding site located at a distance of approximately 15 Å from the first site away from the central enzyme cavity has not been understood as yet. It might be that the second site perform the regulatory role in enzyme functioning.

X-ray diffraction study of the complex of the enzyme SAICAR synthase with the reaction product

The three-dimensional structure of the complex of the enzyme SAICAR synthase with the product of the enzymatic reaction, SAICAR, was solved and refined by methods of protein crystallography. The SAICAR-binding site in the active site of the enzyme was found. The amino-acid residues providing the binding of the reaction product with the protein were revealed. These residues were compared with those involved in the substrate binding in the complex with AICAR and succinic acid studied earlier.

Crystallization and preliminary X-ray investigation of phosphoribosylaminoimidazolesuccinocarboxamide synthase from the yeast Saccharomyces cerevisiae

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.