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PubMed2006

Structure of the catalytic nucleotide-binding subunit A of A-type ATP synthase from Pyrococcus horikoshii reveals a novel domain related to the peripheral stalk.

H(+)-transporting ATP synthase is a multi-subunit enzyme involved in the production of ATP, which is an essential molecule for living organisms as a source of energy. Archaeal A-type ATPase (A-ATPase) is thought to act as a functional ATP synthase in archaea and is thought to have chimeric properties of F-ATPase and V-ATPase. Previous structural studies of F-ATPase indicated that the major nucleotide-binding subunits alpha and beta consist of three domains. The catalytic nucleotide-binding subunit A of V/A-ATPase contains an insertion of about 90 residues which is absent from the F(1)-ATPase beta subunit. Here, the first X-ray structure of the catalytic nucleotide-binding subunit A of an A(1)-ATPase is described, determined at 2.55 A resolution. A(1)-ATPase subunit A from Pyrococcus horikoshii consists of four domains. A novel domain, including part of the insertion, corresponds to the 'knob-like structure' observed in electron microscopy of A(1)-ATPase. Based on the structure, it is highly likely that this inserted domain is related to the peripheral stalk common to the A- and V-ATPases. The arrangement of this inserted domain suggests that this region plays an important role in A-ATPase as well as in V-ATPase.

Amino Acid Sequence↗
PubMed1998

Purification, crystallization and preliminary X-ray crystallographic analysis of Pyrococcus furiosus DNA polymerase.

DNA polymerase gene from the hyperthermophilic Archaeon Pyrococcus furiosus has been cloned and the protein overexpressed in Escherichia coli to produce an active enzyme. The purified protein was crystallized from 0.08 M ammonium sulfate, 0.05 M Na-cacodylate, pH 6.5, 0.15%(v/v) NP40, 0.05%(v/v) Tween 20 and 4.5%(w/v) polyethylene glycol 6000 by the vapour-diffusion method. The orthorhombic crystals had unit-cell dimensions of a = 92.5, b = 125.4, c = 192.1 A; alpha = beta = gamma = 90 degrees. The crystals diffracted beyond 4 A on a 1.08 A synchrotron radiation source.

Cloning, Molecular↗
PubMed1998

Preliminary crystallographic studies of triosephosphate isomerase (TIM) from the hyperthermophilic Archaeon Pyrococcus woesei.

Recombinant triosephosphate isomerase (TIM) from a hyperthermophilic Archaeon, Pyrococcus woesei, has been crystallized. Three crystal forms have been obtained: monoclinic, orthorhombic and hexagonal. The monoclinic crystals belong to space group P21 with cell dimensions a = 79.1, b = 89.2, c = 145.4 A and beta = 92.8 degrees, and diffract to at least 2.6 A. The orthorhombic crystals belong to space group P21212 with a = 89.4, b = 155.9, c = 79.5 A, and diffract to 2.9 A. Diffraction from the hexagonal form showed extensive disorder. The monoclinic form contains two tetramers in the asymmetric unit, which are in the same orientation but related by a pseudo-centering. The orthorhombic form contains one tetramer in the asymmetric unit which is in approximately the same orientation as in the monoclinic form. Knowledge of the structure of this hyperthermostable TIM, which is tetrameric in contrast to dimeric forms previously observed, will add to our understanding of protein thermostability.

Bacterial Proteins↗
PubMed2003

Neutron diffraction studies on rubredoxin from Pyrococcus furiosus.

Single-crystal neutron diffraction data up to a resolution of 1.5 A have been collected at room temperature on two forms of rubredoxin using the BIX-3 diffractometer at the JRR-3 reactor of the Japan Atomic Energy Research Institute (JAERI). Rubredoxin is a small iron-sulfur redox protein with 53 amino acid residues, and the source of this particular protein is the hyperthermophile Pyrococcus furiosus, a microorganism that normally lives at temperatures near that of boiling water. Data were collected on crystals of the wild-type protein and on a mutant in which three of the residues have been replaced. In this paper we will be describing several sets of results arising from these high-resolution neutron structure determinations: (a) the H/D exchange pattern of the N-H bonds of the main backbone, which give information about which regions of the molecule are more exposed to solvent; (b) the orientations of some of the O-D bonds in the protein, information which is often not obtainable from X-ray results; (c) the structure and appearance of water molecules in the protein crystals; and (d) some structural features which may help rationalize the remarkable thermal stability of the wild-type protein from this intriguing microorganism

Binding Sites↗
PubMed2004

High-throughput production of Pyrococcus furiosus proteins: considerations for metalloproteins.

Free-living prokaryotic organisms contain all of the proteins required for the basic biochemical processes of life. As part of the Southeastern Collaboratory for Structural Genomics (SECSG), Pyrococcus furiosus is being used as a model system for developing a high-throughput protein expression and purification protocol. Its 1.9 million basepair genome encodes approximately 2200 putative proteins, less than 25% of which show similarity to any structurally characterized protein in the Protein Data Bank. The overall goal of the structural genomics initiative is to determine, in total, all existing protein folds. The immediate objective of this work is to obtain recombinant forms of all P. furiosus proteins in their functional states for structural determination. Proteins successfully produced by overexpression in another organism such as the bacterium Escherichia coli typically contain a single subunit, are soluble and do not contain (complex) cofactors. Analyses of the P. furiosus genome suggest that perhaps only a quarter of the genes encode proteins that would fall into this category. The hypothesis is that lack of the appropriate cofactor or of the partner protein(s) necessary to form a complex are major reasons why many recombinant proteins are insoluble. This work describes development of the production pipeline with attention to prediction and incorporation of cofactors.

Cloning, Molecular↗
PubMed2004

Crystallization of leucyl-tRNA synthetase complexed with tRNALeu from the archaeon Pyrococcus horikoshii.

All five tRNALeu isoacceptors from the archaeon Pyrococcus horikoshii have been transcribed in vitro and purified. The leucyl-tRNA synthetase (LeuRS) from P. horikoshii was overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNALeu isoacceptors were attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNALeu isoacceptor with the anticodon CAA was used. Electrophoretic analyses revealed that the crystals contain both LeuRS and tRNALeu, suggesting that they are LeuRS-tRNALeu complex crystals. A data set diffracting to 3.3 A resolution was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 118.18, b = 120.55, c = 231.13 A. The asymmetric unit is expected to contain two complexes of LeuRS-tRNALeu, with a corresponding crystal volume per protein weight of 2.9 A3 Da(-1) and a solvent content of 57.3%.

Archaeal Proteins↗
PubMed2004

Crystallization and preliminary crystallographic analysis of the nickel-responsive regulator NikR from Pyrococcus horikoshii.

The nickel-responsive repressor from Pyrococcus horikoshii OT3 (PhNikR) has been crystallized in the apo form (PhNikR-apo) and two nickel-bound forms (PhNikR-Ni-1 and PhNikR-Ni-2). The PhNikR-apo crystals belong to space group P2(1), with unit-cell parameters a = 75.78, b = 54.32, c = 77.28 A, beta = 116.07 degrees, and diffract to 2.2 A. The PhNikR-Ni-1 crystals belong to space group P4(1)2(1)2, with unit-cell parameters a = b = 99.89, c = 97.98 A, and diffract to 3.0 A and the PhNikR-Ni-2 crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 109.95, c = 79.0 A, and diffract to 2.1 A. The crystals obtained were suitable for detailed structural studies.

Archaeal Proteins↗
PubMed2004

Purification, crystallization and preliminary crystallographic analysis of the vacuole-type ATPase subunit E from Pyrococcus horikoshii OT3.

The vacuole-type ATPases in eukaryotic cells translocate protons across various biological membranes including the vacuolar membrane by consuming ATP molecules. The E subunit of the multisubunit complex V-ATPase from Pyrococcus horikoshii OT3, which has a molecular weight of 22.88 kDa, has been cloned, overexpressed in Escherichia coli, purified and crystallized by the microbatch method using PEG 4000 as a precipitant at 296 K. A data set to 1.85 A resolution with 98.8% completeness and an Rmerge of 6.5% was collected from a single flash-cooled crystal using synchrotron radiation. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 52.196, b = 55.317, c = 77.481 A, and is most likely to contain one molecule per asymmetric unit.

Archaeal Proteins↗
PubMed2005

Purification, crystallization and preliminary crystallographic analysis of the biotin-protein ligase from Pyrococcus horikoshii OT3.

Biotin-protein ligase is an enzyme that catalyzes the ATP-dependent biotinylation of a specific lysine residue in acetyl-CoA carboxylase. The biotin-protein ligase from Pyrococcus horikoshii OT3 has been cloned, overexpressed and purified. Crystallization was performed by the microbatch method or the vapour-diffusion method using PEG 2000 as a precipitant at 295 K. X-ray diffraction data have been collected to 1.6 A resolution from a native crystal and to 1.55 A resolution from a selenomethionine-derivative crystal for multiple anomalous dispersion phasing using synchrotron radiation at 100 K. The native crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 38.601, b = 78.264, c = 70.147 A, beta = 101.48 degrees. Assuming a homodimer per asymmetric unit gives a VM value of 2.14 A3 Da(-1) and a solvent content of 42.5%. Cocrystals with biotin, ADP and biotinyl-5'-AMP were prepared and diffraction data sets were collected to 1.6, 1.6 and 1.45 A resolution, respectively.

Bacterial Proteins↗
PubMed2005

Expression, purification, crystallization and preliminary crystallographic analysis of a deblocking aminopeptidase from Pyrococcus horikoshii.

The deblocking aminopeptidase (DAP) of Pyrococcus horikoshii is a hyperthermophilic exoprotease that cleaves the N-terminal amino acid of peptide substrates with a putative deblocking activity for acylated peptides. DAP has been found to be homologous to a tetrahedral aminopeptidase from the halophilic Haloarcula marismortui. The latter enzyme is a dodecameric complex and has been revealed to be a self-compartmentalized protease whose central cavity harbouring the catalytic site is accessible through several channels of different size, unlike all other known proteolytic complexes. Three paralogues of DAP have been identified in P. horikoshii, with about 40% identity between them. Each of them has been overexpressed in Escherichia coli, purified and crystallized in the native and selenomethionine-substituted states. The results indicate that they form two kinds of assemblies, of 12 and of 24 subunits, with a molecular weight of approximately 400 and approximately 800 kDa, respectively. Crystals of the different variants of DAP and in their different oligomeric states diffract up to a resolution of 3 A.

Aminopeptidases↗
PubMed2005

Cloning, purification, crystallization and preliminary crystallographic analysis of a hypothetical acetyltransferase from Pyrococcus furiosus.

The GCN5-related N-acetyltransferase (GNAT) superfamily has a primordial role in cellular processes such as transcription initiation and regulation by histone acetylation, aminoglycoside resistance and melatonin metabolism. To date, no acetyltransferase from the archaeal domain of life has been studied. This paper describes the cloning, expression, purification and crystallization of a Pyrococcus furiosus hypothetical acetyltransferase PfGNAT (MW = 22 007 Da). The crystals belong to space group P622, with one molecule in the asymmetric unit and unit-cell parameters a = b = 82.6, c = 105.92 A, alpha = beta = 90, gamma = 120 degrees. Crystals diffract X-rays to 3.0 A resolution on a synchrotron-radiation source. Determination of this structure will provide new insights into the substrate-specificity of this acetyltransferase and the thermal stability of the N-acetyltransferase domain.

Acetyltransferases↗
PubMed2005

Overproduction and preliminary crystallographic study of a human kynurenine aminotransferase II homologue from Pyrococcus horikoshii OT3.

The Pyrococcus horikoshii OT3 genome contains a gene encoding a human kynurenine aminotransferase II (KAT II) homologue, which consists of 428 amino-acid residues and shows an amino-acid sequence identity of 30% to human KAT II. This gene was overexpressed in Escherichia coli and the recombinant protein (Ph-KAT II) was purified. Gel-filtration chromatography showed that Ph-KAT II exists as a homodimer. Ph-KAT II exhibited enzymatic activity that catalyzes the transamination of L-kynurenine to produce kynurenic acid. Crystals of Ph-KAT II were grown using the sitting-drop vapour-diffusion method and native X-ray diffraction data were collected to 2.2 A resolution using synchrotron radiation from station BL44XU at SPring-8. The crystals belong to the centred orthorhombic space group C222(1), with unit-cell parameters a = 71.75, b = 86.84, c = 137.30 A. Assuming one molecule per asymmetric unit, the VM value was 2.19 A3 Da(-1) and the solvent content was 43.3%.

Archaeal Proteins↗
PubMed2005

Crystallization and preliminary X-ray crystallographic analysis of a conserved domain in plants and prokaryotes from Pyrococcus horikoshii OT3.

A plant- and prokaryote-conserved domain (PPC) has previously been found in AT-hook motif nuclear localized protein 1 (AHL1) localized in the nuclear matrix of Arabidopsis thaliana (AtAHL1). AtAHL1 has a DNA-binding function. Mutation analyses of AtAHL1 has previously revealed that the hydrophobic region of the PPC domain is essential for its nuclear localization. In this study, the PPC of the hyperthermophilic archaebacterium Pyrococcus horikoshii (PhPPC) was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 53.69, c = 159.2 A. Data were obtained at 100 K, with diffraction being observed to a resolution of 1.7 A. A complete data set from crystals of the SeMet-substituted protein was also obtained.

Amino Acid Motifs↗
PubMed2005

Crystallization and preliminary X-ray analysis of hyperthermophilic L-threonine dehydrogenase from the archaeon Pyrococcus horikoshii.

Recombinant L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii was prepared using an Escherichia coli expression system. The hyperthermostable L-threonine dehydrogenase consists of 348 amino acids with a molecular weight of 37.7 kDa. The enzyme was crystallized by the hanging-drop vapour-diffusion method at 277 K and preliminary X-ray crystallographic analysis was carried out. Diffraction data were collected to 2.20 A resolution under cryogenic conditions. P. horikoshii L-threonine dehydrogenase crystals belong to space group I4(1)22, with unit-cell parameters a = b = 143.84, c = 304.13 A. The presence of three subunits of the enzyme per asymmetric unit was estimated to give a Matthews coefficient (VM) of 3.5 A3 Da(-1) and a solvent content of 64.7%(v/v).

Alcohol Oxidoreductases↗
PubMed2005

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

The crystallization and preliminary X-ray diffraction analysis of the chitin-binding domain of chitinase from a hyperthermophilic archaeon, Pyrococcus furiosus, are reported. The recombinant protein was prepared using an Escherichia coli overexpression system and was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected to 1.70 A resolution. The crystal belonged to space group P4(3)2(1)2 or P4(1)2(1)2. The unit-cell parameters were determined to be a = b = 48.8, c = 85.0 A.

Chitin↗
PubMed2005

Crystallization and preliminary X-ray diffraction analysis of a flavoenzyme amine dehydrogenase/oxidase from Pyrococcus furiosus DSM 3638.

A flavoprotein amine dehydrogenase/oxidase with subunit molecular weights of 54.8 kDa (alpha-subunit) and 42.4 kDa (beta-subunit) and specificity for L-proline was cloned from the genomic DNA of the hyperthermophilic marine archaeon Pyrococcus furiosus DSM 3638. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The enzyme was crystallized using the sitting-drop vapour-diffusion technique. Diffraction data from two different crystal forms were collected to 3.3 and 3.6 A, respectively, using synchrotron radiation. Both crystals belonged to space group P1, with unit-cell parameters a = 91.3, b = 136.3, c = 203.8 A, alpha = 94.5, beta = 99.4, gamma = 102.7 degrees and a = 93.7, b = 116.3, c = 126.9 A, alpha = 97.3, beta = 99.9, gamma = 104.6 degrees.

Crystallization↗
PubMed2005

Purification, crystallization and initial X-ray crystallographic analysis of the putative GTPase PH0525 from Pyrococcus horikoshii OT3.

GTPases are involved in diverse cellular functions including cell proliferation, cytoskeleton organization and intracellular traffic. The putative GTPase PH0525 from Pyrococcus horikoshii OT3 has been overexpressed in Escherichia coli and purified. Two distinct crystal forms were grown by the microbatch method at 291 K using a very high protein concentration (80 mg ml(-1)). Native data sets extending to resolutions of 2.3 and 2.4 A have been collected and processed in space groups P2(1) and C222(1), respectively. Assuming the presence of one monomer per asymmetric unit gives VM values of 2.6 and 2.4 A3 Da(-1) for the P2(1) and C222(1) forms, respectively, which is consistent with dynamic light-scattering experiments, which show a monomeric state of the protein in solution.

Amino Acid Motifs↗
PubMed2005

Expression, purification, crystallization and preliminary crystallographic analysis of a stand-alone RAM domain with hydrolytic activity from the hyperthermophile Pyrococcus furiosus.

The RAM domain is one of several ligand-binding modules present in prokaryotes that are presumed to regulate the transcription of specific genes. To date, no hydrolytic activity has been reported for such modules. Curiously, a stand-alone RAM domain in Pyrococcus furiosus was isolated during a screen for hydrolytic activity against chromogenic esters. The gene encoding this protein was cloned and expressed in Escherichia coli and crystallized after a single purification step. X-ray diffraction data from the crystals were obtained to a resolution of 2.8 A using a conventional X-ray source. The cocrystallization of the recombinant protein with 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNP) and phenylmethylsulfonyl fluoride (PMSF) produced crystals that yielded data to 2.2 and 2.8 A, respectively, using synchrotron radiation. Both the untreated and EPNP-treated crystals crystallize isomorphously in space group C2 and contain three dimers in the asymmetric unit. The PMSF-treated crystals also belong to this space group and have almost identical packing density, but show dramatically different unit-cell parameters.

Anisotropy↗