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At least 379 records · Page 21Linked to original sources
PubMed2005

Cloning, purification and crystallization of a Walker-type Pyrococcus abyssi ATPase family member.

Several ATPase proteins play essential roles in the initiation of chromosomal DNA replication in archaea. Walker-type ATPases are defined by their conserved Walker A and B motifs, which are associated with nucleotide binding and ATP hydrolysis. A family of 28 ATPase proteins with non-canonical Walker A sequences has been identified by a bioinformatics study of comparative genomics in Pyrococcus genomes. A high-throughput structural study on P. abyssi has been started in order to establish the structure of these proteins. 16 genes have been cloned and characterized. Six out of the seven soluble constructs were purified in Escherichia coli and one of them, PABY2304, has been crystallized. X-ray diffraction data were collected from selenomethionine-derivative crystals using synchrotron radiation. The crystals belong to the orthorhombic space group C2, with unit-cell parameters a = 79.41, b = 48.63, c = 108.77 A, and diffract to beyond 2.6 A resolution.

Adenosine Triphosphatases↗
PubMed2005

Cloning, purification, crystallization and preliminary crystallographic analysis of a penicillin-binding protein homologue from Pyrococcus abyssi.

The genome of the hyperthermophilic archaeon Pyrococcus abyssi contains a gene (pab0087) encoding a penicillin-binding protein (PBP) homologue. This sequence consists of 447 residues and shows significant sequence similarity to low-molecular-weight PBPs and class C beta-lactamases. The Pab0087 protein was overexpressed, purified and crystallized. Diffraction data from two different crystal forms were collected to 2.7 and 2.0 A resolution. Both crystals belong to space group C2, with unit-cell parameters a = 160.59, b = 135.74, c = 113.02 A, beta = 117.36 degrees and a = 166.97, b = 131.25, c = 189.39 A, beta = 113.81 degrees , respectively. The asymmetric unit contains four and eight molecules, respectively, with fourfold non-crystallographic symmetry.

Cloning, Molecular↗
PubMed2005

Overexpression, purification and crystallization of an archaeal DNA ligase from Pyrococcus furiosus.

DNA ligases seal single-strand breaks in double-stranded DNA and their function is essential to maintain the integrity of the genome during various aspects of DNA metabolism, such as replication, excision repair and recombination. DNA-strand breaks are frequently generated as reaction intermediates in these events and the sealing of these breaks depends solely on the proper function of DNA ligase. Crystals of the archaeal DNA ligase from Pyrococcus furiosus were obtained using 6.6%(v/v) ethanol as a precipitant and diffracted X-rays to 1.7 A resolution. They belong to the monoclinic space group P2(1), with unit-cell parameters a = 61.1, b = 88.3, c = 63.4 A, beta = 108.9 degrees. The asymmetric unit contains one ligase molecule.

Catalytic Domain↗
PubMed2005

Crystallization and preliminary X-ray analysis of PH1566, a putative ribosomal RNA-processing factor from the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

A putative ribosomal RNA-processing factor consisting of two KH domains from Pyrococcus horikoshii OT3 (PH1566; 25 kDa) was crystallized by the sitting-drop vapour-diffusion method using PEG 3000 as the precipitant. The crystals diffracted X-rays to beyond 2.0 A resolution using a synchrotron-radiation source. The space group of the crystals was determined as primitive orthorhombic P2(1)2(1)2(1), with unit-cell parameters a = 45.9, b = 47.4, c = 95.7 A. The crystals contain one molecule in the asymmetric unit (VM = 2.5 A3 Da(-1)) and have a solvent content of 50%.

Archaeal Proteins↗
PubMed2006

Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase from Pyrococcus horikoshii.

Galactokinase (EC 2.7.1.6) catalyzes the ATP-dependent phosphorylation of alpha-D-galactose to alpha-D-galactose-1-phosphate, in an additional metabolic branch of glycolysis. The apo-form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with alpha-D-galactose and an ATP analogue. Diffraction data sets were collected to 1.24 A resolution for the apo form and to 1.7 A for the ternary complex form using synchrotron radiation. The apo-form crystals belong to space group C2, with unit-cell parameters a = 108.08, b = 38.91, c = 81.57 A, beta = 109.8 degrees. The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.

Apoenzymes↗
PubMed2006

Stoichiometric complex formation by proliferating cell nuclear antigen (PCNA) and its interacting protein: purification and crystallization of the DNA polymerase and PCNA monomer mutant complex from Pyrococcus furiosus.

Replicative DNA polymerase interacts with processivity factors, the beta-subunit of DNA polymerase III or proliferating cell nuclear antigen (PCNA), in order to function with a long template DNA. The archaeal replicative DNA polymerase from Pyrococcus furiosus interacts with PCNA via its PCNA-interacting protein (PIP) motif at the C-terminus. The PCNA homotrimeric ring contains one PIP interacting site on each monomer and since the ring can accommodate up to three molecules simultaneously, formation of a stable stoichiometric complex of PCNA with its interacting protein has been difficult to control in vitro. A stable complex of the DNA polymerase with PCNA, using a PCNA monomer mutant, has been purified and crystallized. The best ordered crystal diffracted to 3.0 A resolution using synchrotron radiation. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 A.

Crystallization↗
PubMed2006

Purification, crystallization and preliminary crystallographic analysis of RecA superfamily ATPase PH0284 from Pyrococcus horikoshii OT3.

Circadian (daily) protein clocks are found in cyanobacteria, where a complex of the KaiA, KaiB and KaiC proteins generates circadian rhythms. The 28.09 kDa KaiC homologue PH0284 protein from Pyrococcus horikoshii OT3 was cloned and expressed and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data from the crystal were collected to 2.0 angstroms resolution using synchrotron radiation at 100 K. The crystal belongs to the trigonal space group P3(2)21, with unit-cell parameters a = b = 96.06, c = 298.90 angstroms. Assuming the presence of one hexamer in the asymmetric unit gives a V(M) value of 2.36 angstroms3 Da(-1) and a solvent content of 47.9%. A cocrystal with ATP was prepared and a diffraction data set was collected at 2.3 angstroms resolution.

Adenosine Triphosphatases↗
PubMed2006

Purification, crystallization and preliminary X-ray crystallographic analysis of the archaeal phosphoglycerate mutase PH0037 from Pyrococcus horikoshii OT3.

Phosphoglycerate mutases catalyze the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in glycolysis and gluconeogenesis pathways. The archaeal phosphoglycerate mutase PH0037 from Pyrococcus horikoshii OT3 has been overexpressed in Escherichia coli and purified. Crystals were obtained using the oil-microbatch method at 291 K. A native data set extending to a resolution of 2.2 angstroms has been collected and processed in space group R32. Assuming the presence of a dimer in the asymmetric unit, the V(M) value is calculated to be 3.0 angstroms3 Da(-1), consistent with the dynamic light-scattering experiment result, which shows a dimeric state of the protein in solution. Molecular-replacement trials using the crystal structure of Bacilllus stearothermophilus phosphoglycerate mutase as a search model did not provide a satisfactory solution, indicating substantially different structures of these two phophoglycerate mutases.

Archaeal Proteins↗
PubMed2006

Crystallization and X-ray diffraction analysis of a catalytic domain of hyperthermophilic chitinase from Pyrococcus furiosus.

The crystallization and preliminary X-ray diffraction analysis of a catalytic domain of chitinase (PF1233 gene) from the hyperthermophilic archaeon Pyrococcus furiosus is reported. The recombinant protein, prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at the undulator beamline BL44XU at SPring-8 to a resolution of 1.50 angstroms. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 90.0, b = 92.8, c = 107.2 angstroms.

Amino Acid Sequence↗
PubMed2006

The structure of Pyrococcus horikoshii 2'-5' RNA ligase at 1.94 A resolution reveals a possible open form with a wider active-site cleft.

Bacterial and archaeal 2'-5' RNA ligases, members of the 2H phosphoesterase superfamily, catalyze the linkage of the 5' and 3' exons via a 2'-5'-phosphodiester bond during tRNA-precursor splicing. The crystal structure of the 2'-5' RNA ligase PH0099 from Pyrococcus horikoshii OT3 was solved at 1.94 A resolution (PDB code 1vgj). The molecule has a bilobal alpha+beta arrangement with two antiparallel beta-sheets constituting a V-shaped active-site cleft, as found in other members of the 2H phosphoesterase superfamily. The present structure was significantly different from that determined previously at 2.4 A resolution (PDB code 1vdx) in the active-site cleft; the entrance to the cleft is wider and the active site is easily accessible to the substrate (RNA precursor) in our structure. Structural comparison with the 2'-5' RNA ligase from Thermus thermophilus HB8 also revealed differences in the RNA precursor-binding region. The structural differences in the active-site residues (tetrapeptide motifs H-X-T/S-X) between the members of the 2H phosphoesterase superfamily are discussed.

Crystallization↗
PubMed2006

Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3.

6-Pyruvoyl tetrahydrobiopterin synthase (PTPS) catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. PH0634, a 13.51 kDa archaeal PTPS homologue from Pyrococcus horikoshii OT3, was overexpressed as native and selenomethionine-substituted protein and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data were collected to 2.1 A resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 A. Threefold noncrystallographic symmetry was identified from self-rotation calculations. Assuming the presence of a trimer in the asymmetric unit, the solvent content is 45% (V(M) = 2.24 A3 Da(-1)). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 A.

Archaeal Proteins↗
PubMed2006

Structure of the catalytic domain of the hyperthermophilic chitinase from Pyrococcus furiosus.

The crystal structure of the catalytic domain of a chitinase from the hyperthermophilic archaeon Pyrococcus furiosus (AD2(PF-ChiA)) has been determined at 1.5 A resolution. This is the first structure of the catalytic domain of an archaeal chitinase. The overall structure of AD2(PF-ChiA) is a TIM-barrel fold with a tunnel-like active site that is a common feature of family 18 chitinases. Although the catalytic residues (Asp522, Asp524 and Glu526) are conserved, comparison of the conserved residues and structures with those of other homologous chitinases indicates that the catalytic mechanism of PF-ChiA is different from that of family 18 chitinases.

Amino Acid Sequence↗
PubMed2006

Purification, crystallization and preliminary crystallographic analysis of the putative thiamine-biosynthesis protein PH1313 from Pyrococcus horikoshii OT3.

The putative thiamine-biosynthesis protein PH1313 from Pyrococcus horikoshii OT3 has been overexpressed and purified. Crystallization was performed by the oil-microbatch method using 28%(v/v) 2-methyl-2,4-pentanediol as a precipitant at 291 K. A native X-ray diffraction data set at 1.9 A resolution and a single anomalous dispersion data set from a selenomethionine-derivative crystal at 2.1 A resolution were collected using synchrotron radiation at 100 K. The native crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 71.7, b = 71.2, c = 141.8 A.

Crystallization↗
PubMed2002

Crystal structure of the ADP-dependent glucokinase from Pyrococcus horikoshii at 2.0-A resolution: a large conformational change in ADP-dependent glucokinase.

Although ATP is the most common phosphoryl group donor for kinases, some kinases from certain hyperthermophilic archaea such as Pyrococcus horikoshii and Thermococcus litoralis use ADP as the phosphoryl donor. Those are ADP-dependent glucokinases (ADPGK) and phosphofructokinases in their glycolytic pathway. Here, we succeeded in gene cloning the ADPGK from P. horikoshii OT3 (phGK) in Escherichia coli,and in easy preparation of the enzyme, crystallization, and the structure determination of the apo enzyme. Recently, the three-dimensional structure of the ADPGK from T. litoralis (tlGK) in a complex with ADP was reported. The overall structure of two homologous enzymes (56.7%) was basically similar: This means that they consisted of large alpha/beta-domains and small domains. However, a marked adjustment of the two domains, which is a 10-A translation and a 20 degrees rotation from the conserved GG sequence located at the center of the hinge, was observed between the apo-phGK and ADP-tlGK structures. The ADP-binding loop (430-439) was disordered in the apo form. It is suggested that a large conformational change takes place during the enzymatic reaction.

Amino Acid Sequence↗
PubMed2003

Intermolecular ion pairs maintain the toroidal structure of Pyrococcus furiosus PCNA.

Two mutant proliferating cell nuclear antigens from the hyperthermophilic archaeon Pyrococcus furiosus, PfuPCNA(D143A) and PfuPCNA(D143A/D147A), were prepared by site-specific mutagenesis. The results from gel filtration showed that mutations at D143 and D147 drastically affect the stability of the trimeric structure of PfuPCNA. The PfuPCNA(D143A) still retained the activity to stimulate the DNA polymerase reaction, but PfuPCNA(D143A/D147A) lost the activity. Crystal structures of the mutant PfuPCNAs were determined. Although the wild-type PCNA forms a toroidal trimer with intermolecular hydrogen bonds between the N- and C-terminal domains, the mutant PfuPCNAs exist as V-shaped dimers through intermolecular hydrogen bonds between the two C-terminal domains in the crystal. Because the mutated residues are involved in the intermolecular ion pairs through their side chains in the wild-type PfuPCNA, these ion pairs seem to play a key role in maintaining the toroidal structure of the PfuPCNA trimer. The comparison of the crystal structures revealed intriguing conformational flexibility of each domain in the PfuPCNA subunit. This structural versatility of PCNA may be involved in the mechanisms for ring opening and closing.

Amino Acid Substitution↗
PubMed2003

Solution NMR structure of the 30S ribosomal protein S28E from Pyrococcus horikoshii.

We report NMR assignments and solution structure of the 71-residue 30S ribosomal protein S28E from the archaean Pyrococcus horikoshii, target JR19 of the Northeast Structural Genomics Consortium. The structure, determined rapidly with the aid of automated backbone resonance assignment (AutoAssign) and automated structure determination (AutoStructure) software, is characterized by a four-stranded beta-sheet with a classic Greek-key topology and an oligonucleotide/oligosaccharide beta-barrel (OB) fold. The electrostatic surface of S28E exhibits positive and negative patches on opposite sides, the former constituting a putative binding site for RNA. The 13 C-terminal residues of the protein contain a consensus sequence motif constituting the signature of the S28E protein family. Surprisingly, this C-terminal segment is unstructured in solution.

Amino Acid Sequence↗
PubMed2005

The crystal structure of a novel SAM-dependent methyltransferase PH1915 from Pyrococcus horikoshii.

The S-adenosyl-L-methionine (SAM)-dependent methyltransferases represent a diverse and biologically important class of enzymes. These enzymes utilize the ubiquitous methyl donor SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. Here we present the crystal structure of PH1915 from Pyrococcus horikoshii OT3, a predicted SAM-dependent methyltransferase. This protein belongs to the Cluster of Orthologous Group 1092, and the presented crystal structure is the first representative structure of this protein family. Based on sequence and 3D structure analysis, we have made valuable functional insights that will facilitate further studies for characterizing this group of proteins. Specifically, we propose that PH1915 and its orthologs are rRNA- or tRNA-specific methyltransferases.

Binding Sites↗
PubMed2005

Crystal structure of the probable haloacid dehalogenase PH0459 from Pyrococcus horikoshii OT3.

PH0459, from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, is a probable haloacid dehalogenase with a molecular mass of 26,725 Da. Here, we report the 2.0 A crystal structure of PH0459 (PDB ID: 1X42) determined by the multiwavelength anomalous dispersion method. The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by six alpha-helices and three 3(10)-helices. One disulfide bond, Cys186-Cys212, forms a bridge between an alpha-helix and a 3(10)-helix, although PH0459 seems to be an intracellular protein. The subdomain inserted into the core domain has a four-helix bundle structure. The crystal packing suggests that PH0459 exists as a monomer. A structural homology search revealed that PH0459 resembles the l-2-haloacid dehalogenases l-DEX YL from Pseudomonas sp. YL and DhlB from Xanthobacter autotrophicus GJ10. A comparison of the active sites suggested that PH0459 probably has haloacid dehalogenase activity, but its substrate specificity may be different. In addition, the disulfide bond in PH0459 probably facilitates the structural stabilization of the neighboring region in the monomeric form, although the corresponding regions in l-DEX YL and DhlB may be stabilized by dimerization. Since heat-stable dehalogenases can be used for the detoxification of halogenated aliphatic compounds, PH0459 will be a useful target for biotechnological research.

Amino Acid Sequence↗