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Sample records for pyrococcus horikoshii tryptophanyl-trna

  1. A Self-compartmentalizing Hexamer Serine Protease from Pyrococcus Horikoshii

    PubMed Central

    Menyhárd, Dóra K.; Kiss-Szemán, Anna; Tichy-Rács, Éva; Hornung, Balázs; Rádi, Krisztina; Szeltner, Zoltán; Domokos, Klarissza; Szamosi, Ilona; Náray-Szabó, Gábor; Polgár, László; Harmat, Veronika

    2013-01-01

    Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been under debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated “check-in” system, first passing through a pore on the hexamer surface and then turning to enter through an even smaller opening at the monomers' domain interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases, a residue of the catalytic apparatus is positioned near an amylogenic β-edge, which needs to be protected to prevent aggregation, and we found that different oligopeptidases use different strategies to achieve such an end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states. PMID:23632025

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

    SciTech Connect

    Sun, W.; Xu, X.; Pavlova, M.; Edwards, A.; Joachimiak, A.; Savchenko, A.; Christendat, D.; Biosciences Division; Univ. of Toronto; Univ. Health Network

    2005-01-01

    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.

  3. A self-compartmentalizing hexamer serine protease from Pyrococcus horikoshii: substrate selection achieved through multimerization.

    PubMed

    Menyhárd, Dóra K; Kiss-Szemán, Anna; Tichy-Rács, Éva; Hornung, Balázs; Rádi, Krisztina; Szeltner, Zoltán; Domokos, Klarissza; Szamosi, Ilona; Náray-Szabó, Gábor; Polgár, László; Harmat, Veronika

    2013-06-14

    Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been under debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated "check-in" system, first passing through a pore on the hexamer surface and then turning to enter through an even smaller opening at the monomers' domain interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases, a residue of the catalytic apparatus is positioned near an amylogenic β-edge, which needs to be protected to prevent aggregation, and we found that different oligopeptidases use different strategies to achieve such an end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states. PMID:23632025

  4. Pyrococcus horikoshii TET2 Peptidase Assembling Process and Associated Functional Regulation*

    PubMed Central

    Appolaire, Alexandre; Rosenbaum, Eva; Durá, M. Asunción; Colombo, Matteo; Marty, Vincent; Savoye, Marjolaine Noirclerc; Godfroy, Anne; Schoehn, Guy; Girard, Eric; Gabel, Frank; Franzetti, Bruno

    2013-01-01

    Tetrahedral (TET) aminopeptidases are large polypeptide destruction machines present in prokaryotes and eukaryotes. Here, the rules governing their assembly into hollow 12-subunit tetrahedrons are addressed by using TET2 from Pyrococcus horikoshii (PhTET2) as a model. Point mutations allowed the capture of a stable, catalytically active precursor. Small angle x-ray scattering revealed that it is a dimer whose architecture in solution is identical to that determined by x-ray crystallography within the fully assembled TET particle. Small angle x-ray scattering also showed that the reconstituted PhTET2 dodecameric particle displayed the same quaternary structure and thermal stability as the wild-type complex. The PhTET2 assembly intermediates were characterized by analytical ultracentrifugation, native gel electrophoresis, and electron microscopy. They revealed that PhTET2 assembling is a highly ordered process in which hexamers represent the main intermediate. Peptide degradation assays demonstrated that oligomerization triggers the activity of the TET enzyme toward large polypeptidic substrates. Fractionation experiments in Pyrococcus and Halobacterium cells revealed that, in vivo, the dimeric precursor co-exists together with assembled TET complexes. Taken together, our observations explain the biological significance of TET oligomerization and suggest the existence of a functional regulation of the dimer-dodecamer equilibrium in vivo. PMID:23696647

  5. Molecular characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3.

    PubMed

    Okochi, Mina; Matsuzaki, Hiroki; Nomura, Tomoko; Ishii, Noriyuki; Yohda, Masafumi

    2005-04-01

    The group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 (PhCPN) and its functional cooperation with the cognate prefoldin were investigated. PhCPN existed as a homo-oligomer in a double-ring structure, which protected the citrate synthase of a porcine heart from thermal aggregation at 45 degrees C, and did the same on the isopropylmalate dehydrogenase (IPMDH) of a thermophilic bacterium, Thermus thermophilus HB8, at 90 degrees C. PhCPN also enhanced the refolding of green fluorescent protein (GFP), which had been unfolded by low pH, in an ATP-dependent manner. Unexpectedly, functional cooperation between PhCPN and Pyrococcus prefoldin (PhPFD) in the refolding of GFP was not observed. Instead, cooperation between PhCPN and PhPFD was observed in the refolding of IPMDH unfolded with guanidine hydrochloride. Although PhCPN alone was not effective in the refolding of IPMDH, the refolding efficiency was enhanced by the cooperation of PhCPN with PhPFD. PMID:15538645

  6. Substrate recognition of N,N'-diacetylchitobiose deacetylase from Pyrococcus horikoshii.

    PubMed

    Nakamura, Tsutomu; Yonezawa, Yasushige; Tsuchiya, Yuko; Niiyama, Mayumi; Ida, Kurumi; Oshima, Maki; Morita, Junji; Uegaki, Koichi

    2016-09-01

    Enzymes of carbohydrate esterase (CE) family 14 catalyze hydrolysis of N-acetyl groups at the non-reducing end of the N-acetylglucosamine (GlcNAc) residue of chitooligosaccharides or related compounds. N,N'-diacetylchitobiose deacetylase (Dac) belongs to the CE-14 family and plays a role in the chitinolytic pathway in archaea by deacetylating N,N'-diacetylchitobiose (GlcNAc2), which is the end product of chitinase. In this study, we revealed the structural basis of reaction specificity in CE-14 deacetylases by solving a crystal structure of Dac from Pyrococcus horikoshii (Ph-Dac) in complex with a novel reaction intermediate analog. We developed 2-deoxy-2-methylphosphoramido-d-glucose (MPG) as the analog of the tetrahedral oxyanion intermediate of the monosaccharide substrate GlcNAc. The crystal structure of Ph-Dac in complex with MPG demonstrated that Arg92, Asp115, and His152 side chains interact with hydroxyl groups of the glucose moiety of the non-reducing-end GlcNAc residue. The amino acid residues responsible for recognition of the MPG glucose moiety are spatially conserved in other CE-14 deacetylases. Molecular dynamics simulation of the structure of the Ph-Dac-GlcNAc2 complex indicated that the reducing GlcNAc residue is placed in a large intermolecular cleft and is not involved with specific interactions with the enzyme. This observation was consistent with results indicating that Ph-Dac displayed similar kinetic parameters for both GlcNAc and GlcNAc2. This study provides the structural basis of reaction-site specificity of Dac and related CE-14 enzymes. PMID:27456364

  7. Cloning, expression, purification, crystallization and preliminary X-ray diffraction data of the Pyrococcus horikoshii RadA intein

    PubMed Central

    Lyskowski, Andrzej; Oeemig, Jesper S.; Jaakkonen, Anniina; Rommi, Katariina; DiMaio, Frank; Zhou, Dongwen; Kajander, Tommi; Baker, David; Wlodawer, Alexander; Goldman, Adrian; Iwaï, Hideo

    2011-01-01

    The RadA intein from the hyperthermophilic archaebacterium Pyrococcus horikoshii was cloned, expressed and purified for subsequent structure determination. The protein crystallized rapidly in several conditions. The best crystals, which diffracted to 1.75 Å resolution, were harvested from drops consisting of 0.1 M HEPES pH 7.5, 3.0 M NaCl and were cryoprotected with Paratone-N before flash-cooling. The collected data were processed in the orthorhombic space group P212121, with unit-cell parameters a = 58.1, b = 67.4, c = 82.9 Å. Molecular replacement with Rosetta using energy- and density-guided structure optimization provided the initial solution, which is currently under refinement. PMID:21543876

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

    SciTech Connect

    Lokanath, Neratur K.; Ukita, Yoko; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-01-01

    The E subunit of vacuole-type ATPase from P. horikoshii OT3 was overexpressed, purified and crystallized. The native crystals diffracted X-rays to 1.85 Å resolution. 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 Å resolution with 98.8% completeness and an R{sub merge} of 6.5% was collected from a single flash-cooled crystal using synchrotron radiation. The crystal belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 52.196, b = 55.317, c = 77.481 Å, and is most likely to contain one molecule per asymmetric unit.

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

    SciTech Connect

    Bagautdinov, Bagautdin; Kunishima, Naoki

    2006-04-01

    RecA superfamily ATPase PH0284 from P. horikoshii OT3 was overexpressed, purified, crystallized and cocrystallized with ATP. Both crystal forms belong to the trigonal space group P3{sub 2}21 and diffract X-rays to 2.0 and 2.3 Å resolution, respectively. 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 Å resolution using synchrotron radiation at 100 K. The crystal belongs to the trigonal space group P3{sub 2}21, with unit-cell parameters a = b = 96.06, c = 298.90 Å. Assuming the presence of one hexamer in the asymmetric unit gives a V{sub M} value of 2.36 Å{sup 3} Da{sup −1} and a solvent content of 47.9%. A cocrystal with ATP was prepared and a diffraction data set was collected at 2.3 Å resolution.

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

    SciTech Connect

    Bagautdinov, Bagautdin; Sugahara, Mitsuaki; Kunishima, Naoki

    2007-01-01

    An archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue from P. horikoshii OT3 was overexpressed as native and selenomethionine-substituted protein, purified and crystallized. The native and selenomethionine-derivative crystals are isomorphous and diffract X-rays to 2.1 and 2.9 Å resolution, respectively. 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 Å resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 Å. 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{sub M} = 2.24 Å{sup 3} Da{sup −1}). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 Å.

  11. Overexpression, purification, crystallization and preliminary crystallographic studies of a hyperthermophilic adenylosuccinate synthetase from Pyrococcus horikoshii OT3

    PubMed Central

    Wang, Xiaoying; Akasaka, Ryogo; Takemoto, Chie; Morita, Satoshi; Yamaguchi, Machiko; Terada, Takaho; Shirozu, Mikako; Yokoyama, Shigeyuki; Chen, Shilin; Si, Shuyi; Xie, Yong

    2011-01-01

    Adenylosuccinate synthetase (AdSS) is a ubiquitous enzyme that catalyzes the first committed step in the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP) in the purine-biosynthetic pathway. Although AdSS from the vast majority of organisms is 430–457 amino acids in length, AdSS sequences isolated from thermophilic archaea are 90–120 amino acids shorter. In this study, crystallographic studies of a short AdSS sequence from Pyrococcus horikoshii OT3 (PhAdSS) were performed in order to reveal the unusual structure of AdSS from thermophilic archaea. Crystals of PhAdSS were obtained by the microbatch-under-oil method and X-ray diffraction data were collected to 2.50 Å resolution. The crystal belonged to the trigonal space group P3212, with unit-cell parameters a = b = 57.2, c = 107.9 Å. There was one molecule per asymmetric unit, giving a Matthews coefficient of 2.17 Å3 Da−1 and an approximate solvent content of 43%. In contrast, the results of native polyacrylamide gel electrophoresis and analytical ultracentrifugation showed that the recombinant PhAdSS formed a dimer in solution. PMID:22139164

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

    PubMed

    Bagautdinov, Bagautdin; Sugahara, Mitsuaki; Kunishima, Naoki

    2007-01-01

    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. PMID:17183164

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

    PubMed

    Bagautdinov, Bagautdin; Kunishima, Naoki

    2006-04-01

    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. PMID:16582499

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

    PubMed Central

    Bagautdinov, Bagautdin; Sugahara, Mitsuaki; Kunishima, Naoki

    2007-01-01

    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 Å resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 Å. 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 Å3 Da−1). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 Å. PMID:17183164

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

    PubMed

    Bagautdinov, Bagautdin; Kuroishi, Chizu; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-02-01

    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. PMID:16510991

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

    PubMed Central

    Bagautdinov, Bagautdin; Kuroishi, Chizu; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-01-01

    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 Å resolution from a native crystal and to 1.55 Å 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 P21, with unit-cell parameters a = 38.601, b = 78.264, c  =  70.147 Å, β = 101.48°. Assuming a homodimer per asymmetric unit gives a V M value of 2.14 Å3 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 Å resolution, respectively. PMID:16510991

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

    PubMed Central

    Bagautdinov, Bagautdin; Kunishima, Naoki

    2006-01-01

    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 Å resolution using synchrotron radiation at 100 K. The crystal belongs to the trigonal space group P3221, with unit-cell parameters a = b = 96.06, c = 298.90 Å. Assuming the presence of one hexamer in the asymmetric unit gives a V M value of 2.36 Å3 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 Å resolution. PMID:16582499

  18. ADP-dependent 6-Phosphofructokinase from Pyrococcus horikoshii OT3: STRUCTURE DETERMINATION AND BIOCHEMICAL CHARACTERIZATION OF PH1645

    SciTech Connect

    Currie, Mark A.; Merino, Felipe; Skarina, Tatiana; Wong, Andrew H.Y.; Singer, Alexander; Brown, Greg; Savchenko, Alexei; Caniuguir, Andrés; Guixé, Victoria; Yakunin, Alexander F.; Jia, Zongchao

    2009-12-01

    Some hyperthermophilic archaea use a modified glycolytic pathway that employs an ADP-dependent glucokinase (ADP-GK) and an ADP-dependent phosphofructokinase (ADP-PFK) or, in the case of Methanococcus jannaschii, a bifunctional ADP-dependent glucophosphofructokinase (ADP-GK/PFK). The crystal structures of three ADP-GKs have been determined. However, there is no structural information available for ADP-PFKs or the ADP-GK/PFK. Here, we present the first crystal structure of an ADP-PFK from Pyrococcus horikoshii OT3 (PhPFK) in both apo- and AMP-bound forms determined to 2.0-{angstrom} and 1.9-{angstrom} resolution, respectively, along with biochemical characterization of the enzyme. The overall structure of PhPFK maintains a similar large and small {alpha}/{beta} domain structure seen in the ADP-GK structures. A large conformational change accompanies binding of phosphoryl donor, acceptor, or both, in all members of the ribokinase superfamily characterized thus far, which is believed to be critical to enzyme function. Surprisingly, no such conformational change was observed in the AMP-bound PhPFK structure compared with the apo structure. Through comprehensive site-directed mutagenesis of the substrate binding pocket we identified residues that were critical for both substrate recognition and the phosphotransfer reaction. The catalytic residues and many of the substrate binding residues are conserved between PhPFK and ADP-GKs; however, four key residues differ in the sugar-binding pocket, which we have shown determine the sugar-binding specificity. Using these results we were able to engineer a mutant PhPFK that mimics the ADP-GK/PFK and is able to phosphorylate both fructose 6-phosphate and glucose.

  19. Crystal structures of biotin protein ligase from Pyrococcus horikoshii OT3 and its complexes: structural basis of biotin activation.

    PubMed

    Bagautdinov, Bagautdin; Kuroishi, Chizu; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-10-21

    Biotin protein ligase (EC 6.3.4.15) catalyses the synthesis of an activated form of biotin, biotinyl-5'-AMP, from substrates biotin and ATP followed by biotinylation of the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase. The three-dimensional structure of biotin protein ligase from Pyrococcus horikoshii OT3 has been determined by X-ray diffraction at 1.6A resolution. The structure reveals a homodimer as the functional unit. Each subunit contains two domains, a larger N-terminal catalytic domain and a smaller C-terminal domain. The structural feature of the active site has been studied by determination of the crystal structures of complexes of the enzyme with biotin, ADP and the reaction intermediate biotinyl-5'-AMP at atomic resolution. This is the first report of the liganded structures of biotin protein ligase with nucleotide and biotinyl-5'-AMP. The structures of the unliganded and the liganded forms are isomorphous except for an ordering of the active site loop upon ligand binding. Catalytic binding sites are suitably arranged to minimize the conformational changes required during the reaction, as the pockets for biotin and nucleotide are located spatially adjacent to each other in a cleft of the catalytic domain and the pocket for biotinyl-5'-AMP binding mimics the combination of those of the substrates. The exact locations of the ligands and the active site residues allow us to propose a general scheme for the first step of the reaction carried out by biotin protein ligase in which the positively charged epsilon-amino group of Lys111 facilitates the nucleophilic attack on the ATP alpha-phosphate group by the biotin carboxyl oxygen atom and stabilizes the negatively charged intermediates. PMID:16169557

  20. Improving the Catalytic Activity of Hyperthermophilic Pyrococcus horikoshii Prolidase for Detoxification of Organophosphorus Nerve Agents over a Broad Range of Temperatures

    PubMed Central

    Theriot, Casey M.; Semcer, Rebecca L.; Shah, Saumil S.; Grunden, Amy M.

    2011-01-01

    Prolidases hydrolyze Xaa-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus (OP) compounds, including the nerve agents soman and sarin. Ph1prol (PH0974) has previously been isolated and characterized from Pyrococcus horikoshii and was shown to have higher catalytic activity over a broader pH range, higher affinity for metal, and increased thermostability compared to P. furiosus prolidase, Pfprol (PF1343). To obtain a better enzyme for OP nerve agent decontamination and to investigate the structural factors that may influence protein thermostability and thermoactivity, randomly mutated Ph1prol enzymes were prepared. Four Ph1prol mutants (A195T/G306S-, Y301C/K342N-, E127G/E252D-, and E36V-Ph1prol) were isolated which had greater thermostability and improved activity over a broader range of temperatures against Xaa-Pro dipeptides and OP nerve agents compared to wild type Pyrococcus prolidases. PMID:22162664

  1. Improving the catalytic activity of hyperthermophilic Pyrococcus horikoshii prolidase for detoxification of organophosphorus nerve agents over a broad range of temperatures.

    PubMed

    Theriot, Casey M; Semcer, Rebecca L; Shah, Saumil S; Grunden, Amy M

    2011-01-01

    Prolidases hydrolyze Xaa-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus (OP) compounds, including the nerve agents soman and sarin. Ph1prol (PH0974) has previously been isolated and characterized from Pyrococcus horikoshii and was shown to have higher catalytic activity over a broader pH range, higher affinity for metal, and increased thermostability compared to P. furiosus prolidase, Pfprol (PF1343). To obtain a better enzyme for OP nerve agent decontamination and to investigate the structural factors that may influence protein thermostability and thermoactivity, randomly mutated Ph1prol enzymes were prepared. Four Ph1prol mutants (A195T/G306S-, Y301C/K342N-, E127G/E252D-, and E36V-Ph1prol) were isolated which had greater thermostability and improved activity over a broader range of temperatures against Xaa-Pro dipeptides and OP nerve agents compared to wild type Pyrococcus prolidases. PMID:22162664

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

    SciTech Connect

    Bagautdinov, Bagautdin; Kuroishi, Chizu; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-02-01

    The biotin–protein ligase from P. horikoshii OT3 was overexpressed, purified, crystallized and cocrystallized with biotin, ADP and biotinyl-5′-AMP. The crystals belong to space group P2{sub 1} and diffract to beyond 1.6 Å resolution.

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

    SciTech Connect

    Lin, Linyen; Nakano, Hiroaki; Uchiyama, Susumu; Fujimoto, Satoru; Matsunaga, Sachihiro; Nakamura, Shota; Kobayashi, Yuji; Ohkubo, Tadayasu; Fukui, Kiichi

    2005-04-01

    A plant- and prokaryote-conserved domain (PPC) has been crystallized. The crystal diffracted to 1.7 Å resolution and belonged to space group P6{sub 3}22. 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{sub 3}22, with unit-cell parameters a = b = 53.69, c = 159.2 Å. Data were obtained at 100 K, with diffraction being observed to a resolution of 1.7 Å. A complete data set from crystals of the SeMet-substituted protein was also obtained.

  4. Expression, purification, crystallization and preliminary X-ray analysis of the KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

    PubMed Central

    Kang, Hee-Jin; Kubota, Keiko; Miyazono, Ken-ichi; Tanokura, Masaru

    2011-01-01

    KaiC is the central protein in the circadian rhythm in cyanobacteria. The 28 kDa KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of PH0187 were obtained using a reservoir solution consisting of 1.0 M ammonium phosphate monobasic and 0.1 M sodium citrate tribasic pH 5.3 (the final pH value of the reservoir solution was 4.8) and diffracted X-rays to 2.75 Å resolution. The crystal of PH0187 belonged to space group P6322, with unit-cell parameters a = b = 239.1, c = 106.5 Å. The crystal contained four PH0187 molecules in the asymmetric unit. PMID:21206047

  5. Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3.

    PubMed

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2007-04-01

    Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein-protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPDeltaN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*-BCCPDeltaN76 and BPL**-BCCPDeltaN76 complexes as well as crystals of BPL*, BPL** and BCCPDeltaN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*-BCCPDeltaN76 and BPL**-BCCPDeltaN76 crystals were collected at 100 K to 2.7 and 2.0 A resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2(1), with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 A, beta = 95.9 degrees . Assuming two subunits of the complex per asymmetric unit gives a V(M) value of 2.45 A(3) Da(-1) and a solvent content of 50%. PMID:17401210

  6. Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3

    PubMed Central

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2007-01-01

    Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein–protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPΔN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*–BCCPΔN76 and BPL**–BCCPΔN76 complexes as well as crystals of BPL*, BPL** and BCCPΔN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals were collected at 100 K to 2.7 and 2.0 Å resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P21, with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 Å, β = 95.9°. Assuming two subunits of the complex per asymmetric unit gives a V M value of 2.45 Å3 Da−1 and a solvent content of 50%. PMID:17401210

  7. Identification and characterization of a thermostable bifunctional enzyme with phosphomannose isomerase and sugar-1-phosphate nucleotidylyltransferase activities from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3.

    PubMed

    Akutsu, Jun-ichi; Zhang, Zilian; Morita, Rihito; Kawarabayasi, Yutaka

    2015-11-01

    Mannosylglycerate is known as a compatible solute, and plays important roles for salinity adaptation and high temperature stability of microorganisms. In the gene cluster for the mannosylglycerate biosynthetic pathway predicted from the genomic data of Pyrococcus horikoshii OT3, the PH0925 protein was found as a putative bifunctional enzyme with phosphomannose isomerase (PMI) and mannose-1-phosphate guanylyltransferase (Man-1-P GTase) activities, which can synthesize GDP-mannose when accompanied by a phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme (PH0923). The recombinant PH0925 protein, expressed in E. coli, exhibited both expected PMI and Man-1-P GTase activities, as well as absolute thermostability; 95 °C was the optimum reaction temperature. According to the guanylyltransferase activity (GTase) of the PH0925 protein, it was found that the protein can catalyze glucose-1-phosphate (Glc-1-P) and glucosamine-1-phosphate (GlcN-1-P) in addition to Man-1-P. The analyses of C-terminus-truncated forms of the PH0925 protein indicated that sugar-1-phosphate nucleotidylyltransferase (Sugar-1-P NTase) activity was located in the region from the N-terminus to the 345th residue, and that the C-terminal 114 residue region of the PH0925 protein inhibited the Man-1-P GTase activity. Conversely, the PMI activity was abolished by deletion of the C-terminal 14 residues. This is the first report of a thermostable enzyme with both PMI and multiple Sugar-1-P NTase activities. PMID:26290359

  8. The structures of the CutA1 proteins from Thermus thermophilus and Pyrococcus horikoshii: characterization of metal-binding sites and metal-induced assembly

    PubMed Central

    Bagautdinov, Bagautdin

    2014-01-01

    CutA1 (copper tolerance A1) is a widespread cytoplasmic protein found in archaea, bacteria, plants and animals, including humans. In Escherichia coli it is implicated in divalent metal tolerance, while the mammalian CutA1 homologue has been proposed to mediate brain enzyme acetylcholinesterase activity and copper homeostasis. The X-ray structures of CutA1 from the thermophilic bacterium Thermus thermophilus (TtCutA1) with and without bound Na+ at 1.7 and 1.9 Å resolution, respectively, and from the hyperthermophilic archaeon Pyrococcus horikoshii (PhCutA1) in complex with Na+ at 1.8 Å resolution have been determined. Both are short and rigid proteins of about 12 kDa that form intertwined compact trimers in the crystal and solution. The main difference in the structures is a wide-type β-bulge on top of the TtCutA1 trimer. It affords a mechanism for lodging a single-residue insertion in the middle of β2 while preserving the interprotomer main-chain hydrogen-bonding network. The liganded forms of the proteins provide new structural information about the metal-binding sites and CutA1 assembly. The Na+–TtCutA1 structure unveils a dodecameric assembly with metal ions in the trimer–trimer interfaces and the lateral clefts of the trimer. For Na+–PhCutA1, the metal ion associated with six waters in an octahedral geometry. The structures suggest that CutA1 may contribute to regulating intracellular metal homeostasis through various binding modes. PMID:24699729

  9. The structures of the CutA1 proteins from Thermus thermophilus and Pyrococcus horikoshii: characterization of metal-binding sites and metal-induced assembly.

    PubMed

    Bagautdinov, Bagautdin

    2014-04-01

    CutA1 (copper tolerance A1) is a widespread cytoplasmic protein found in archaea, bacteria, plants and animals, including humans. In Escherichia coli it is implicated in divalent metal tolerance, while the mammalian CutA1 homologue has been proposed to mediate brain enzyme acetylcholinesterase activity and copper homeostasis. The X-ray structures of CutA1 from the thermophilic bacterium Thermus thermophilus (TtCutA1) with and without bound Na(+) at 1.7 and 1.9 Å resolution, respectively, and from the hyperthermophilic archaeon Pyrococcus horikoshii (PhCutA1) in complex with Na(+) at 1.8 Å resolution have been determined. Both are short and rigid proteins of about 12 kDa that form intertwined compact trimers in the crystal and solution. The main difference in the structures is a wide-type β-bulge on top of the TtCutA1 trimer. It affords a mechanism for lodging a single-residue insertion in the middle of β2 while preserving the interprotomer main-chain hydrogen-bonding network. The liganded forms of the proteins provide new structural information about the metal-binding sites and CutA1 assembly. The Na(+)-TtCutA1 structure unveils a dodecameric assembly with metal ions in the trimer-trimer interfaces and the lateral clefts of the trimer. For Na(+)-PhCutA1, the metal ion associated with six waters in an octahedral geometry. The structures suggest that CutA1 may contribute to regulating intracellular metal homeostasis through various binding modes. PMID:24699729

  10. Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3

    SciTech Connect

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2007-04-01

    A truncated form of biotin carboxyl carrier protein containing the C-terminal half fragment (BCCPΔN76) and the biotin protein ligase (BPL) with the mutation R48A (BPL*) or the double mutation R48A K111A (BPL**) were successfully cocrystallized in the presence of ATP and biotin. The BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals belong to space group P2{sub 1} and diffract X-rays to 2.7 and 2.0 Å resolution, respectively. Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein–protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPΔN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*–BCCPΔN76 and BPL**–BCCPΔN76 complexes as well as crystals of BPL*, BPL** and BCCPΔN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals were collected at 100 K to 2.7 and 2.0 Å resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2{sub 1}, with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 Å, β = 95.9°. Assuming two subunits of the complex per asymmetric unit gives a V{sub M} value of 2.45 Å{sup 3} Da{sup −1} and a solvent content of 50%.

  11. Expression, high-pressure refolding, purification, crystallization and preliminary X-ray analysis of a novel single-strand-specific 3′–5′ exonuclease PhoExo I from Pyrococcus horikoshii OT3

    PubMed Central

    Miyazono, Ken-ichi; Tsutsumi, Kanae; Ishino, Yoshizumi; Tanokura, Masaru

    2014-01-01

    PhoExo I is a single-strand-specific 3′–5′ exonuclease from Pyrococcus horikoshii OT3 and is thought to be involved in a Thermococcales-specific DNA-repair pathway. The recombinant PhoExo I protein was produced as inclusion bodies in Escherichia coli cells. Solubilization of the inclusion bodies was performed by the high-pressure refolding method and highly purified protein was subjected to crystallization by the sitting-drop vapour-diffusion method at 20°C. A crystal of PhoExo I was obtained in a reservoir solution consisting of 0.1 M Tris–HCl pH 8.9, 27% PEG 6000 and diffracted X-rays to 1.52 Å resolution. The crystal of PhoExo I belonged to space group H32, with unit-cell parameters a = b = 112.07, c = 202.28 Å. The crystal contained two PhoExo I molecules in the asymmetric unit. PMID:25084386

  12. Expression, high-pressure refolding, purification, crystallization and preliminary X-ray analysis of a novel single-strand-specific 3'-5' exonuclease PhoExo I from Pyrococcus horikoshii OT3.

    PubMed

    Miyazono, Ken-ichi; Tsutsumi, Kanae; Ishino, Yoshizumi; Tanokura, Masaru

    2014-08-01

    PhoExo I is a single-strand-specific 3'-5' exonuclease from Pyrococcus horikoshii OT3 and is thought to be involved in a Thermococcales-specific DNA-repair pathway. The recombinant PhoExo I protein was produced as inclusion bodies in Escherichia coli cells. Solubilization of the inclusion bodies was performed by the high-pressure refolding method and highly purified protein was subjected to crystallization by the sitting-drop vapour-diffusion method at 20°C. A crystal of PhoExo I was obtained in a reservoir solution consisting of 0.1 M Tris-HCl pH 8.9, 27% PEG 6000 and diffracted X-rays to 1.52 Å resolution. The crystal of PhoExo I belonged to space group H32, with unit-cell parameters a = b = 112.07, c = 202.28 Å. The crystal contained two PhoExo I molecules in the asymmetric unit. PMID:25084386

  13. Structure of Quinolinate Synthase from Pyrococcus horikoshii in the Presence of Its Product, Quinolinic Acid.

    PubMed

    Esakova, Olga A; Silakov, Alexey; Grove, Tyler L; Saunders, Allison H; McLaughlin, Martin I; Yennawar, Neela H; Booker, Squire J

    2016-06-15

    Quinolinic acid (QA) is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) and its derivatives in all organisms that synthesize the molecule de novo. In most prokaryotes, it is formed from the condensation of dihydroxyacetone phosphate (DHAP) and aspartate-enamine by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique, non-cysteinyl-ligated, iron ion (Fea), which is proposed to bind the hydroxyl group of a postulated intermediate in the last step of the reaction to facilitate a dehydration. However, direct evidence for this role in catalysis has yet to be provided. Herein, we present the structure of NadA in the presence of the product of its reaction, QA. We find that N1 and the C7 carboxylate group of QA ligate to Fea in a bidentate fashion, which is confirmed by Hyperfine Sublevel Correlation (HYSCORE) spectroscopy. This binding mode would place the C5 hydroxyl group of the postulated final intermediate distal to Fea and virtually incapable of coordinating to it. The structure shows that three strictly conserved amino acids, Glu198, Tyr109, and Tyr23, are in close proximity to the bound product. Substitution of these amino acids with Gln, Phe, and Phe, respectively, leads to complete loss of activity. PMID:27224840

  14. Operon prediction in Pyrococcus furiosus

    PubMed Central

    Tran, Thao T.; Dam, Phuongan; Su, Zhengchang; Poole, Farris L.; Adams, Michael W. W.; Zhou, G. Tong; Xu, Ying

    2007-01-01

    Identification of operons in the hyperthermophilic archaeon Pyrococcus furiosus represents an important step to understanding the regulatory mechanisms that enable the organism to adapt and thrive in extreme environments. We have predicted operons in P.furiosus by combining the results from three existing algorithms using a neural network (NN). These algorithms use intergenic distances, phylogenetic profiles, functional categories and gene-order conservation in their operon prediction. Our method takes as inputs the confidence scores of the three programs, and outputs a prediction of whether adjacent genes on the same strand belong to the same operon. In addition, we have applied Gene Ontology (GO) and KEGG pathway information to improve the accuracy of our algorithm. The parameters of this NN predictor are trained on a subset of all experimentally verified operon gene pairs of Bacillus subtilis. It subsequently achieved 86.5% prediction accuracy when applied to a subset of gene pairs for Escherichia coli, which is substantially better than any of the three prediction programs. Using this new algorithm, we predicted 470 operons in the P.furiosus genome. Of these, 349 were validated using DNA microarray data. PMID:17148478

  15. Pyrococcus furiosus strains and methods of using same

    DOEpatents

    Lipscomb, Gina L; Farkas, Joel Andrew; Adams, Michael W. W.; Westpheling, Janet

    2015-01-06

    Provided herein are methods for transforming a Pyrococcus furiosus with a polynucleotide. In one embodiment, the method includes contacting a P. furiosus with a polynucleotide under conditions suitable for uptake of the polynucleotide by the P. furiosus, and identifying transformants at a frequency of, for instance, at least 10.sup.3 transformants per microgram DNA. Also provided are isolated Pyrococcus furiosus having the characteristics of Pyrococcus furiosus COM1, and plasmids that include an origin of replication that functions in a Pyrococcus furiosus. The plasmid is stable in a recipient P. furiosus without selection for more than 100 generations and is structurally unchanged after replication in P. furiosus for more than 100 generations.

  16. Draft Whole-Genome Sequence of the Type Strain Bacillus horikoshii DSM 8719.

    PubMed

    Hernández-González, Ismael L; Olmedo-Álvarez, Gabriela

    2016-01-01

    Members of the Bacillus genus have been extensively studied because of their ability to produce enzymes with high biotechnological value. Here, we report the draft of the whole-genome sequence of the type strain Bacillus horikoshii DSM 8719, an alkali-tolerant strain. PMID:27417833

  17. Draft Whole-Genome Sequence of the Type Strain Bacillus horikoshii DSM 8719

    PubMed Central

    Hernández-González, Ismael L.

    2016-01-01

    Members of the Bacillus genus have been extensively studied because of their ability to produce enzymes with high biotechnological value. Here, we report the draft of the whole-genome sequence of the type strain Bacillus horikoshii DSM 8719, an alkali-tolerant strain. PMID:27417833

  18. Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus.

    PubMed

    Wu, Chang-Hao; McTernan, Patrick M; Walter, Mary E; Adams, Michael W W

    2015-01-01

    Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity's growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophile Pyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed. PMID:26543406

  19. Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus

    PubMed Central

    Wu, Chang-Hao; McTernan, Patrick M.; Walter, Mary E.; Adams, Michael W. W.

    2015-01-01

    Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity's growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophile Pyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed. PMID:26543406

  20. Neutron diffraction studies on rubredoxin from Pyrococcus furiosus.

    PubMed

    Bau, Robert

    2004-01-01

    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 PMID:14646139

  1. Structure of hyperthermophilic β-glucosidase from Pyrococcus furiosus.

    PubMed

    Kado, Yuji; Inoue, Tsuyoshi; Ishikawa, Kazuhiko

    2011-12-01

    Three categories of cellulases, endoglucanases, cellobiohydrolases and β-glucosidases, are commonly used in the process of cellulose saccharification. In particular, the activity and characteristics of hyperthermophilic β-glucosidase make it promising in industrial applications of biomass. In this paper, the crystal structure of the hyperthermophilic β-glucosidase from Pyrococcus furiosus (BGLPf) was determined at 2.35 Å resolution in a new crystal form. The structure showed that there is one tetramer in the asymmetric unit and that the dimeric molecule exhibits a structure that is stable towards sodium dodecyl sulfate (SDS). The dimeric molecule migrated in reducing SDS polyacrylamide gel electrophoresis (SDS-PAGE) buffer even after boiling at 368 K. Energy calculations demonstrated that one of the two dimer interfaces acquired the largest solvation free energy. Structural comparison and sequence alignment with mesophilic β-glucosidase A from Clostridium cellulovorans (BGLACc) revealed that the elongation at the C-terminal end forms a hydrophobic patch at the dimer interface that might contribute to hyperthermostability. PMID:22139147

  2. Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus

    DOE PAGESBeta

    Wu, Chang-Hao; McTernan, Patrick M.; Walter, Mary E.; Adams, Michael W. W.

    2015-01-01

    Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity’s growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophile Pyrococcus furiosus ,more » a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed.« less

  3. Expression, Purification, and Characterisation of Dehydroquinate Synthase from Pyrococcus furiosus

    PubMed Central

    Negron, Leonardo; Patchett, Mark L.; Parker, Emily J.

    2011-01-01

    Dehydroquinate synthase (DHQS) catalyses the second step of the shikimate pathway to aromatic compounds. DHQS from the archaeal hyperthermophile Pyrococcus furiosus was insoluble when expressed in Escherichia coli but was partially solubilised when KCl was included in the cell lysis buffer. A purification procedure was developed, involving lysis by sonication at 30°C followed by a heat treatment at 70°C and anion exchange chromatography. Purified recombinant P. furiosus DHQS is a dimer with a subunit Mr of 37,397 (determined by electrospray ionisation mass spectrometry) and is active over broad pH and temperature ranges. The kinetic parameters are KM (3-deoxy-D-arabino-heptulosonate 7-phosphate) 3.7 μM and kcat 3.0 sec−1 at 60°C and pH 6.8. EDTA inactivates the enzyme, and enzyme activity is restored by several divalent metal ions including (in order of decreasing effectiveness) Cd2+, Co2+, Zn2+, and Mn2+. High activity of a DHQS in the presence of Cd2+ has not been reported for enzymes from other sources, and may be related to the bioavailability of Cd2+ for P. furiosus. This study is the first biochemical characterisation of a DHQS from a thermophilic source. Furthermore, the characterisation of this hyperthermophilic enzyme was carried out at elevated temperatures using an enzyme-coupled assay. PMID:21603259

  4. Structure of hyperthermophilic β-glucosidase from Pyrococcus furiosus

    PubMed Central

    Kado, Yuji; Inoue, Tsuyoshi; Ishikawa, Kazuhiko

    2011-01-01

    Three categories of cellulases, endoglucanases, cellobiohydrolases and β-glucosidases, are commonly used in the process of cellulose saccharification. In particular, the activity and characteristics of hyperthermophilic β-glucosidase make it promising in industrial applications of biomass. In this paper, the crystal structure of the hyperthermophilic β-glucosidase from Pyrococcus furiosus (BGLPf) was determined at 2.35 Å resolution in a new crystal form. The structure showed that there is one tetramer in the asymmetric unit and that the dimeric molecule exhibits a structure that is stable towards sodium dodecyl sulfate (SDS). The dimeric molecule migrated in reducing SDS polyacrylamide gel electrophoresis (SDS–PAGE) buffer even after boiling at 368 K. Energy calculations demonstrated that one of the two dimer interfaces acquired the largest solvation free energy. Structural comparison and sequence alignment with mesophilic β-glucosidase A from Clostridium cellulovorans (BGLACc) revealed that the elongation at the C-terminal end forms a hydrophobic patch at the dimer interface that might contribute to hyperthermostability. PMID:22139147

  5. Analysis of the complete genome sequence of the archaeon Pyrococcus chitonophagus DSM 10152 (formerly Thermococcus chitonophagus).

    PubMed

    Papadimitriou, Konstantinos; Baharidis, Panagiotis K; Georgoulis, Anastasios; Engel, Marion; Louka, Maria; Karamolegkou, Georgia; Tsoka, Aggeliki; Blom, Jochen; Pot, Bruno; Malecki, Piotr; Rypniewski, Wojciech; Huber, Harald; Schloter, Michael; Vorgias, Constantinos

    2016-05-01

    Here we analyze the first complete genome sequence of Pyrococcus chitonophagus. The archaeon was previously suggested to belong to the Thermococcus rather than the Pyrococcus genus. Whole genome phylogeny as well as whole proteome comparisons using all available complete genomes in Thermococcales clearly showed that the species belongs to the Pyrococcus genus. P. chitonophagus was originally isolated from a hydrothermal vent site and it has been described to effectively degrade chitin debris, and therefore is considered to play a major role in the sea water ecology and metabolic activity of microbial consortia within hot sea water ecosystems. Indeed, an obvious feature of the P. chitonophagus genome is that it carries proteins showing complementary activities for chitin degradation, i.e. endo- and exo-chitinase, diacetylchitobiose deacetylase and exo-β-D glucosaminidase activities. This finding supports the hypothesis that compared to other Thermococcales species P. chitonophagus is adapted to chitin degradation. PMID:27016195

  6. Comparative Analysis of Barophily-Related Amino Acid Content in Protein Domains of Pyrococcus abyssi and Pyrococcus furiosus

    PubMed Central

    Yafremava, Liudmila S.; Di Giulio, Massimo; Caetano-Anollés, Gustavo

    2013-01-01

    Amino acid substitution patterns between the nonbarophilic Pyrococcus furiosus and its barophilic relative P. abyssi confirm that hydrostatic pressure asymmetry indices reflect the extent to which amino acids are preferred by barophilic archaeal organisms. Substitution patterns in entire protein sequences, shared protein domains defined at fold superfamily level, domains in homologous sequence pairs, and domains of very ancient and very recent origin now provide further clues about the environment that led to the genetic code and diversified life. The pyrococcal proteomes are very similar and share a very early ancestor. Relative amino acid abundance analyses showed that biases in the use of amino acids are due to their shared fold superfamilies. Within these repertoires, only two of the five amino acids that are preferentially barophilic, aspartic acid and arginine, displayed this preference significantly and consistently across structure and in domains appearing in the ancestor. The more primordial asparagine, lysine and threonine displayed a consistent preference for nonbarophily across structure and in the ancestor. Since barophilic preferences are already evident in ancient domains that are at least ~3 billion year old, we conclude that barophily is a very ancient trait that unfolded concurrently with genetic idiosyncrasies in convergence towards a universal code. PMID:24187517

  7. Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes

    PubMed Central

    Fukui, Toshiaki; Atomi, Haruyuki; Kanai, Tamotsu; Matsumi, Rie; Fujiwara, Shinsuke; Imanaka, Tadayuki

    2005-01-01

    The genus Thermococcus, comprised of sulfur-reducing hyperthermophilic archaea, belongs to the order Thermococcales in Euryarchaeota along with the closely related genus Pyrococcus. The members of Thermococcus are ubiquitously present in natural high-temperature environments, and are therefore considered to play a major role in the ecology and metabolic activity of microbial consortia within hot-water ecosystems. To obtain insight into this important genus, we have determined and annotated the complete 2,088,737-base genome of Thermococcus kodakaraensis strain KOD1, followed by a comparison with the three complete genomes of Pyrococcus spp. A total of 2306 coding DNA sequences (CDSs) have been identified, among which half (1165 CDSs) are annotatable, whereas the functions of 41% (936 CDSs) cannot be predicted from the primary structures. The genome contains seven genes for probable transposases and four virus-related regions. Several proteins within these genetic elements show high similarities to those in Pyrococcus spp., implying the natural occurrence of horizontal gene transfer of such mobile elements among the order Thermococcales. Comparative genomics clarified that 1204 proteins, including those for information processing and basic metabolisms, are shared among T. kodakaraensis and the three Pyrococcus spp. On the other hand, among the set of 689 proteins unique to T. kodakaraensis, there are several intriguing proteins that might be responsible for the specific trait of the genus Thermococcus, such as proteins involved in additional pyruvate oxidation, nucleotide metabolisms, unique or additional metal ion transporters, improved stress response system, and a distinct restriction system. PMID:15710748

  8. Pyrococcus Furiosus Genome Supplementary Data from the Adams Laboratory at the University of Georgia

    DOE Data Explorer

    Adams, Michael W.W.; Weinberg, Michael V.; Schut, Gerrit J.; Brehm, Scott; Datta, Susmitta; Zhou, J.

    The research in the Adams Laboratory focuses on the physiology of hyperthermophilic organisms with an emphasis on metal-containing enzymes in the hyperthermophilic marine archaeon Pyrococcus furiosus. Three of the many articles from this University of Georgia lab have supplementary materials that are available on the Adams Lab website. All three sets of data are Open Reading Frames (ORFs) used for DNA microarray experiments and the changes in signal intensities. The full citations for the three articles are: 1) Weinberg, M. V., Schut, G. J., Brehm, S., Datta, S. and Adams, M. W. W. (2005) Cold shock of a hyperthermophilic archaeon: Pyrococcus furiosus exhibits multiple responses to a suboptimal growth temperature with a key role for membrane-bound glycoproteins. J Bacteriol. 187, 336-348; 2) Schut, G. J., Brehm, S. D., Datta, S. and Adams, M. W. W. (2003) "Whole genome DNA microarray analysis of a hyperthermophile and an archaeon: Pyrococcus furiosus grown on carbohydrates or peptides" J. Bacteriol. 185, 3935-3947; Schut, G. J., Zhou, J. and Adams, M. W. W. (2001) "DNA microarray analysis of the hyperthermophilic archaeon Pyrococcus furiosus evidence for a new type of sulfur-reducing enzyme" J. Bacteriol. 183, 7027-7036. Note that these articles are copyrighted by the Journal of Bacteriology.

  9. High hydrostatic pressure adaptive strategies in an obligate piezophile Pyrococcus yayanosii

    PubMed Central

    Michoud, Grégoire; Jebbar, Mohamed

    2016-01-01

    Pyrococcus yayanosii CH1, as the first and only obligate piezophilic hyperthermophilic microorganism discovered to date, extends the physical and chemical limits of life on Earth. It was isolated from the Ashadze hydrothermal vent at 4,100 m depth. Multi-omics analyses were performed to study the mechanisms used by the cell to cope with high hydrostatic pressure variations. In silico analyses showed that the P. yayanosii genome is highly adapted to its harsh environment, with a loss of aromatic amino acid biosynthesis pathways and the high constitutive expression of the energy metabolism compared with other non-obligate piezophilic Pyrococcus species. Differential proteomics and transcriptomics analyses identified key hydrostatic pressure-responsive genes involved in translation, chemotaxis, energy metabolism (hydrogenases and formate metabolism) and Clustered Regularly Interspaced Short Palindromic Repeats sequences associated with Cellular apoptosis susceptibility proteins. PMID:27250364

  10. High hydrostatic pressure adaptive strategies in an obligate piezophile Pyrococcus yayanosii.

    PubMed

    Michoud, Grégoire; Jebbar, Mohamed

    2016-01-01

    Pyrococcus yayanosii CH1, as the first and only obligate piezophilic hyperthermophilic microorganism discovered to date, extends the physical and chemical limits of life on Earth. It was isolated from the Ashadze hydrothermal vent at 4,100 m depth. Multi-omics analyses were performed to study the mechanisms used by the cell to cope with high hydrostatic pressure variations. In silico analyses showed that the P. yayanosii genome is highly adapted to its harsh environment, with a loss of aromatic amino acid biosynthesis pathways and the high constitutive expression of the energy metabolism compared with other non-obligate piezophilic Pyrococcus species. Differential proteomics and transcriptomics analyses identified key hydrostatic pressure-responsive genes involved in translation, chemotaxis, energy metabolism (hydrogenases and formate metabolism) and Clustered Regularly Interspaced Short Palindromic Repeats sequences associated with Cellular apoptosis susceptibility proteins. PMID:27250364

  11. High hydrostatic pressure adaptive strategies in an obligate piezophile Pyrococcus yayanosii

    NASA Astrophysics Data System (ADS)

    Michoud, Grégoire; Jebbar, Mohamed

    2016-06-01

    Pyrococcus yayanosii CH1, as the first and only obligate piezophilic hyperthermophilic microorganism discovered to date, extends the physical and chemical limits of life on Earth. It was isolated from the Ashadze hydrothermal vent at 4,100 m depth. Multi-omics analyses were performed to study the mechanisms used by the cell to cope with high hydrostatic pressure variations. In silico analyses showed that the P. yayanosii genome is highly adapted to its harsh environment, with a loss of aromatic amino acid biosynthesis pathways and the high constitutive expression of the energy metabolism compared with other non-obligate piezophilic Pyrococcus species. Differential proteomics and transcriptomics analyses identified key hydrostatic pressure-responsive genes involved in translation, chemotaxis, energy metabolism (hydrogenases and formate metabolism) and Clustered Regularly Interspaced Short Palindromic Repeats sequences associated with Cellular apoptosis susceptibility proteins.

  12. Characterization of amylolytic enzyme activities associated with the hyperthermophilic archaebacterium Pyrococcus furiosus

    SciTech Connect

    Brown, S.H.; Costantino, H.R.; Kelly, R.M. Univ. of Maryland, Baltimore )

    1990-07-01

    The hyperthermophilic archaebacterium Pyrococcus furiosus produces several amylolytic enzymes in response to the presence of complex carbohydrates in the growth medium. These enzyme activities, {alpha}-glucosidase, pullulanase, and {alpha}-amylase, were detected in both cell extracts and culture supernatants. All activities were characterized by temperature optima of at least 100{degree}C as well as a high degree of thermostability. The existence of this collection of activities in P. furiosus suggests that polysaccharide availability in its growth environment is a significant aspect of the niche from which it was isolated.

  13. Phosphate and arsenate removal efficiency by thermostable ferritin enzyme from Pyrococcus furiosus using radioisotopes.

    PubMed

    Sevcenco, Ana-Maria; Paravidino, Monica; Vrouwenvelder, Johannes S; Wolterbeek, Hubert Th; van Loosdrecht, Mark C M; Hagen, Wilfred R

    2015-06-01

    Oxo-anion binding properties of the thermostable enzyme ferritin from Pyrococcus furiosus were characterized with radiography. Radioisotopes (32)P and (76)As present as oxoanions were used to measure the extent and the rate of their absorption by the ferritin. Thermostable ferritin proved to be an excellent system for rapid phosphate and arsenate removal from aqueous solutions down to residual concentrations at the picomolar level. These very low concentrations make thermostable ferritin a potential tool to considerably mitigate industrial biofouling by phosphate limitation or to remove arsenate from drinking water. PMID:25817554

  14. Structure of a double hexamer of the Pyrococcus furiosus minichromosome maintenance protein N-terminal domain.

    PubMed

    Meagher, Martin; Enemark, Eric J

    2016-07-01

    The crystal structure of the N-terminal domain of the Pyrococcus furiosus minichromosome maintenance (MCM) protein as a double hexamer is described. The MCM complex is a ring-shaped helicase that unwinds DNA at the replication fork of eukaryotes and archaea. Prior to replication initiation, the MCM complex assembles as an inactive double hexamer at specific sites of DNA. The presented structure is highly consistent with previous MCM double-hexamer structures and shows two MCM hexamers with a head-to-head interaction mediated by the N-terminal domain. Minor differences include a diminished head-to-head interaction and a slightly reduced inter-hexamer rotation. PMID:27380371

  15. Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein

    SciTech Connect

    O'Brien, Kevin M.; Schufreider, Ann K.; McGill, Melissa A.; O'Brien, Kathryn M.; Reitter, Julie N.; Mills, Kenneth V.

    2010-12-17

    Research highlights: {yields} The Pyrococcus abyssi lon protease intein promotes efficient protein splicing. {yields} Inteins with mutations that interfere with individual steps of splicing do not promote unproductive side reactions. {yields} The intein splices with Lys in place of the highly conserved penultimate His. {yields} The intein is flanked by a Gly-rich region at its C terminus that may increase the efficiency of the third step of splicing, Asn cyclization coupled to peptide bond cleavage. -- Abstract: Protein splicing is a post-translational process by which an intervening polypeptide, the intein, excises itself from the flanking polypeptides, the exteins, coupled to ligation of the exteins. The lon protease of Pyrococcus abyssi (Pab) is interrupted by an intein. When over-expressed as a fusion protein in Escherichia coli, the Pab lon protease intein can promote efficient protein splicing. Mutations that block individual steps of splicing generally do not lead to unproductive side reactions, suggesting that the intein tightly coordinates the splicing process. The intein can splice, although it has Lys in place of the highly conserved penultimate His, and mutants of the intein in the C-terminal region lead to the accumulation of stable branched-ester intermediate.

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

    SciTech Connect

    Nishida, Hirokazu; Tsuchiya, Daisuke; Ishino, Yoshizumi; Morikawa, Kosuke

    2005-12-01

    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 Å resolution. 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 Å resolution. They belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 61.1, b = 88.3, c = 63.4 Å, β = 108.9°. The asymmetric unit contains one ligase molecule.

  17. Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes.

    PubMed

    Gaspin, C; Cavaillé, J; Erauso, G; Bachellerie, J P

    2000-04-01

    Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into

  18. Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei

    PubMed Central

    Ghasemi, Amir; Ghafourian, Sobhan; Vafaei, Sedighe; Mohebi, Reza; Farzi, Maryam; Taherikalani, Morovat; Sadeghifard, Nourkhoda

    2015-01-01

    Objectives In an attempt α-amylase gene from Pyrococcus woesei was amplified and cloned into a pTYB2 vector to generate the recombinant plasmid pTY- α-amylase. Methods Escherichia coli BL21 used as a host and protein expression was applied using IPTG. SDS-PAGE assay demonstrated the 100 kDa protein. Amylolytic activity of proteins produced by transformed E. coli cells was detected by zymography, and the rate of active α-amylase with and without the intein tag in both soluble conditions and as inclusion bodies solubilized by 4M urea were measured. Results Amylolytic activity of ∼185,000 U/L of bacterial culture was observed from the soluble form of the protein using this system. Conclusion These results indicate that this expression system was appropriate for the production of thermostable α-amylase. PMID:26835242

  19. Bioenergetic studies of sulfur reduction in the hyperthermophilic archaebacteria Pyrodictium brockii and Pyrococcus furiosus

    SciTech Connect

    Schicho, R.N.

    1992-01-01

    The central focus of this work was the investigation of the central energy generating pathways of two hyperthermophilic sulfidogenic archaebacteria, Pyrodictium brockii and Pyrococcus furiosus. A potential application of these organisms in the desulfurization of coals was investigated. An effective ;means of removing elemental sulfur (S[degrees]) was developed. Analytical and processing applications are discussed. The rates of sulfur removal by the hyperthermophiles were 5 fold those measured of the mesophile, Thiobacillus thiooxidans. The primary energy generating pathway of Pyrodicutium brockii has been termed hydrogen-sulfur autotrophy and is characterized by the oxidation of H[sub 2] and reduction of S[degrees]. The goals of this part of the work were to quantify the stoichiometry of this organism and to estimate Y[sub s[sup MAX

  20. Role of Polysulfides in Reduction of Elemental Sulfur by the Hyperthermophilic Archaebacterium Pyrococcus furiosus†

    PubMed Central

    Blumentals, I. I.; Itoh, M.; Olson, G. J.; Kelly, R. M.

    1990-01-01

    Polysulfides formed through the breakdown of elemental sulfur or other sulfur compounds were found to be reduced to H2S by the hyperthermophilic archaebacterium Pyrococcus furiosus during growth. Metabolism of polysulfides by the organism was dissimilatory, as no incorporation of 35S-labeled elemental sulfur was detected. However, [35S]cysteine and [35S]methionine were incorporated into cellular protein. Contact between the organism and elemental sulfur is not necessary for metabolism. The sulfide generated from metabolic reduction of polysulfides dissociates to a strong nucleophile, HS−, which in turn opens up the S8 elemental sulfur ring. In addition to H2S, P. furiosus cultures produced methyl mercaptan in a growth-associated fashion. PMID:16348181

  1. Heterologous Production of an Energy-Conserving Carbon Monoxide Dehydrogenase Complex in the Hyperthermophile Pyrococcus furiosus.

    PubMed

    Schut, Gerrit J; Lipscomb, Gina L; Nguyen, Diep M N; Kelly, Robert M; Adams, Michael W W

    2016-01-01

    Carbon monoxide (CO) is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a CO dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na(+)/H(+) antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na(+) motive force that is used to conserve energy by a Na(+)-dependent ATP synthase. Herein we used a bacterial artificial chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100°C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80°C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms. PMID:26858706

  2. Heterologous Production of an Energy-Conserving Carbon Monoxide Dehydrogenase Complex in the Hyperthermophile Pyrococcus furiosus

    PubMed Central

    Schut, Gerrit J.; Lipscomb, Gina L.; Nguyen, Diep M. N.; Kelly, Robert M.; Adams, Michael W. W.

    2016-01-01

    Carbon monoxide (CO) is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a CO dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na+/H+ antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na+ motive force that is used to conserve energy by a Na+-dependent ATP synthase. Herein we used a bacterial artificial chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100°C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80°C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms. PMID:26858706

  3. Heterologous production of an energy-conserving carbon monoxide dehydrogenase complex in the hyperthermophile Pyrococcus furiosus

    DOE PAGESBeta

    Schut, Gerrit J.; Lipscomb, Gina L.; Nguyen, Diep M. N.; Kelly, Robert M.; Adams, Michael W. W.

    2016-01-29

    In this study, carbon monoxide (CO) is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a CO dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na+/H+ antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na+ motive force that is used to conserve energy by a Na+-dependent ATP synthase. Herein we used a bacterial artificialmore » chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100° C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80° C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms.« less

  4. Preliminary neutron crystallographic analysis of selectively CH3-protonated, deuterated rubredoxin from Pyrococcus furiosus

    SciTech Connect

    Weiss, Kevin L; Meilleur, Flora; Blakeley, Matthew; Myles, Dean A A

    2008-01-01

    Neutron crystallography is used to locate hydrogen atoms in biological materials and can distinguish between negatively scattering hydrogen and positively scattering deuterium substituted positions in isomorphous neutron structures. Recently, Hauptman and Langs (2003) have shown that neutron diffraction data can be used to solve macromolecular structures by direct methods and that solution is aided by the presence of negatively scattering hydrogen atoms in the structure. Selective labeling protocols allow the design and production of H/D-labeled macromolecular structures in which the ratio of hydrogen to deuterium atoms can be precisely controlled. We have applied methyl-selective labeling protocols to introduce (1H-delta methyl)-leucine and (1H-gamma methyl)-valine into deuterated rubredoxin from Pyrococcus furiosus (PfRd). Here we report on the production, crystallization, and preliminary neutron analysis of the selectively CH3-protonated, deuterated PfRd sample, which provided a high quality neutron data set extending to 1.75 resolution at the new LADI-III instrument at the Insititut Laue-Langevin. Preliminary analysis of neutron density maps allows unambiguous assignment of the positions of hydrogen atoms at the methyl groups of the valine and leucine residues in the otherwise deuterated rubredoxin structure.

  5. Characterization of the glycolytic enzyme enolase which is abundant in the hyperthermophilic archaeon, Pyrococcus furiosus

    SciTech Connect

    Peak, M.J.; Peak, J.G.; Stevens, F.J.; Blamey, J.; Mai, X.; Zhou, Z.H.; Adams, M.W.W.

    1993-12-31

    High enolase activity, as measured by the conversion of 2-phosphoglycerate to phosphoenolphyruvate, was found in the cytoplasm of Pyrococcus (an anaerobic, hyperthermophilic archaeon that grows optimally at 100{degree}C). In this organism, the enzyme probably functions in a sugar fermentation pathway. The enzyme was purified to homogeneity. It had a temperature optimum of >90 {degree}C, and a pH optimum of 8.1. The enzyme was extremely thermostable with a half time for inactivation at 100{degree}C of 40 min. In contrast, an enolase from yeast was inactivated in 1 min at 88{degree}C. Both the P. furiosus and yeast enzymes required a metal ion for activity, but whereas the yeast enzyme has an absolute requirement for Mg{sup ++} the P. furiosus enolase was equally active in the presence of Mn{sup ++}. Both enzymes were competitively inhibited by citrate. P. furiosus enolase, as for mesophilic enolases, probably has a homodimeric structure with subunit M{sub r} greater than 45,000. A highly conserved sequence of eight amino acids in the N-terminal region was found in enolases from P. furiosus and a wide range of other organisms including bacteria, yeast, birds, and mammals.

  6. Complete saccharification of β-glucan using hyperthermophilic endocellulase and β-glucosidase from Pyrococcus furiosus.

    PubMed

    Kataoka, Misumi; Ishikawa, Kazuhiko

    2014-01-01

    Hyperthermophilic cellulase is an industrially important enzyme for biomass saccharification at high temperature. Two hyperthermophilic cellulases from the hyperthermophile Pyrococcus furiosus, endocellulase (EGPf) and β-glucosidase (BGLPf), exhibit optimal activity at 90-105 °C and a combination of two enzymes can hydrolyze a wide range of β-linked substrates. EGPf cleaves the β(1→4) bond of various substrates containing either only the β(1→4) linkage or β(1→3),(1→4) mixed-linkages. In contrast, BGLPf preferentially hydrolyzes the β(1→3) linkage over the β(1→4) linkage of disaccharides. β-Glucans are polysaccharides of D-glucose monomers formed by β(1→3),(1→4) mixed-linkage bonds. They occur most commonly as cellulose in plants, in the bran of cereal grains, the cell wall of baker's yeast, and in certain fungi, mushrooms, and bacteria. We reveal that β-glucan can be completely degraded to glucose at high temperature with a combination of EGPf and BGLPf. PMID:25209501

  7. Multiple crystal forms of N,N'-diacetylchitobiose deacetylase from Pyrococcus furiosus.

    PubMed

    Nakamura, Tsutomu; Niiyama, Mayumi; Hashimoto, Wakana; Ida, Kurumi; Abe, Manabu; Morita, Junji; Uegaki, Koichi

    2015-06-01

    Native N,N'-diacetylchitobiose deacetylase from Pyrococcus furiosus (Pf-Dac) and its selenomethionine derivative (Se-Pf-Dac) were crystallized and analyzed in the presence and absence of cadmium ion. The four crystal structures fell into three different crystal-packing groups, with the cadmium-free Pf-Dac and Se-Pf-Dac belonging to the same space group, with homologous unit-cell parameters. The crystal structures in the presence of cadmium contained distorted octahedral cadmium complexes coordinated by three chlorides, two O atoms and an S or Se atom from the N-terminal methionine or selenomethionine, respectively. The N-terminal cadmium complex was involved in crystal contacts between symmetry-related molecules through hydrogen bonding to the N-termini. While all six N-termini of Se-Pf-Dac were involved in cadmium-complex formation, only two of the Pf-Dac N-termini participated in complex formation in the Cd-containing crystal, resulting in different crystal forms. These differences are discussed in light of the higher stability of the Cd-Se bond than the Cd-S bond. This work provides an example of the contribution of cadmium towards determining protein crystal quality and packing depending on the use of the native protein or the selenomethionine derivative. PMID:26057790

  8. Characterization of recombinant glutamine synthetase from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

    PubMed Central

    Adul Rahman, R N; Jongsareejit, B; Fujiwara, S; Imanaka, T

    1997-01-01

    The glnA gene encoding glutamine synthetase was cloned from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1, and its nucleotide sequence was determined. The glnA gene was expressed in Escherichia coli ME8459 (glnA mutant strain), and the protein was purified to homogeneity and shown to be functional in a dodecameric from (637,000 Da), exhibiting both transferase and synthetase activities. However, kinetic studies indicated that the enzyme possessed low biosynthetic activity, suggesting that the reaction was biased towards glutamate production. The optimum temperature for both activities was 60 degrees C, which was lower than the optimal growth temperature of KOD1. Recombinant KOD1 GlnA exhibited different optimum pHs depending on the reaction employed (pH 7.8 for the synthetase reaction and pH 7.2 for the transferase reaction). Of the various nucleoside triphosphates tested, GTP as well as ATP was involved in the synthetase reaction. PMID:9172372

  9. A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens.

    PubMed

    Wang, Ping; Wang, Peili; Tian, Jian; Yu, Xiaoxia; Chang, Meihui; Chu, Xiaoyu; Wu, Ningfeng

    2016-01-01

    Extracellular α-amylase from Pyrococcus furiosus (PFA) shows great starch-processing potential for industrial application due to its thermostability, long half-life and optimal activity at low pH; however, it is difficult to produce in large quantities. In contrast, α-amylase from Bacillus amyloliquefaciens (BAA) can be produced in larger quantities, but shows lower stability at high temperatures and low pH. Here, we describe a BAA protein expression pattern-mimicking strategy to express PFA in B. amyloliquefaciens using the expression and secretion elements of BAA, including the codon usage bias and mRNA structure of gene, promoter, signal peptide, host and cultivation conditions. This design was assessed to be successful by comparing the various genes (mpfa and opfa), promoters (PamyA and P43), and strains (F30, F31, F32 and F30-∆amyA). The final production of PFA yielded 2714 U/mL, about 3000- and 14-fold that reportedly produced in B. subtilis or E. coli, respectively. The recombinant PFA was optimally active at ~100 °C and pH 5 and did not require Ca(2+) for activity or thermostability, and >80% of the enzyme activity was retained after treatment at 100 °C for 4 h. PMID:26916714

  10. Characterization of UDP amino sugars as major phosphocompounds in the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed Central

    Ramakrishnan, V; Teng, Q; Adams, M W

    1997-01-01

    The archaeon Pyrococcus furiosus is a strictly anaerobic heterotroph that grows optimally at 100 degrees C by the fermentation of carbohydrates. It is known to contain high concentrations of novel intracellular solutes such as beta-mannosylglycerate and di-myo-inositol 1,1'-phosphate (DIP) (L. O. Martins and H. Santos, Appl. Environ. Microbiol. 61:3299-3303, 1995). Here, 31P nuclear magnetic resonance (NMR) spectroscopy was used to show that this organism also accumulates another type of phospho compound, as revealed by a major multiplet signal in the pyrophosphate region. The compounds were purified from cell extracts of P. furiosus by anion-exchange and gel filtration chromatographic procedures and were structurally analyzed by 1H, 13C, and 31P NMR spectroscopy. They were identified as two uridylated amino sugars, UDP N-acetylglucosamine and UDP N-acetylgalactosamine. Unambiguous characterizations and complete assignments of 1H and 13C resonances from such sugars have not been previously reported. In vitro 31P NMR spectroscopic analyses showed that, in contrast to DIP, which is maintained at a constant intracellular concentration (approximately 32 mM) throughout the growth phase of P. furiosus, the UDP amino sugars accumulated (to approximately 14 mM) only during the late log phase. The possible biochemical roles of these compounds in P. furiosus are discussed. PMID:9045806

  11. A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens

    PubMed Central

    Wang, Ping; Wang, Peili; Tian, Jian; Yu, Xiaoxia; Chang, Meihui; Chu, Xiaoyu; Wu, Ningfeng

    2016-01-01

    Extracellular α-amylase from Pyrococcus furiosus (PFA) shows great starch-processing potential for industrial application due to its thermostability, long half-life and optimal activity at low pH; however, it is difficult to produce in large quantities. In contrast, α-amylase from Bacillus amyloliquefaciens (BAA) can be produced in larger quantities, but shows lower stability at high temperatures and low pH. Here, we describe a BAA protein expression pattern-mimicking strategy to express PFA in B. amyloliquefaciens using the expression and secretion elements of BAA, including the codon usage bias and mRNA structure of gene, promoter, signal peptide, host and cultivation conditions. This design was assessed to be successful by comparing the various genes (mpfa and opfa), promoters (PamyA and P43), and strains (F30, F31, F32 and F30-∆amyA). The final production of PFA yielded 2714 U/mL, about 3000- and 14-fold that reportedly produced in B. subtilis or E. coli, respectively. The recombinant PFA was optimally active at ~100 °C and pH 5 and did not require Ca2+ for activity or thermostability, and >80% of the enzyme activity was retained after treatment at 100 °C for 4 h. PMID:26916714

  12. A cryo-crystallographic time course for peroxide reduction by rubrerythrin from Pyrococcus furiosus

    SciTech Connect

    Dillard, Bret D.; Demick, Jonathan M.; Adams, Michael W.W.; Lanzilotta, William N.

    2011-09-06

    High-resolution crystal structures of Pyrococcus furiosus rubrerythrin (PfRbr) in the resting (all-ferrous) state and at time points following exposure of the crystals to hydrogen peroxide are reported. This approach was possible because of the relativity slow turnover of PfRbr at room temperature. To this end, we were able to perform time-dependent peroxide treatment of the fully reduced enzyme, under strictly anaerobic conditions, in the crystalline state. In this work we demonstrate, for the first time, that turnover of a thermophilic rubrerythrin results in approximately 2-{angstrom} movement of one iron atom in the diiron site from a histidine to a carboxylate ligand. These results confirm that, despite the domain-swapped architecture, the hyperthermophilic rubrerythrins also utilize the classic combination of iron sites together with redox-dependent iron toggling to selectively reduce hydrogen peroxide over dioxygen. In addition, we have identified previously unobserved intermediates in the reaction cycle and observed structural changes that may explain the enzyme precipitation observed for the all-iron form of PfRbr upon oxidation to the all-ferric state.

  13. A novel endonuclease that may be responsible for damaged DNA base repair in Pyrococcus furiosus

    PubMed Central

    Shiraishi, Miyako; Ishino, Sonoko; Yamagami, Takeshi; Egashira, Yuriko; Kiyonari, Shinichi; Ishino, Yoshizumi

    2015-01-01

    DNA is constantly damaged by endogenous and environmental influences. Deaminated adenine (hypoxanthine) tends to pair with cytosine and leads to the A:T→G:C transition mutation during DNA replication. Endonuclease V (EndoV) hydrolyzes the second phosphodiester bond 3′ from deoxyinosine in the DNA strand, and was considered to be responsible for hypoxanthine excision repair. However, the downstream pathway after EndoV cleavage remained unclear. The activity to cleave the phosphodiester bond 5′ from deoxyinosine was detected in a Pyrococcus furiosus cell extract. The protein encoded by PF1551, obtained from the mass spectrometry analysis of the purified fraction, exhibited the corresponding cleavage activity. A putative homolog from Thermococcus kodakarensis (TK0887) showed the same activity. Further biochemical analyses revealed that the purified PF1551 and TK0887 proteins recognize uracil, xanthine and the AP site, in addition to hypoxanthine. We named this endonuclease Endonuclease Q (EndoQ), as it may be involved in damaged base repair in the Thermococcals of Archaea. PMID:25694513

  14. Microwave-Assisted Synthesis of Glycoconjugates by Transgalactosylation with Recombinant Thermostable β-Glycosidase from Pyrococcus.

    PubMed

    Henze, Manja; Merker, Dorothee; Elling, Lothar

    2016-01-01

    The potential of the hyperthermophilic β-glycosidase from Pyrococcus woesei (DSM 3773) for the synthesis of glycosides under microwave irradiation (MWI) at low temperatures was investigated. Transgalactosylation reactions with β-N-acetyl-d-glucosamine as acceptor substrate (GlcNAc-linker-tBoc) under thermal heating (TH, 85 °C) and under MWI at 100 and 300 W resulted in the formation of (Galβ(1,4)GlcNAc-linker-tBoc) as the main product in all reactions. Most importantly, MWI at temperatures far below the temperature optimum of the hyperthermophilic glycosidase led to higher product yields with only minor amounts of side products β(1,6-linked disaccharide and trisaccharides). At high acceptor concentrations (50 mM), transgalactosylation reactions under MWI at 300 W gave similar product yields when compared to TH at 85 °C. In summary, we demonstrate that MWI is useful as a novel experimental set-up for the synthesis of defined galacto-oligosaccharides. In conclusion, glycosylation reactions under MWI at low temperatures have the potential as a general strategy for regioselective glycosylation reactions of hyperthermophilic glycosidases using heat-labile acceptor or donor substrates. PMID:26861292

  15. Molecular and biochemical characterization of the ADP-dependent phosphofructokinase from the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed

    Tuininga, J E; Verhees, C H; van der Oost, J; Kengen, S W; Stams, A J; de Vos, W M

    1999-07-23

    Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively. PMID:10409652

  16. Complete genome sequence of the hyperthermophilic archaeon Pyrococcus sp. strain ST04, isolated from a deep-sea hydrothermal sulfide chimney on the Juan de Fuca Ridge.

    PubMed

    Jung, Jong-Hyun; Lee, Ju-Hoon; Holden, James F; Seo, Dong-Ho; Shin, Hakdong; Kim, Hae-Yeong; Kim, Wooki; Ryu, Sangryeol; Park, Cheon-Seok

    2012-08-01

    Pyrococcus sp. strain ST04 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a deep-sea hydrothermal sulfide chimney on the Endeavour Segment of the Juan de Fuca Ridge in the northeastern Pacific Ocean. To further understand the distinct characteristics of this archaeon at the genome level (polysaccharide utilization at high temperature and ATP generation by a Na(+) gradient), the genome of strain ST04 was completely sequenced and analyzed. Here, we present the complete genome sequence analysis results of Pyrococcus sp. ST04 and report the major findings from the genome annotation, with a focus on its saccharolytic and metabolite production potential. PMID:22843576

  17. Complete Genome Sequence of the Hyperthermophilic Archaeon Pyrococcus sp. Strain ST04, Isolated from a Deep-Sea Hydrothermal Sulfide Chimney on the Juan de Fuca Ridge

    PubMed Central

    Jung, Jong-Hyun; Lee, Ju-Hoon; Holden, James F.; Seo, Dong-Ho; Shin, Hakdong; Kim, Hae-Yeong; Kim, Wooki; Ryu, Sangryeol

    2012-01-01

    Pyrococcus sp. strain ST04 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a deep-sea hydrothermal sulfide chimney on the Endeavour Segment of the Juan de Fuca Ridge in the northeastern Pacific Ocean. To further understand the distinct characteristics of this archaeon at the genome level (polysaccharide utilization at high temperature and ATP generation by a Na+ gradient), the genome of strain ST04 was completely sequenced and analyzed. Here, we present the complete genome sequence analysis results of Pyrococcus sp. ST04 and report the major findings from the genome annotation, with a focus on its saccharolytic and metabolite production potential. PMID:22843576

  18. Identification and Characterization of an Archaeal Kojibiose Catabolic Pathway in the Hyperthermophilic Pyrococcus sp. Strain ST04

    PubMed Central

    Jung, Jong-Hyun; Seo, Dong-Ho; Holden, James F.

    2014-01-01

    A unique gene cluster responsible for kojibiose utilization was identified in the genome of Pyrococcus sp. strain ST04. The proteins it encodes hydrolyze kojibiose, a disaccharide product of glucose caramelization, and form glucose-6-phosphate (G6P) in two steps. Heterologous expression of the kojibiose-related enzymes in Escherichia coli revealed that two genes, Py04_1502 and Py04_1503, encode kojibiose phosphorylase (designated PsKP, for Pyrococcus sp. strain ST04 kojibiose phosphorylase) and β-phosphoglucomutase (PsPGM), respectively. Enzymatic assays show that PsKP hydrolyzes kojibiose to glucose and β-glucose-1-phosphate (β-G1P). The Km values for kojibiose and phosphate were determined to be 2.53 ± 0.21 mM and 1.34 ± 0.04 mM, respectively. PsPGM then converts β-G1P into G6P in the presence of 6 mM MgCl2. Conversion activity from β-G1P to G6P was 46.81 ± 3.66 U/mg, and reverse conversion activity from G6P to β-G1P was 3.51 ± 0.13 U/mg. The proteins are highly thermostable, with optimal temperatures of 90°C for PsKP and 95°C for PsPGM. These results indicate that Pyrococcus sp. strain ST04 converts kojibiose into G6P, a substrate of the glycolytic pathway. This is the first report of a disaccharide utilization pathway via phosphorolysis in hyperthermophilic archaea. PMID:24391053

  19. Identification and characterization of an archaeal kojibiose catabolic pathway in the hyperthermophilic Pyrococcus sp. strain ST04.

    PubMed

    Jung, Jong-Hyun; Seo, Dong-Ho; Holden, James F; Park, Cheon-Seok

    2014-03-01

    A unique gene cluster responsible for kojibiose utilization was identified in the genome of Pyrococcus sp. strain ST04. The proteins it encodes hydrolyze kojibiose, a disaccharide product of glucose caramelization, and form glucose-6-phosphate (G6P) in two steps. Heterologous expression of the kojibiose-related enzymes in Escherichia coli revealed that two genes, Py04_1502 and Py04_1503, encode kojibiose phosphorylase (designated PsKP, for Pyrococcus sp. strain ST04 kojibiose phosphorylase) and β-phosphoglucomutase (PsPGM), respectively. Enzymatic assays show that PsKP hydrolyzes kojibiose to glucose and β-glucose-1-phosphate (β-G1P). The Km values for kojibiose and phosphate were determined to be 2.53 ± 0.21 mM and 1.34 ± 0.04 mM, respectively. PsPGM then converts β-G1P into G6P in the presence of 6 mM MgCl2. Conversion activity from β-G1P to G6P was 46.81 ± 3.66 U/mg, and reverse conversion activity from G6P to β-G1P was 3.51 ± 0.13 U/mg. The proteins are highly thermostable, with optimal temperatures of 90°C for PsKP and 95°C for PsPGM. These results indicate that Pyrococcus sp. strain ST04 converts kojibiose into G6P, a substrate of the glycolytic pathway. This is the first report of a disaccharide utilization pathway via phosphorolysis in hyperthermophilic archaea. PMID:24391053

  20. Structures of the superoxide reductase from Pyrococcus furiosus in the oxidized and reduced states.

    PubMed

    Yeh, A P; Hu, Y; Jenney, F E; Adams, M W; Rees, D C

    2000-03-14

    Superoxide reductase (SOR) is a blue non-heme iron protein that functions in anaerobic microbes as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to hydrogen peroxide [Jenney, F. E., Jr., Verhagen, M. F. J. M., Cui, X. , and Adams, M. W. W. (1999) Science 286, 306-309]. Crystal structures of SOR from the hyperthermophilic archaeon Pyrococcus furiosus have been determined in the oxidized and reduced forms to resolutions of 1.7 and 2.0 A, respectively. SOR forms a homotetramer, with each subunit adopting an immunoglobulin-like beta-barrel fold that coordinates a mononuclear, non-heme iron center. The protein fold and metal center are similar to those observed previously for the homologous protein desulfoferrodoxin from Desulfovibrio desulfuricans [Coelho, A. V., Matias, P., Fülöp, V., Thompson, A., Gonzalez, A., and Carrondo, M. A. (1997) J. Bioinorg. Chem. 2, 680-689]. Each iron is coordinated to imidazole nitrogens of four histidines in a planar arrangement, with a cysteine ligand occupying an axial position normal to this plane. In two of the subunits of the oxidized structure, a glutamate carboxylate serves as the sixth ligand to form an overall six-coordinate, octahedral coordinate environment. In the remaining two subunits, the sixth coordination site is either vacant or occupied by solvent molecules. The iron centers in all four subunits of the reduced structure exhibit pentacoordination. The structures of the oxidized and reduced forms of SOR suggest a mechanism by which superoxide accessibility may be controlled and define a possible binding site for rubredoxin, the likely physiological electron donor to SOR. PMID:10704199

  1. Engineering Hyperthermophilic Archaeon Pyrococcus furiosus to Overproduce Its Cytoplasmic [NiFe]-Hydrogenase*

    PubMed Central

    Chandrayan, Sanjeev K.; McTernan, Patrick M.; Hopkins, R. Christopher; Sun, Junsong; Jenney, Francis E.; Adams, Michael W. W.

    2012-01-01

    The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H2 production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production. PMID:22157005

  2. Identification of membrane proteins in the hyperthermophilic archaeon Pyrococcus furiosus using proteomics and prediction programs.

    SciTech Connect

    Holden, J. F.; Poole, F. L.; Tollaksen, S. L.; Giometti, C. S.; Lim, H.; Yates, J. R.; Adams, M. W. W.; Biosciences Division; Univ. of Georgia; The Scnpps Research Inst.

    2001-01-01

    Cell-free extracts from the hyperthermophilic archaeon Pyrococcus furiosus were separated into membrane and cytoplasmic fractions and each was analyzed by 2D-gel electrophoresis. A total of 66 proteins were identified, 32 in the membrane fraction and 34 in the cytoplasmic fraction. Six prediction programs were used to predict the subcellular locations of these proteins. Three were based on signal-peptides (SignalP, TargetP, and SOSUISignal) and three on transmembrane-spanning a-helices (TSEG, SOSUI, and PRED-TMR2). A consensus of the six programs predicted that 23 of the 32 proteins (72%) from the membrane fraction should be in the membrane and that all of the proteins from the cytoplasmic fraction should be in the cytoplasm. Two membrane-associated proteins predicted to be cytoplasmic by the programs are also predicted to consist primarily of transmembrane-spanning {beta}-sheets using porin protein models, suggesting that they are, in fact, membrane components. An ATPase subunit homolog found in the membrane fraction, although predicted to be cytoplasmic, is most likely complexed with other ATPase subunits in the membrane fraction. An additional three proteins predicted to be cytoplasmic but found in the membrane fraction, may be cytoplasmic contaminants. These include a chaperone homolog that may have attached to denatured membrane proteins during cell fractionation. Omitting these three proteins would boost the membrane-protein predictability of the models to near 80%. A consensus prediction using all six programs for all 2242 ORFs in the P. furiosus genome estimates that 24% of the ORF products are found in the membrane. However, this is likely to be a minimum value due to the programs' inability to recognize certain membrane-related proteins, such as subunits associated with membrane complexes and porin-type proteins.

  3. Highly thermostable RadA protein from the archaeon Pyrococcus woesei enhances specificity of simplex and multiplex PCR assays.

    PubMed

    Stefanska, Aleksandra; Gaffke, Lidia; Kaczorowska, Anna-Karina; Plotka, Magdalena; Dabrowski, Slawomir; Kaczorowski, Tadeusz

    2016-05-01

    The radA gene of the hyperthermophilic archaeon Pyrococcus woesei (Thermococcales) was cloned and overexpressed in Escherichia coli. The 1050-bp gene codes for a 349-amino-acid polypeptide with an M r of 38,397 which shows 100 % positional amino acid identity to Pyrococcus furiosus RadA and 27.1 % to the E. coli RecA protein. Recombinant RadA was overproduced in Escherichia coli as a His-tagged fusion protein and purified to electrophoretic homogeneity using a simple procedure consisting of ammonium sulfate precipitation and metal-affinity chromatography. In solution RadA exists as an undecamer (11-mer). The protein binds both to ssDNA and dsDNA. RadA has been found to be highly thermostable, it remains almost unaffected by a 4-h incubation at 94 °C. The addition of the RadA protein to either simplex or multiplex PCR assays, significantly improves the specificity of DNA amplification by eliminating non-specific products. Among applications tested the RadA protein proved to be useful in allelic discrimination assay of HADHA gene associated with long-chain 3-hydroxylacyl-CoA dehydrogenase deficiency that in infancy may lead to hypotonia, serious heart and liver problems and even sudden death. PMID:26337425

  4. Improving the catalytic activity of hyperthermophilic Pyrococcus prolidases for detoxification of organophosphorus nerve agents over a broad range of temperatures.

    PubMed

    Theriot, Casey M; Du, Xuelian; Tove, Sherry R; Grunden, Amy M

    2010-08-01

    Prolidase isolated from the hyperthermophilic archaeon Pyrococcus furiosus has potential for application for decontamination of organophosphorus compounds in certain pesticides and chemical warfare agents under harsh conditions. However, current applications that use an enzyme-based cocktail are limited by poor long-term enzyme stability and low reactivity over a broad range of temperatures. To obtain a better enzyme for OP nerve agent decontamination and to investigate structural factors that influence protein thermostability and thermoactivity, randomly mutated P. furiosus prolidases were prepared by using XL1-red-based mutagenesis and error-prone PCR. An Escherichia coli strain JD1 (lambdaDE3) (auxotrophic for proline [DeltaproA] and having deletions in pepQ and pepP dipeptidases with specificity for proline-containing dipeptides) was constructed for screening mutant P. furiosus prolidase expression plasmids. JD1 (lambdaDE3) cells were transformed with mutated prolidase expression plasmids and plated on minimal media supplemented with 50 muM Leu-Pro as the only source of proline. By using this positive selection, Pyrococcus prolidase mutants with improved activity over a broader range of temperatures were isolated. The activities of the mutants over a broad temperature range were measured for both Xaa-Pro dipeptides and OP nerve agents, and the thermoactivity and thermostability of the mutants were determined. PMID:20422176

  5. Crystallization and preliminary X-ray analysis of a RecB-family nuclease from the archaeon Pyrococcus abyssi

    SciTech Connect

    Ren, Bin; Kuhn, Joëlle; Meslet-Cladiere, Laurence; Myllykallio, Hannu; Ladenstein, Rudolf

    2007-05-01

    A RecB-like nuclease from the archaeon Pyrococcus abyssi was expressed, purified and crystallized. The crystals belong to the orthorhombic space group C222{sub 1} with a = 81.5, b = 159.8, c = 100.8 Å, and a native data set was collected to 2.65 Å resolution. Nucleases are required to process and repair DNA damage in living cells. One of the best studied nucleases is the RecB protein, which functions in Escherichia coli as a component of the RecBCD enzyme complex that amends double-strand breaks in DNA. Although archaea do not contain the RecBCD complex, a RecB-like nuclease from Pyrococcus abyssi has been cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant. The crystals belong to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 81.5, b = 159.8, c = 100.8 Å. Self-rotation function and native Patterson map calculations revealed that there is a dimer in the asymmetric unit with its local twofold axis running parallel to the crystallographic twofold screw axis. The crystals diffracted to about 2 Å and a complete native data set was collected to 2.65 Å resolution.

  6. Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics

    PubMed Central

    Yuan, Hui; Peng, Li; Han, Zhong; Xie, Juan-Juan; Liu, Xi-Peng

    2015-01-01

    Hyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of P. furiosus proteins at whole genome level, we constructed expression plasmids of each P. furiosus gene using a ligase-independent cloning method, which was based on amplifying target gene and vector by PCR using phosphorothioate-modified primers and digesting PCR products by λ exonuclease. Our cloning method had a positive clone percentage of ≥ 80% in 96-well plate cloning format. Small-scale expression experiment showed that 55 out of 80 genes were efficiently expressed in Escherichia coli Strain Rosetta 2(DE3)pLysS. In summary, this recombinant expression library of P. furiosus provides a platform for functional and structural studies, as well as developing novel industrial enzymes. Our cloning scheme is adaptable to constructing recombinant expression library of other sequenced organisms. PMID:26441878

  7. Anaerobic desulfurization of ground rubber with the thermophilic archaeon Pyrococcus furiosus--a new method for rubber recycling.

    PubMed

    Bredberg, K; Persson, J; Christiansson, M; Stenberg, B; Holst, O

    2001-01-01

    The anaerobic sulfur-reducing archaeon Pyrococcus furiosus was investigated regarding its capacity to desulfurize rubber material. The microorganism's sensitivity towards common rubber elastomers and additives was tested and several were shown to be toxic to P. furiosus. The microorganism was shown to utilize sulfur in vulcanized natural rubber and an increase in cell density was obtained when cultivated in the presence of spent tire rubber. Ethanol-leached cryo-ground tire rubber treated with P. furiosus for 10 days was vulcanized together with virgin rubber material (15% w/w) and the mechanical properties of the resulting material were determined. The increase in the stress at break value and the decrease in swell ratio and stress relaxation rate obtained for material containing microbially treated rubber (compared to untreated material) show the positive effects of microbial desulfurization on rubber. PMID:11234957

  8. Studies on Hydrogen Production by Photosynthetic Bacteria after Anaerobic Fermentation of Starch by a Hyperthermophile, Pyrococcus furiosus

    NASA Astrophysics Data System (ADS)

    Sugitate, Toshihiro; Fukatsu, Makoto; Ishimi, Katsuhiro; Kohno, Hideki; Wakayama, Tatsuki; Nakamura, Yoshihiro; Miyake, Jun; Asada, Yasuo

    In order to establish the sequential hydrogen production from waste starch using a hyperthermophile, Pyrococcus furiosus, and a photosynthetic bacterium, basic studies were done. P. furiosus produced hydrogen and acetate by anaerobic fermentation at 90°C. A photosynthetic bacterium, Rhodobacter sphaeroides RV, was able to produce hydrogen from acetate under anaerobic and light conditions at 30°C. However, Rb. sphaeroides RV was not able to produce hydrogen from acetate in the presence of sodium chloride that was essential for the growth and hydrogen production of P. furiosus although it produced hydrogen from lactate at a reduced rate with 1% sodium chloride. A newly isolated strain, CST-8, from natural environment was, however, able to produce hydrogen from acetate, especially with 3 mM L-alanine and in the presence of 1% sodium chloride. The sequential hydrogen production with P. furiosus and salt-tolerant photosynthetic bacteria could be probable at least in the laboratory experiment scale.

  9. Cell-free transcription at 95 degrees: thermostability of transcriptional components and DNA topology requirements of Pyrococcus transcription.

    PubMed Central

    Hethke, C; Bergerat, A; Hausner, W; Forterre, P; Thomm, M

    1999-01-01

    Cell-free transcription of archaeal promoters is mediated by two archaeal transcription factors, aTBP and TFB, which are orthologues of the eukaryotic transcription factors TBP and TFIIB. Using the cell-free transcription system described for the hyperthermophilic Archaeon Pyrococcus furiosus by Hethke et al., the temperature limits and template topology requirements of archaeal transcription were investigated. aTBP activity was not affected after incubation for 1 hr at 100 degrees. In contrast, the half-life of RNA polymerase activity was 23 min and that of TFB activity was 3 min. The half-life of a 328-nt RNA product was 10 min at 100 degrees. Best stability of RNA was observed at pH 6, at 400 mm K-glutamate in the absence of Mg(2+) ions. Physiological concentrations of K-glutamate were found to stabilize protein components in addition, indicating that salt is an important extrinsic factor contributing to thermostability. Both RNA and proteins were stabilized by the osmolyte betaine at a concentration of 1 m. The highest activity for RNA synthesis at 95 degrees was obtained in the presence of 1 m betaine and 400 mm K-glutamate. Positively supercoiled DNA, which was found to exist in Pyrococcus cells, can be transcribed in vitro both at 70 degrees and 90 degrees. However, negatively supercoiled DNA was the preferred template at all temperatures tested. Analyses of transcripts from plasmid topoisomers harboring the glutamate dehydrogenase promoter and of transcription reactions conducted in the presence of reverse gyrase indicate that positive supercoiling of DNA inhibits transcription from this promoter. PMID:10430563

  10. Purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed Central

    Cheng, T. C.; Ramakrishnan, V.; Chan, S. I.

    1999-01-01

    A novel metallocarboxypeptidase (PfuCP) has been purified to homogeneity from the hyperthermophilic archaeon, Pyrococcus furiosus, with its intended use in C-terminal ladder sequencing of proteins and peptides at elevated temperatures. PfuCP was purified in its inactive state by the addition of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) to purification buffers, and the activity was restored by the addition of divalent cobalt (K, = 24 +/- 4 microM at 80 degrees C). The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) had no effect on the activity. The molecular mass of monomeric PfuCP is 59 kDa as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 58 kDa by SDS-PAGE analysis. In solution, PfuCP exists as a homodimer of approximately 128 kDa as determined by gel filtration chromatography. The activity of PfuCP exhibits a temperature optimum exceeding 90 degrees C under ambient pressure, and a narrow pH optimum of 6.2-6.6. Addition of Co2+ to the apoPfuCP at room temperature does not alter its far-UV circular dichroism (CD) or its intrinsic fluorescence spectrum. Even when the CoPfuCP is heated to 80 degrees C, its far-UV CD shows a minimal change in the global conformation and the intrinsic fluorescence of aromatic residues shows only a partial quenching. Changes in the intrinsic fluorescence appear essentially reversible with temperature. Finally, the far-UV CD and intrinsic fluorescence data suggest that the overall structure of the holoenzyme is extremely thermostable. However, the activities of both the apo and holo enzyme exhibit a similar second-order decay over time, with 50% activity remaining after approximately 40 min at 80 degrees C. The N-blocked synthetic dipeptide, N-carbobenzoxy-Ala-Arg (ZAR), was used in the purification assay. The kinetic parameters at 80 degrees C with 0.4 mM CoCl2 were: Km, 0.9 +/- 0.1 mM; Vmax, 2,300 +/- 70 U mg(-1); and turn over number

  11. Cloning, expression, and molecular characterization of the gene encoding an extremely thermostable [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed Central

    Heltzel, A; Smith, E T; Zhou, Z H; Blamey, J M; Adams, M W

    1994-01-01

    The gene for ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, sequenced, and expressed in Escherichia coli. The coding region confirmed the determined amino acid sequence. Putative archaeon-type transcriptional regulatory elements were identified. The fdxA gene appears to be an independent transcriptional unit. Recombinant ferredoxin was indistinguishable from the protein purified from P. furiosus in its thermal stability and in the potentiometric and spectroscopic properties of its [4Fe-4S] cluster. PMID:8045914

  12. Microwave-Assisted Synthesis of Glycoconjugates by Transgalactosylation with Recombinant Thermostable β-Glycosidase from Pyrococcus

    PubMed Central

    Henze, Manja; Merker, Dorothee; Elling, Lothar

    2016-01-01

    The potential of the hyperthermophilic β-glycosidase from Pyrococcus woesei (DSM 3773) for the synthesis of glycosides under microwave irradiation (MWI) at low temperatures was investigated. Transgalactosylation reactions with β-N-acetyl-d-glucosamine as acceptor substrate (GlcNAc-linker-tBoc) under thermal heating (TH, 85 °C) and under MWI at 100 and 300 W resulted in the formation of (Galβ(1,4)GlcNAc-linker-tBoc) as the main product in all reactions. Most importantly, MWI at temperatures far below the temperature optimum of the hyperthermophilic glycosidase led to higher product yields with only minor amounts of side products β(1,6-linked disaccharide and trisaccharides). At high acceptor concentrations (50 mM), transgalactosylation reactions under MWI at 300 W gave similar product yields when compared to TH at 85 °C. In summary, we demonstrate that MWI is useful as a novel experimental set-up for the synthesis of defined galacto-oligosaccharides. In conclusion, glycosylation reactions under MWI at low temperatures have the potential as a general strategy for regioselective glycosylation reactions of hyperthermophilic glycosidases using heat-labile acceptor or donor substrates. PMID:26861292

  13. Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Covalent Immobilization

    PubMed Central

    Branco, Roberta V.; Gutarra, Melissa L. E.; Freire, Denise M. G.; Almeida, Rodrigo V.; Palomo, Jose M.

    2015-01-01

    A recombinant thermostable lipase (Pf2001Δ60) from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL) was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL) and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment) (glyoxyl-DTT PFUL) and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment) (glyoxyl PFUL). The enzyme's properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity), whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity). Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity (E = 22) and enantiomeric excess (ee (%) = 91). PMID:25839031

  14. Characterization of the Recombinant Thermostable Lipase (Pf2001) from Pyrococcus furiosus: Effects of Thioredoxin Fusion Tag and Triton X-100

    PubMed Central

    Alquéres, Sylvia Maria Campbell; Branco, Roberta Vieira; Freire, Denise Maria Guimarães; Alves, Tito Lívio Moitinho; Martins, Orlando Bonifácio; Almeida, Rodrigo Volcan

    2011-01-01

    In this work, the lipase from Pyrococcus furiosus encoded by ORF PF2001 was expressed with a fusion protein (thioredoxin) in Escherichia coli. The purified enzymes with the thioredoxin tag (TRX−PF2001Δ60) and without the thioredoxin tag (PF2001Δ60) were characterized, and various influences of Triton X-100 were determined. The optimal temperature for both enzymes was 80°C. Although the thioredoxin presence did not influence the optimum temperature, the TRX−PF2001Δ60 presented specific activity twice lower than the enzyme PF2001Δ60. The enzyme PF2001Δ60 was assayed using MUF-acetate, MUF-heptanoate, and MUF-palmitate. MUF-heptanoate was the preferred substrate of this enzyme. The chelators EDTA and EGTA increased the enzyme activity by 97 and 70%, respectively. The surfactant Triton X-100 reduced the enzyme activity by 50% and lowered the optimum temperature to 60°C. However, the thermostability of the enzyme PF2001Δ60 was enhanced with Triton X-100. PMID:21760993

  15. Neutron crystallographic study on rubredoxin from Pyrococcus furiosus by BIX-3, a single-crystal diffractometer for biomacromolecules.

    PubMed

    Kurihara, Kazuo; Tanaka, Ichiro; Chatake, Toshiyuki; Adams, Michael W W; Jenney, Francis E; Moiseeva, Natalia; Bau, Robert; Niimura, Nobuo

    2004-08-01

    The structure of a partially deuterated rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus, an organism that grows optimally at 100 degrees C, was determined by using the neutron single-crystal diffractometer dedicated for biological macromolecules (BIX-3) at the JRR-3M reactor of the Japan Atomic Energy Research Institute. Data were collected at room temperature up to a resolution of 1.5 A, and the completeness factor of the data set was 81.9%. The model contains 306 H and 50 D atoms. A total of 37 hydration water molecules were identified, with 15 having all three atoms fully located and the remaining D2O molecules partially defined. The model has been refined to final agreement factors of R = 18.6% and Rfree = 21.7%. Several orientations of the O-D bonds of side chains, whose assignments from x-ray data were previously ambiguous, were clearly visible in the neutron structure. Although most backbone N-H bonds had undergone some degree of H/D exchange throughout the rubredoxin molecule, 5 H atom positions still had distinctly negative (H) peaks. The neutron Fourier maps clearly showed the details of an extensive set of H bonds involving the ND3+ terminus that may contribute to the unusual thermostability of this molecule. PMID:15272083

  16. N-Linked Glycans Are Assembled on Highly Reduced Dolichol Phosphate Carriers in the Hyperthermophilic Archaea Pyrococcus furiosus

    PubMed Central

    Chang, Michelle M.; Imperiali, Barbara; Eichler, Jerry; Guan, Ziqiang

    2015-01-01

    In all three domains of life, N-glycosylation begins with the assembly of glycans on phosphorylated polyisoprenoid carriers. Like eukaryotes, archaea also utilize phosphorylated dolichol for this role, yet whereas the assembled oligosaccharide is transferred to target proteins from dolichol pyrophosphate in eukaryotes, archaeal N-linked glycans characterized to date are derived from a dolichol monophosphate carrier, apart from a single example. In this study, glycan-charged dolichol phosphate from the hyperthermophile Pyrococcus furiosus was identified and structurally characterized. Normal and reverse phase liquid chromatography-electrospray ionization mass spectrometry revealed the existence of dolichol phosphate charged with the heptasaccharide recently described in in vitro studies of N-glycosylation on this species. As with other described archaeal dolichol phosphates, the α- and ω-terminal isoprene subunits of the P. furiosus lipid are saturated, in contrast to eukaryal phosphodolichols that present only a saturated α-position isoprene subunit. Interestingly, an additional 1-4 of the 12-14 isoprene subunits comprising P. furiosus dolichol phosphate are saturated, making this lipid not only the longest archaeal dolichol phosphate described to date but also the most highly saturated. PMID:26098850

  17. Characterization of Ten Heterotetrameric NDP-Dependent Acyl-CoA Synthetases of the Hyperthermophilic Archaeon Pyrococcus furiosus

    DOE PAGESBeta

    Scott, Joseph W.; Poole, Farris L.; Adams, Michael W. W.

    2014-01-01

    Tmore » he hyperthermophilic archaeon Pyrococcus furiosus grows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified from P. furiosus and have α 2 β 2 structures but the genome contains genes encoding three additional α -subunits.he ten possible combinations of α and β genes were expressed in E. coli and each resulted in stable and active α 2 β 2 isoenzymes.he α -subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids.he β -subunit determined preference for adenine or guanine nucleotides.he GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes.ranscriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs of P. furiosus and other members of thehermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.« less

  18. An In Silico Approach for Characterization of an Aminoglycoside Antibiotic-Resistant Methyltransferase Protein from Pyrococcus furiosus (DSM 3638)

    PubMed Central

    Oany, Arafat Rahman; Jyoti, Tahmina Pervin; Ahmad, Shah Adil Ishtiyaq

    2014-01-01

    Pyrococcus furiosus is a hyperthermophilic archaea. A hypothetical protein of this archaea, PF0847, was selected for computational analysis. Basic local alignment search tool and multiple sequence alignment (MSA) tool were employed to search for related proteins. Both the secondary and tertiary structure prediction were obtained for further analysis. Three-dimensional model was assessed by PROCHECK and QMEAN6 programs. To get insights about the physical and functional associations of the protein, STRING network analysis was performed. Binding of the SAM (S-adenosyl-l-methionine) ligand with our protein, fetched from an antibiotic-related methyltransferase (PDB code: 3P2K: D), showed high docking energy and suggested the function of the protein as methyltransferase. Finally, we tried to look for a specific function of the proposed methyltransferase, and binding of the geneticin bound to the eubacterial 16S rRNA A-site (PDB code: 1MWL) in the active site of the PF0847 gave us the indication to predict the protein responsible for aminoglycoside antibiotic resistance. PMID:24683305

  19. Changes in the catalytic properties of Pyrococcus furiosus thermostable amylase by mutagenesis of the substrate binding sites.

    PubMed

    Yang, Sung-Jae; Min, Byoung-Chul; Kim, Young-Wan; Jang, Sang-Mok; Lee, Byong-Hoon; Park, Kwan-Hwa

    2007-09-01

    Pyrococcus furiosus thermostable amylase (TA) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of four residues (His414, Gly415, Met439, and Asp440) in the function of P. furiosus TA by using site-directed mutagenesis and kinetic analysis. A variant form of P. furiosus TA containing two mutations (H414N and G415E) exhibited strongly enhanced alpha-(1,4)-transglycosylation activity, resulting in the production of a series of maltooligosaccharides that were longer than the initial substrates. In contrast, the variant enzymes with single mutations (H414N or G415E) showed a substrate preference similar to that of the wild-type enzyme. Other mutations (M439W and D440H) reversed the substrate preference of P. furiosus TA from CDs to maltooligosaccharides. Relative substrate preferences for maltoheptaose over beta-CD, calculated by comparing k(cat)/K(m) ratios, of 1, 8, and 26 for wild-type P. furiosus TA, P. furiosus TA with D440H, and P. furiosus TA with M439W and D440H, respectively, were found. Our results suggest that His414, Gly415, Met439, and Asp440 play important roles in substrate recognition and transglycosylation. Therefore, this study provides information useful in engineering glycoside hydrolase family 13 enzymes. PMID:17630303

  20. DNA targeting by the type I-G and type I-A CRISPR–Cas systems of Pyrococcus furiosus

    PubMed Central

    Elmore, Joshua; Deighan, Trace; Westpheling, Jan; Terns, Rebecca M.; Terns, Michael P.

    2015-01-01

    CRISPR–Cas systems silence plasmids and viruses in prokaryotes. CRISPR–Cas effector complexes contain CRISPR RNAs (crRNAs) that include sequences captured from invaders and direct CRISPR-associated (Cas) proteins to destroy corresponding invader nucleic acids. Pyrococcus furiosus (Pfu) harbors three CRISPR–Cas immune systems: a Cst (Type I-G) system with an associated Cmr (Type III-B) module at one locus, and a partial Csa (Type I-A) module (lacking known invader sequence acquisition and crRNA processing genes) at another locus. The Pfu Cmr complex cleaves complementary target RNAs, and Csa systems have been shown to target DNA, while the mechanism by which Cst complexes silence invaders is unknown. In this study, we investigated the function of the Cst as well as Csa system in Pfu strains harboring a single CRISPR–Cas system. Plasmid transformation assays revealed that the Cst and Csa systems both function by DNA silencing and utilize similar flanking sequence information (PAMs) to identify invader DNA. Silencing by each system specifically requires its associated Cas3 nuclease. crRNAs from the 7 shared CRISPR loci in Pfu are processed for use by all 3 effector complexes, and Northern analysis revealed that individual effector complexes dictate the profile of mature crRNA species that is generated. PMID:26519471

  1. Expression and Characterization of a Novel Glycerophosphodiester Phosphodiesterase from Pyrococcus furiosus DSM 3638 That Possesses Lysophospholipase D Activity.

    PubMed

    Wang, Fanghua; Lai, Linhui; Liu, Yanhua; Yang, Bo; Wang, Yonghua

    2016-01-01

    Glycerophosphodiester phosphodiesterases (GDPD) are enzymes which degrade various glycerophosphodiesters to produce glycerol-3-phosphate and the corresponding alcohol moiety. Apart from this, a very interesting finding is that this enzyme could be used in the degradation of toxic organophosphorus esters, which has resulted in much attention on the biochemical and application research of GDPDs. In the present study, a novel GDPD from Pyrococcus furiosus DSM 3638 (pfGDPD) was successfully expressed in Escherichia coli and biochemically characterized. This enzyme hydrolyzed bis(p-nitrophenyl) phosphate, one substrate analogue of organophosphorus diester, with an optimal reaction temperature 55 °C and pH 8.5. The activity of pfGDPD was strongly dependent on existing of bivalent cations. It was strongly stimulated by Mn(2+) ions, next was Co(2+) and Ni(2+) ions. Further investigations were conducted on its substrate selectivity towards different phospholipids. The results indicated that except of glycerophosphorylcholine (GPC), this enzyme also possessed lysophospholipase D activity toward both sn1-lysophosphatidylcholine (1-LPC) and sn2-lysophosphatidylcholine (2-LPC). Higher activity was found for 1-LPC than 2-LPC; however, no hydrolytic activity was found for phosphatidylcholine (PC). Molecular docking based on the 3D-modeled structure of pfGDPD was conducted in order to provide a structural foundation for the substrate selectivity. PMID:27248999

  2. Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic Archaeon Pyrococcus furiosus

    PubMed Central

    Adams, Michael W. W.; Holden, James F.; Menon, Angeli Lal; Schut, Gerrit J.; Grunden, Amy M.; Hou, Chun; Hutchins, Andrea M.; Jenney, Francis E.; Kim, Chulhwan; Ma, Kesen; Pan, Guangliang; Roy, Roopali; Sapra, Rajat; Story, Sherry V.; Verhagen, Marc F. J. M.

    2001-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S0). Growth rates were highest on media containing peptides and S0, with or without maltose. Growth did not occur on the peptide medium without S0. S0 had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S0 with or without maltose were the same, suggesting that S0 is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S0 in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S0-reducing enzyme in this organism and the mechanism of the S0 dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites. PMID:11133967

  3. Structures of the Signal Recognition Particle Receptor From the Archaeon Pyrococcus Furiosus: Implications for the Targeting Step at the Membrane

    SciTech Connect

    Egea, P.F.; Tsuruta, H.; Leon, G.P.de; Napetschnig, J.; Walter, P.; Stroud, R.M.

    2009-05-18

    In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP {center_dot} magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP {center_dot} SR targeting complexes.

  4. Expression and Characterization of a Novel Glycerophosphodiester Phosphodiesterase from Pyrococcus furiosus DSM 3638 That Possesses Lysophospholipase D Activity

    PubMed Central

    Wang, Fanghua; Lai, Linhui; Liu, Yanhua; Yang, Bo; Wang, Yonghua

    2016-01-01

    Glycerophosphodiester phosphodiesterases (GDPD) are enzymes which degrade various glycerophosphodiesters to produce glycerol-3-phosphate and the corresponding alcohol moiety. Apart from this, a very interesting finding is that this enzyme could be used in the degradation of toxic organophosphorus esters, which has resulted in much attention on the biochemical and application research of GDPDs. In the present study, a novel GDPD from Pyrococcus furiosus DSM 3638 (pfGDPD) was successfully expressed in Escherichia coli and biochemically characterized. This enzyme hydrolyzed bis(p-nitrophenyl) phosphate, one substrate analogue of organophosphorus diester, with an optimal reaction temperature 55 °C and pH 8.5. The activity of pfGDPD was strongly dependent on existing of bivalent cations. It was strongly stimulated by Mn2+ ions, next was Co2+ and Ni2+ ions. Further investigations were conducted on its substrate selectivity towards different phospholipids. The results indicated that except of glycerophosphorylcholine (GPC), this enzyme also possessed lysophospholipase D activity toward both sn1-lysophosphatidylcholine (1-LPC) and sn2-lysophosphatidylcholine (2-LPC). Higher activity was found for 1-LPC than 2-LPC; however, no hydrolytic activity was found for phosphatidylcholine (PC). Molecular docking based on the 3D-modeled structure of pfGDPD was conducted in order to provide a structural foundation for the substrate selectivity. PMID:27248999

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

    SciTech Connect

    Delfosse, Vanessa; Hugonnet, Jean-Emmanuel; Sougakoff, Wladimir; Mayer, Claudine

    2005-11-01

    The crystallization of a hypothetical penicillin-binding protein from the archaeon P. abyssi in space group C2 by hanging-drop vapour diffusion is reported. 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 β-lactamases. The Pab0087 protein was overexpressed, purified and crystallized. Diffraction data from two different crystal forms were collected to 2.7 and 2.0 Å resolution. Both crystals belong to space group C2, with unit-cell parameters a = 160.59, b = 135.74, c = 113.02 Å, β = 117.36° and a = 166.97, b = 131.25, c = 189.39 Å, β = 113.81°, respectively. The asymmetric unit contains four and eight molecules, respectively, with fourfold non-crystallographic symmetry.

  6. Flagella of Pyrococcus furiosus: multifunctional organelles, made for swimming, adhesion to various surfaces, and cell-cell contacts.

    PubMed

    Näther, Daniela J; Rachel, Reinhard; Wanner, Gerhard; Wirth, Reinhard

    2006-10-01

    Pyrococcus furiosus ("rushing fireball") was named for the ability of this archaeal coccus to rapidly swim at its optimal growth temperature, around 100 degrees C. Early electron microscopic studies identified up to 50 cell surface appendages originating from one pole of the coccus, which have been called flagella. We have analyzed these putative motility organelles and found them to be composed primarily (>95%) of a glycoprotein that is homologous to flagellins from other archaea. Using various electron microscopic techniques, we found that these flagella can aggregate into cable-like structures, forming cell-cell connections between ca. 5% of all cells during stationary growth phase. P. furiosus cells could adhere via their flagella to carbon-coated gold grids used for electron microscopic analyses, to sand grains collected from the original habitat (Porto di Levante, Vulcano, Italy), and to various other surfaces. P. furiosus grew on surfaces in biofilm-like structures, forming microcolonies with cells interconnected by flagella and adhering to the solid supports. Therefore, we concluded that P. furiosus probably uses flagella for swimming but that the cell surface appendages also enable this archaeon to form cable-like cell-cell connections and to adhere to solid surfaces. PMID:16980494

  7. An Extended Network of Genomic Maintenance in the Archaeon Pyrococcus abyssi Highlights Unexpected Associations between Eucaryotic Homologs

    PubMed Central

    Pluchon, Pierre-François; Fouqueau, Thomas; Crezé, Christophe; Laurent, Sébastien; Briffotaux, Julien; Hogrel, Gaëlle; Palud, Adeline; Henneke, Ghislaine; Godfroy, Anne; Hausner, Winfried; Thomm, Michael; Nicolas, Jacques; Flament, Didier

    2013-01-01

    In Archaea, the proteins involved in the genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of eukaryotes. Characterizations of components of the eukaryotic-type replication machinery complex provided many interesting insights into DNA replication in both domains. In contrast, DNA repair processes of hyperthermophilic archaea are less well understood and very little is known about the intertwining between DNA synthesis, repair and recombination pathways. The development of genetic system in hyperthermophilic archaea is still at a modest stage hampering the use of complementary approaches of reverse genetics and biochemistry to elucidate the function of new candidate DNA repair gene. To gain insights into genomic maintenance processes in hyperthermophilic archaea, a protein-interaction network centred on informational processes of Pyrococcus abyssi was generated by affinity purification coupled with mass spectrometry. The network consists of 132 interactions linking 87 proteins. These interactions give insights into the connections of DNA replication with recombination and repair, leading to the discovery of new archaeal components and of associations between eucaryotic homologs. Although this approach did not allow us to clearly delineate new DNA pathways, it provided numerous clues towards the function of new molecular complexes with the potential to better understand genomic maintenance processes in hyperthermophilic archaea. Among others, we found new potential partners of the replication clamp and demonstrated that the single strand DNA binding protein, Replication Protein A, enhances the transcription rate, in vitro, of RNA polymerase. This interaction map provides a valuable tool to explore new aspects of genome integrity in Archaea and also potentially in Eucaryotes. PMID:24244547

  8. Structural basis of thermal stability of the tungsten cofactor synthesis protein MoaB from Pyrococcus furiosus.

    PubMed

    Havarushka, Nastassia; Fischer-Schrader, Katrin; Lamkemeyer, Tobias; Schwarz, Guenter

    2014-01-01

    Molybdenum and tungsten cofactors share a similar pterin-based scaffold, which hosts an ene-dithiolate function being essential for the coordination of either molybdenum or tungsten. The biosynthesis of both cofactors involves a multistep pathway, which ends with the activation of the metal binding pterin (MPT) by adenylylation before the respective metal is incorporated. In the hyperthermophilic organism Pyrococcus furiosus, the hexameric protein MoaB (PfuMoaB) has been shown to catalyse MPT-adenylylation. Here we determined the crystal structure of PfuMoaB at 2.5 Å resolution and identified key residues of α3-helix mediating hexamer formation. Given that PfuMoaB homologues from mesophilic organisms form trimers, we investigated the impact on PfuMoaB hexamerization on thermal stability and activity. Using structure-guided mutagenesis, we successfully disrupted the hexamer interface in PfuMoaB. The resulting PfuMoaB-H3 variant formed monomers, dimers and trimers as determined by size exclusion chromatography. Circular dichroism spectroscopy as well as chemical cross-linking coupled to mass spectrometry confirmed a wild-type-like fold of the protomers as well as inter-subunits contacts. The melting temperature of PfuMoaB-H3 was found to be reduced by more than 15 °C as determined by differential scanning calorimetry, thus demonstrating hexamerization as key determinant for PfuMoaB thermal stability. Remarkably, while a loss of activity at temperatures higher than 50 °C was observed in the PfuMoaB-H3 variant, at lower temperatures, we determined a significantly increased catalytic activity. The latter suggests a gain in conformational flexibility caused by the disruption of the hexamerization interface. PMID:24465852

  9. A thermostable hybrid cluster protein from Pyrococcus furiosus: effects of the loss of a three helix bundle subdomain.

    PubMed

    Overeijnder, Marieke L; Hagen, Wilfred R; Hagedoorn, Peter-Leon

    2009-06-01

    Pyrococcus furiosus hybrid cluster protein (HCP) was expressed in Escherichia coli, purified, and characterized. This is the first archaeal and thermostable HCP to be isolated. Compared with the protein sequences of previously characterized HCPs from mesophiles, the protein sequence of P. furiosus HCP exhibits a deletion of approximately 13 kDa as a single amino acid stretch just after the N-terminal cysteine motif, characteristic for class-III HCPs from (hyper)thermophilic archaea and bacteria. The protein was expressed as a thermostable, soluble homodimeric protein. Hydroxylamine reductase activity of P. furiosus HCP showed a K(m) value of 0.40 mM and a k(cat) value of 3.8 s(-1) at 70 degrees C and pH 9.0. Electron paramagnetic resonance spectroscopy showed evidence for the presence of a spin-admixed, S = 3/2 [4Fe-4S](+) cubane cluster and of the hybrid cluster. The cubane cluster of P. furiosus HCP is presumably coordinated by a CXXC-X(7)-C-X(5)-C motif close to the N-terminus, which is similar to the CXXC-X(8)-C-X(5)-C motif of the Desulfovibrio desulfuricans and Desulfovibrio vulgaris HCPs. Amino acid sequence alignment and homology modeling of P. furiosus HCP reveal that the deletion results in a loss of one of the two three-helix bundles of domain 1. Clearly the loss of one of the three-helix bundles of domain 1 does not diminish the hydroxylamine reduction activity and the incorporation of the iron-sulfur clusters. PMID:19241093

  10. Improving dideoxynucleotide-triphosphate utilisation by the hyper-thermophilic DNA polymerase from the archaeon Pyrococcus furiosus

    PubMed Central

    Evans, Steven J.; Fogg, Mark J.; Mamone, Anthony; Davis, Maria; Pearl, Laurence H.; Connolly, Bernard A.

    2000-01-01

    Polymerases from the Pol-I family which are able to efficiently use ddNTPs have demonstrated a much improved performance when used to sequence DNA. A number of mutations have been made to the gene coding for the Pol-II family DNA polymerase from the archaeon Pyrococcus furiosus with the aim of improving ddNTP utilisation. ‘Rational’ alterations to amino acids likely to be near the dNTP binding site (based on sequence homologies and structural information) did not yield the desired level of selectivity for ddNTPs. However, alteration at four positions (Q472, A486, L490 and Y497) gave rise to variants which incorporated ddNTPs better than the wild type, allowing sequencing reactions to be carried out at lowered ddNTP:dNTP ratios. Wild-type Pfu–Pol required a ddNTP:dNTP ratio of 30:1; values of 5:1 (Q472H), 1:3 (L490W), 1:5 (A486Y) and 5:1 (Y497A) were found with the four mutants; A486Y representing a 150-fold improvement over the wild type. A486, L490 and Y497 are on an α-helix that lines the dNTP binding groove, but the side chains of the three amino acids point away from this groove; Q472 is in a loop that connects this α-helix to a second long helix. None of the four amino acids can contact the dNTP directly. Therefore, the increased selectivity for ddNTPs is likely to arise from two factors: (i) small overall changes in conformation that subtly alter the nucleotide triphosphate binding site such that ddNTPs become favoured; (ii) interference with a conformational change that may be critical both for the polymerisation step and discrimination between different nucleotide triphosphates. PMID:10666444

  11. A Unique Chitinase with Dual Active Sites and Triple Substrate Binding Sites from the Hyperthermophilic Archaeon Pyrococcus kodakaraensis KOD1

    PubMed Central

    Tanaka, Takeshi; Fujiwara, Shinsuke; Nishikori, Shingo; Fukui, Toshiaki; Takagi, Masahiro; Imanaka, Tadayuki

    1999-01-01

    We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85°C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA. PMID:10583986

  12. A billion-fold range in acidity for the solvent-exposed amides of Pyrococcus furiosus rubredoxin.

    PubMed

    Anderson, Janet S; Hernández, Griselda; Lemaster, David M

    2008-06-10

    The exchange rates of the static solvent-accessible amide hydrogens of Pyrococcus furiosus rubredoxin range from near the diffusion-limited rate to a billion-fold slower for the non-hydrogen-bonded Val 38 (eubacterial numbering). Hydrogen exchange directly monitors the kinetic acidity of the peptide nitrogen. Electrostatic solvation free energies were calculated by Poisson-Boltzmann methods for the individual peptide anions that form during the hydroxide-catalyzed exchange reaction to examine how well the predicted thermodynamic acidities match the experimentally determined kinetic acidities. With the exception of the Ile 12 amide, the differential exchange rate constant for each solvent-exposed amide proton that is not hydrogen bonded to a backbone carbonyl can be predicted within a factor of 6 (10 (0.78)) root-mean-square deviation (rmsd) using the CHARMM22 electrostatic parameter set and an internal dielectric value of 3. Under equivalent conditions, the PARSE parameter set yields a larger rmsd value of 1.28 pH units, while the AMBER parm99 parameter set resulted in a considerably poorer correlation. Either increasing the internal dielectric value to 4 or reducing it to a value of 2 significantly degrades the quality of the prediction. Assigning the excess charge of the peptide anion equally between the peptide nitrogen and the carbonyl oxygen also reduces the correlation to the experimental data. These continuum electrostatic calculations were further analyzed to characterize the specific structural elements that appear to be responsible for the wide range of peptide acidities observed for these solvent-exposed amides. The striking heterogeneity in the potential at sites along the protein-solvent interface should prove germane to the ongoing challenge of quantifying the contribution that electrostatic interactions make to the catalytic acceleration achieved by enzymes. PMID:18479148

  13. Relationship between Glycosyl Hydrolase Inventory and Growth Physiology of the Hyperthermophile Pyrococcus furiosus on Carbohydrate-Based Media

    PubMed Central

    Driskill, Lance E.; Kusy, Kevin; Bauer, Michael W.; Kelly, Robert M.

    1999-01-01

    Utilization of a range of carbohydrates for growth by the hyperthermophile Pyrococcus furiosus was investigated by examining the spectrum of glycosyl hydrolases produced by this microorganism and the thermal labilities of various saccharides. Previously, P. furiosus had been found to grow in batch cultures on several α-linked carbohydrates and cellobiose but not on glucose or other β-linked sugars. Although P. furiosus was not able to grow on any nonglucan carbohydrate or any form of cellulose in this study (growth on oat spelt arabinoxylan was attributed to glucan contamination of this substrate), significant growth at 98°C occurred on β-1,3- and β-1,3–β-1,4-linked glucans. Oligosaccharides generated by digestion with a recombinant laminarinase derived from P. furiosus were the compounds that were most effective in stimulating growth of the microorganism. In several cases, periodic addition of β-glucan substrates to fed-batch cultures limited adverse thermochemical modifications of the carbohydrates (i.e., Maillard reactions and caramelization) and led to significant increases (as much as two- to threefold) in the cell yields. While glucose had only a marginally positive effect on growth in batch culture, the final cell densities nearly tripled when glucose was added by the fed-batch procedure. Nonenzymatic browning reactions were found to be significant at 98°C for saccharides with degrees of polymerization (DP) ranging from 1 to 6; glucose was the most labile compound on a mass basis and the least labile compound on a molar basis. This suggests that for DP of 2 or greater protection of the nonreducing monosaccharide component may be a factor in substrate availability. For P. furiosus, carbohydrate utilization patterns were found to reflect the distribution of the glycosyl hydrolases which are known to be produced by this microorganism. PMID:10049838

  14. Relationship between glycosyl hydrolase inventory and growth physiology of the hyperthermophile Pyrococcus furiosus on carbohydrate-based media.

    PubMed

    Driskill, L E; Kusy, K; Bauer, M W; Kelly, R M

    1999-03-01

    Utilization of a range of carbohydrates for growth by the hyperthermophile Pyrococcus furiosus was investigated by examining the spectrum of glycosyl hydrolases produced by this microorganism and the thermal labilities of various saccharides. Previously, P. furiosus had been found to grow in batch cultures on several alpha-linked carbohydrates and cellobiose but not on glucose or other beta-linked sugars. Although P. furiosus was not able to grow on any nonglucan carbohydrate or any form of cellulose in this study (growth on oat spelt arabinoxylan was attributed to glucan contamination of this substrate), significant growth at 98 degrees C occurred on beta-1,3- and beta-1,3-beta-1,4-linked glucans. Oligosaccharides generated by digestion with a recombinant laminarinase derived from P. furiosus were the compounds that were most effective in stimulating growth of the microorganism. In several cases, periodic addition of beta-glucan substrates to fed-batch cultures limited adverse thermochemical modifications of the carbohydrates (i.e., Maillard reactions and caramelization) and led to significant increases (as much as two- to threefold) in the cell yields. While glucose had only a marginally positive effect on growth in batch culture, the final cell densities nearly tripled when glucose was added by the fed-batch procedure. Nonenzymatic browning reactions were found to be significant at 98 degrees C for saccharides with degrees of polymerization (DP) ranging from 1 to 6; glucose was the most labile compound on a mass basis and the least labile compound on a molar basis. This suggests that for DP of 2 or greater protection of the nonreducing monosaccharide component may be a factor in substrate availability. For P. furiosus, carbohydrate utilization patterns were found to reflect the distribution of the glycosyl hydrolases which are known to be produced by this microorganism. PMID:10049838

  15. Preparation of lactose-free pasteurized milk with a recombinant thermostable β-glucosidase from Pyrococcus furiosus

    PubMed Central

    2013-01-01

    Background Lactose intolerance is a common health concern causing gastrointestinal symptoms and avoidance of dairy products by afflicted individuals. Since milk is a primary source of calcium and vitamin D, lactose intolerant individuals often obtain insufficient amounts of these nutrients which may lead to adverse health outcomes. Production of lactose-free milk can provide a solution to this problem, although it requires use of lactase from microbial sources and increases potential for contamination. Use of thermostable lactase enzymes can overcome this issue by functioning under pasteurization conditions. Results A thermostable β-glucosidase gene from Pyrococcus furiosus was cloned in frame with the Saccharomyces cerecisiae a-factor secretory signal and expressed in Pichia pastoris strain X-33. The recombinant enzyme was purified by a one-step method of weak anion exchange chromatography. The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively. The enzyme activity was not significantly inhibited by Ca2+. We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose. Conclusions The thermostablity of this recombinant β-glucosidase, combined with its neutral pH activity and favorable temperature activity optima, suggest that this enzyme is an ideal candidate for the hydrolysis of lactose in milk, and it would be suitable for application in low-lactose milk production during pasteurization. PMID:24053641

  16. The L7Ae protein binds to two kink-turns in the Pyrococcus furiosus RNase P RNA

    PubMed Central

    Lai, Stella M.; Lai, Lien B.; Foster, Mark P.; Gopalan, Venkat

    2014-01-01

    The RNA-binding protein L7Ae, known for its role in translation (as part of ribosomes) and RNA modification (as part of sn/oRNPs), has also been identified as a subunit of archaeal RNase P, a ribonucleoprotein complex that employs an RNA catalyst for the Mg2+-dependent 5′ maturation of tRNAs. To better understand the assembly and catalysis of archaeal RNase P, we used a site-specific hydroxyl radical-mediated footprinting strategy to pinpoint the binding sites of Pyrococcus furiosus (Pfu) L7Ae on its cognate RNase P RNA (RPR). L7Ae derivatives with single-Cys substitutions at residues in the predicted RNA-binding interface (K42C/C71V, R46C/C71V, V95C/C71V) were modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide. Upon addition of hydrogen peroxide and ascorbate, these L7Ae-tethered nucleases were expected to cleave the RPR at nucleotides proximal to the EDTA-Fe–modified residues. Indeed, footprinting experiments with an enzyme assembled with the Pfu RPR and five protein cofactors (POP5, RPP21, RPP29, RPP30 and L7Ae–EDTA-Fe) revealed specific RNA cleavages, localizing the binding sites of L7Ae to the RPR's catalytic and specificity domains. These results support the presence of two kink-turns, the structural motifs recognized by L7Ae, in distinct functional domains of the RPR and suggest testable mechanisms by which L7Ae contributes to RNase P catalysis. PMID:25361963

  17. Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed

    McTernan, Patrick M; Chandrayan, Sanjeev K; Wu, Chang-Hao; Vaccaro, Brian J; Lancaster, W Andrew; Yang, Qingyuan; Fu, Dax; Hura, Greg L; Tainer, John A; Adams, Michael W W

    2014-07-11

    The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions. PMID:24860091

  18. Structural Basis of Thermal Stability of the Tungsten Cofactor Synthesis Protein MoaB from Pyrococcus furiosus

    PubMed Central

    Havarushka, Nastassia; Fischer-Schrader, Katrin; Lamkemeyer, Tobias; Schwarz, Guenter

    2014-01-01

    Molybdenum and tungsten cofactors share a similar pterin-based scaffold, which hosts an ene-dithiolate function being essential for the coordination of either molybdenum or tungsten. The biosynthesis of both cofactors involves a multistep pathway, which ends with the activation of the metal binding pterin (MPT) by adenylylation before the respective metal is incorporated. In the hyperthermophilic organism Pyrococcus furiosus, the hexameric protein MoaB (PfuMoaB) has been shown to catalyse MPT-adenylylation. Here we determined the crystal structure of PfuMoaB at 2.5 Å resolution and identified key residues of α3-helix mediating hexamer formation. Given that PfuMoaB homologues from mesophilic organisms form trimers, we investigated the impact on PfuMoaB hexamerization on thermal stability and activity. Using structure-guided mutagenesis, we successfully disrupted the hexamer interface in PfuMoaB. The resulting PfuMoaB-H3 variant formed monomers, dimers and trimers as determined by size exclusion chromatography. Circular dichroism spectroscopy as well as chemical cross-linking coupled to mass spectrometry confirmed a wild-type-like fold of the protomers as well as inter-subunits contacts. The melting temperature of PfuMoaB-H3 was found to be reduced by more than 15°C as determined by differential scanning calorimetry, thus demonstrating hexamerization as key determinant for PfuMoaB thermal stability. Remarkably, while a loss of activity at temperatures higher than 50°C was observed in the PfuMoaB-H3 variant, at lower temperatures, we determined a significantly increased catalytic activity. The latter suggests a gain in conformational flexibility caused by the disruption of the hexamerization interface. PMID:24465852

  19. Experimental silicification of the extremophilic Archaea Pyrococcus abyssi and Methanocaldococcus jannaschii: applications in the search for evidence of life in early Earth and extraterrestrial rocks.

    PubMed

    Orange, F; Westall, F; Disnar, J-R; Prieur, D; Bienvenu, N; Le Romancer, M; Défarge, Ch

    2009-09-01

    Hydrothermal activity was common on the early Earth and associated micro-organisms would most likely have included thermophilic to hyperthermophilic species. 3.5-3.3 billion-year-old, hydrothermally influenced rocks contain silicified microbial mats and colonies that must have been bathed in warm to hot hydrothermal emanations. Could they represent thermophilic or hyperthermophilic micro-organisms and if so, how were they preserved? We present the results of an experiment to silicify anaerobic, hyperthermophilic micro-organisms from the Archaea Domain Pyrococcus abyssi and Methanocaldococcus jannaschii, that could have lived on the early Earth. The micro-organisms were placed in a silica-saturated medium for periods up to 1 year. Pyrococcus abyssi cells were fossilized but the M. jannaschii cells lysed naturally after the exponential growth phase, apart from a few cells and cell remains, and were not silicified although their extracellular polymeric substances were. In this first simulated fossilization of archaeal strains, our results suggest that differences between species have a strong influence on the potential for different micro-organisms to be preserved by fossilization and that those found in the fossil record represent probably only a part of the original diversity. Our results have important consequences for biosignatures in hydrothermal or hydrothermally influenced deposits on Earth, as well as on early Mars, as environmental conditions were similar on the young terrestrial planets and traces of early Martian life may have been similarly preserved as silicified microfossils. PMID:19656214

  20. Purification and characterization of two functional forms of intracellular protease PfpI from the hyperthermophilic archaeon Pyrococcus furiosus

    SciTech Connect

    Halio, S.B.; Bauer, M.W.; Kelley, R.M.

    1997-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100{degrees}C by the fermentation of peptides and carbohydrates. From this organism, An intracellular protease was purified, previously designated PfpI (P. furiosus protease I). The protease contains exists in at least two functional conformations, which were purified separately. The predominant form from the purification (designated PfpI-C1) is a hexamer with a molecular mass of 124 {+-} 6 kDa (by gel filtration) and comprises about 90% of the total activity. The minor form (designated PfpI-C2) is trimeric with a molecular mass of 59 {+-} 3 kDa. PfpI-C1 hydrolyzed both basic and hydrophobic residues in the P1 position, indicating trypsin- and chymotrypsin-like specificities, respectively. The temperature optimum for Ala-Ala-Phe-7-amido-4-methylcoumarin (AAF-MCA) hydrolysis was {approximately}85{degrees}C both for purified PfpI-C1 and for proteolytic activity in P. furiosus cell extract. In contrast, the temperature optimum for PfpI prepared by incubating a cell extract of P. furiosus at 98{degrees}C in 1% sodium dodecyl sulfate for 24 h at 95 to 100{degrees}C, designated PfpI-H, was {approximately}100{degrees}C. Moreover, the half-life of activity of PfpI-C1 at 98{degrees}C was less than 30 min, in contrast to a value of more than 33 h measured for PfpI-H. PfpI-C1 appears to be a predominant serine-type protease in cell extracts but is converted in vitro, probably in part by deamination of Asn and Gln residues, to a more thermally stable form (PfpI-H) by prolonged heat treatment. The deamination hypothesis is supported by the differences in the measured pI values of PfpI-C1 (6.1) and PfpI-H (3.8). High levels of potassium phosphate (>0.5 mM) were found to extend the half-life of PfpI-C1 activity towards AAF-MCA by up to 2.5-fold at 90{degrees}C, suggesting that compatible solutes play an important role in the in vivo function of this protease. 43 refs., 6 figs., 2 tabs.

  1. MAGGIE Component 1: Identification and Purification of Native and Recombinant Multiprotein Complexes and Modified Proteins from Pyrococcus furiosus

    SciTech Connect

    Adams, Michael W.; W. W. Adams, Michael

    2014-01-07

    Virtualy all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes (PCs), the composition of which is largely unknown. Structural genomics efforts have demonstrated that less than 25% of the genes in a given prokaryotic genome will yield stable, soluble proteins when expressed using a one-ORF-at-a-time approach. We proposed that much of the remaining 75% of the genes encode proteins that are part of multiprotein complexes or are modified post-translationally, for example, with metals. The problem is that PCs and metalloproteins (MPs) cannot be accurately predicted on a genome-wide scale. The only solution to this dilemma is to experimentally determine PCs and MPs in biomass of a model organism and to develop analytical tools that can then be applied to the biomass of any other organism. In other words, organisms themselves must be analyzed to identify their PCs and MPs: “native proteomes” must be determined. This information can then be utilized to design multiple ORF expression systems to produce recombinant forms of PCs and MPs. Moreover, the information and utility of this approach can be enhanced by using a hyperthermophile, one that grows optimally at 100°C, as a model organism. By analyzing the native proteome at close to 100 °C below the optimum growth temperature, we will trap reversible and dynamic complexes, thereby enabling their identification, purification, and subsequent characterization. The model organism for the current study is Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100°C. It is grown up to 600-liter scale and kg quantities of biomass are available. In this project we identified native PCs and MPs using P. furiosus biomass (with MS/MS analyses to identify proteins by component 4). In addition, we provided samples of abundant native PCs and MPs for structural characterization (using SAXS by component 5). We also designed and evaluated generic bioinformatics and

  2. Purification, crystallization and preliminary X-ray diffraction studies of the soluble domain of the oligosaccharyltransferase STT3 subunit from the thermophilic archaeon Pyrococcus furiosus

    SciTech Connect

    Igura, Mayumi; Maita, Nobuo; Obita, Takayuki; Kamishikiryo, Jun; Maenaka, Katsumi; Kohda, Daisuke

    2007-09-01

    The C-terminal soluble domain of the catalytic subunit (STT3) of the oligosaccharyltransferase from P. furiosus was purified and crystallized. A native crystal and a SeMet derivative have been analyzed using X-ray diffraction. Oligosaccharyltransferase catalyzes the transfer of preassembled oligosaccharides onto asparagine residues in nascent polypeptide chains. The STT3 subunit is thought to bear the catalytic site. The C-terminal domain of the STT3 protein of Pyrococcus furiosus was expressed in Escherichia coli cells. STT3 protein prepared from two different sources, the soluble fraction and the inclusion bodies, produced crystals that diffracted to 2.7 Å. During crystallization screening, cocrystals of P. furiosus STT3 with an E. coli 50S ribosomal protein, L7/L12, were accidentally obtained. This cross-species interaction is not biologically relevant, but may be used to design a built-in polypeptide substrate for the STT3 crystals.

  3. The K-loop, a general feature of the Pyrococcus C/D guide RNAs, is an RNA structural motif related to the K-turn

    PubMed Central

    Nolivos, Sophie; Carpousis, Agamemnon J.; Clouet-d'Orval, Béatrice

    2005-01-01

    The C/D guide RNAs predicted from the genomic sequences of three species of Pyrococcus delineate a family of small non-coding archaeal RNAs involved in the methylation of rRNA and tRNA. The C/D guides assemble into ribonucleoprotein (RNP) that contains the methyltransferase. The protein L7Ae, a key structural component of the RNP, binds to a Kink-turn (K-turn) formed by the C/D motif. The K-turn is a structure that consists of two RNA stems separated by a short asymmetric loop with a characteristic sharp bend (kink) between the two stems. The majority of the pyrococcal C/D guides contain a short 3 nt-spacer between the C′/D′ motifs. We show here that conserved terminal stem–loops formed by the C′/D′ motif of the Pyrococcus C/D RNAs are also L7Ae-binding sites. These stem–loops are related to the K-turn by sequence and structure, but they consist of a single stem closed by a terminal loop. We have named this structure the K-loop. We show that conserved non-canonical base pairs in the stem of the K-loop are necessary for L7Ae binding. For the C/D guides with a 3 nt-spacer we show that the sequence and length is also important. The K-loop could improve the stability of the C/D guide RNAs in Pyrococcal species, which are extreme hyperthermophiles. PMID:16293637

  4. Purification and Characterization of Two Functional Forms of Intracellular Protease PfpI from the Hyperthermophilic Archaeon Pyrococcus furiosus

    PubMed Central

    Halio, S. B.; Bauer, M. W.; Mukund, S.; Adams, M.; Kelly, R. M.

    1997-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100(deg)C by the fermentation of peptides and carbohydrates. From this organism, we have purified to homogeneity an intracellular protease, previously designated PfpI (P. furiosus protease I) (S. B. Halio, I. I. Blumentals, S. A. Short, B. M. Merrill, and R. M. Kelly, J. Bacteriol. 178:2605-2612, 1996). The protease contains a single subunit with a molecular mass of approximately 19 kDa and exists in at least two functional conformations, which were purified separately. The predominant form from the purification (designated PfpI-C1) is a hexamer with a molecular mass of 124 (plusmn) 6 kDa (by gel filtration) and comprises about 90% of the total activity. The minor form (designated PfpI-C2) is trimeric with a molecular mass of 59 (plusmn) 3 kDa. PfpI-C1 hydrolyzed both basic and hydrophobic residues in the P1 position, indicating trypsin- and chymotrypsin-like specificities, respectively. The temperature optimum for Ala-Ala-Phe-7-amido-4-methylcoumarin (AAF-MCA) hydrolysis was (symbl)85(deg)C both for purified PfpI-C1 and for proteolytic activity in P. furiosus cell extract. In contrast, the temperature optimum for PfpI prepared by incubating a cell extract of P. furiosus at 98(deg)C in 1% sodium dodecyl sulfate for 24 h at 95 to 100(deg)C (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1255-1262, 1990), designated PfpI-H, was (symbl)100(deg)C. Moreover, the half-life of activity of PfpI-C1 at 98(deg)C was less than 30 min, in contrast to a value of more than 33 h measured for PfpI-H. PfpI-C1 appears to be a predominant serine-type protease in cell extracts but is converted in vitro, probably in part by deamidation of Asn and Gln residues, to a more thermally stable form (PfpI-H) by prolonged heat treatment. The deamination hypothesis is supported by the differences in the measured pI values of PfpI-C1 (6.1) and PfpI-H (3.8). High levels of potassium phosphate (>0

  5. Hydrogenase of the hyperthermophile Pyrococcus furiosus is an elemental sulfur reductase or sulfhydrogenase: evidence for a sulfur-reducing hydrogenase ancestor.

    PubMed

    Ma, K; Schicho, R N; Kelly, R M; Adams, M W

    1993-06-01

    Microorganisms growing near and above 100 degrees C have recently been discovered near shallow and deep sea hydrothermal vents. Most are obligately dependent upon the reduction of elemental sulfur (S0) to hydrogen sulfide (H2S) for optimal growth, even though S0 reduction readily occurs abiotically at their growth temperatures. The sulfur reductase activity of the anaerobic archaeon Pyrococcus furiosus, which grows optimally at 100 degrees C by a metabolism that produces H2S if S0 is present, was found in the cytoplasm. It was purified anaerobically and was shown to be identical to the hydrogenase that had been previously purified from this organism. Both S0 and polysulfide served as substrates for H2S production, and the S0 reduction activity but not the H2-oxidation activity was enhanced by the redox protein rubredoxin. The H2-oxidizing and S0-reduction activities of the enzyme also showed different responses to pH, temperature, and inhibitors. This bifunctional "sulfhydrogenase" enzyme can, therefore, dispose of the excess reductant generated during fermentation using either protons or polysulfides as the electron acceptor. In addition, purified hydrogenases from both hyperthermophilic and mesophilic representatives of the archaeal and bacterial domains were shown to reduce S0 to H2S. It is suggested that the function of some form of ancestral hydrogenase was S0 reduction rather than, or in addition to, the reduction of protons. PMID:8389482

  6. Solution structure of Pyrococcus furiosus RPP21, a component of the archaeal RNase P holoenzyme, and interactions with its RPP29 protein partner.

    PubMed

    Amero, Carlos D; Boomershine, William P; Xu, Yiren; Foster, Mark

    2008-11-11

    RNase P is the ubiquitous ribonucleoprotein metalloenzyme responsible for cleaving the 5'-leader sequence of precursor tRNAs during their maturation. While the RNA subunit is catalytically active on its own at high monovalent and divalent ion concentrations, four protein subunits are associated with archaeal RNase P activity in vivo: RPP21, RPP29, RPP30, and POP5. These proteins have been shown to function in pairs: RPP21-RPP29 and POP5-RPP30. We have determined the solution structure of RPP21 from the hyperthermophilic archaeon Pyrococcus furiosus ( Pfu) using conventional and paramagnetic NMR techniques. Pfu RPP21 in solution consists of an unstructured N-terminus, two alpha-helices, a zinc binding motif, and an unstructured C-terminus. Moreover, we have used chemical shift perturbations to characterize the interaction of RPP21 with RPP29. The data show that the primary contact with RPP29 is localized to the two helices of RPP21. This information represents a fundamental step toward understanding structure-function relationships of the archaeal RNase P holoenzyme. PMID:18922021

  7. 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

    SciTech Connect

    Nishida, Hirokazu; Matsumiya, Shigeki; Tsuchiya, Daisuke; Ishino, Yoshizumi; Morikawa, Kosuke

    2006-03-01

    A stable stoichiometric complex of archaeal DNA polymerase with proliferating cell nuclear antigen (PCNA) was formed using a PCNA monomer mutant and the complex was successfully crystallized. Replicative DNA polymerase interacts with processivity factors, the β-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 Å resolution using synchrotron radiation. The crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 Å.

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

    SciTech Connect

    Agapay, Ramelito C.; Savvides, Savvas N.; Van Driessche, Gonzalez; Devreese, Bart; Van Beeumen, Jozef; Jongejan, Jaap A. Hagen, Wilfred R.

    2005-10-01

    A P. furiosus stand-alone RAM domain with hydrolytic activity has been cloned and expressed in E. coli. The purified protein was crystallized alone and with EPNP and PMSF, producing crystals that yield diffraction data to resolutions of 2.8, 2.2 and 2.8 Å, respectively. 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 Å 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 Å, 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.

  9. In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

    PubMed Central

    Yan, Z; Fujiwara, S; Kohda, K; Takagi, M; Imanaka, T

    1997-01-01

    The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit. PMID:9023959

  10. Isolation and Characterization of a Second Subunit of Molecular Chaperonin from Pyrococcus kodakaraensis KOD1: Analysis of an ATPase-Deficient Mutant Enzyme

    PubMed Central

    Izumi, Michi; Fujiwara, Shinsuke; Takagi, Masahiro; Kanaya, Shigenori; Imanaka, Tadayuki

    1999-01-01

    The cpkA gene encoding a second (α) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (β subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp95 to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E. coli without requiring energy from ATP hydrolysis. PMID:10103287

  11. Comparative Physiological Studies on Hyperthermophilic Archaea Isolated from Deep-Sea Hot Vents with Emphasis on Pyrococcus Strain GB-D †

    PubMed Central

    Jannasch, Holger W.; Wirsen, Carl O.; Molyneaux, Stephen J.; Langworthy, Thomas A.

    1992-01-01

    Three new sulfur- or non-sulfur-dependent archaeal isolates, including a Pyrococcus strain, from Guaymas Basin hydrothermal vents (Gulf of California; depth, 2,010 m) were characterized and physiologically compared with four known hyperthermophiles, previously isolated from other vent sites, with an emphasis on growth and survival under the conditions particular to the natural habitat. Incubation under in situ pressure (200 atm [1 atm = 101.29 kPa]) did not increase the maximum growth temperature by more than 1°C for any of the organisms but did result in increases in growth rates of up to 15% at optimum growth temperatures. At in situ pressure, temperatures considerably higher than those limiting growth (i.e., > 105°C) were survived best by isolates with the highest maximum growth temperatures, but none of the organisms survived at temperatures of 150°C or higher for 5 min. Free oxygen was toxic to all isolates at growth range temperatures, but at ambient deep-sea temperature (3 to 4°C), the effect varied in different isolates, the non-sulfur-dependent isolate being the most oxygen tolerant. Hyperthermophiles could be isolated from refrigerated and oxygenated samples after 5 years of storage. Cu, Zn, and Pb ions were found to be toxic under nongrowth conditions (absence of organic substrate), with the non-sulfur-dependent isolate again being the most tolerant. PMID:16348799

  12. Exploitation of the S-layer self-assembly system for site directed immobilization of enzymes demonstrated for an extremophilic laminarinase from Pyrococcus furiosus

    PubMed Central

    Tschiggerl, Helga; Breitwieser, Andreas; de Roo, Guy; Verwoerd, Theo; Scḧaffer, Christina; Sleytr, Uwe B.

    2015-01-01

    A fusion protein based on the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and the enzyme laminarinase (LamA) from Pyrococcus furiosus was designed and overexpressed in Escherichia coli. Due to the construction principle, the S-layer fusion protein fully retained the self-assembly capability of the S-layer moiety, while the catalytic domain of LamA remained exposed at the outer surface of the formed protein lattice. The enzyme activity of the S-layer fusion protein monolayer obtained upon recrystallization on silicon wafers, glass slides and different types of polymer membranes was determined colorimetrically and related to the activity of sole LamA that has been immobilized with conventional techniques. LamA aligned within the S-layer fusion protein lattice in a periodic and orientated fashion catalyzed twice the glucose release from the laminarin polysaccharide substrate in comparison to the randomly immobilized enzyme. In combination with the good shelf-life and the high resistance towards temperature and diverse chemicals, these novel composites are regarded a promising approach for site-directed enzyme immobilization. PMID:18035441

  13. 'Super-perfect' enzymes: Structural stabilities and activities of recombinant triose phosphate isomerases from Pyrococcus furiosus and Thermococcus onnurineus produced in Escherichia coli.

    PubMed

    Sharma, Prerna; Guptasarma, Purnananda

    2015-05-01

    Triose phosphate isomerases (TIMs) are considered to be 'kinetically perfect' enzymes, limited in their activity only by the rates of diffusion of substrate and product molecules. Most studies conducted thus far have been on mesophile-derived TIMs. Here, we report studies of two extremophile-derived TIMs produced in Escherichia coli: (i) TonTIM, sourced from the genome of the thermophile archaeon, Thermococcus onnurineus, and (ii) PfuTIM, sourced from the genome of the hyperthermophile archaeon, Pyrococcus furiosus (PfuTIM). Although these enzymes are presumed to have evolved to function optimally at temperatures close to the boiling point of water, we find that TonTIM and PfuTIM display second-order rate-constants of activity (k(cat)/K(m) values) comparable to mesophile-derived TIMs, at 25 °C. At 90 °C, TonTIM and PfuTIM reach maximum velocities of reaction of ∼ 10(6)-10(7) μmol/s/mg, and display k(cat)/K(m) values in the range of ∼ 10(10)-10(11) M(-1) s(-1), which are three orders of magnitude higher than those reported for mesophile TIMs. Further, the two enzymes display no signs of having undergone any structural unfolding at 90 °C. Such enzymes could thus probably be called 'super-perfect' enzymes. PMID:25824038

  14. Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli.

    PubMed Central

    Zwickl, P; Fabry, S; Bogedain, C; Haas, A; Hensel, R

    1990-01-01

    The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103 degrees C) was purified to homogeneity. This enzyme was strictly phosphate dependent, utilized either NAD+ or NADP+, and was insensitive to pentalenolactone like the enzyme from the methanogenic archaebacterium Methanothermus fervidus. The enzyme exhibited a considerable thermostability, with a 44-min half-life at 100 degrees C. The amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase from P. woesei was deduced from the nucleotide sequence of the coding gene. Compared with the enzyme homologs from mesophilic archaebacteria (Methanobacterium bryantii, Methanobacterium formicicum) and an extremely thermophilic archaebacterium (Methanothermus fervidus), the primary structure of the P. woesei enzyme exhibited a strikingly high proportion of aromatic amino acid residues and a low proportion of sulfur-containing residues. The coding gene of P. woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme. Images PMID:2165475

  15. Purification and characterization of an alpha-glucosidase from a hyperthermophilic archaebacterium, Pyrococcus furiosus, exhibiting a temperature optimum of 105 to 115 degrees C.

    PubMed Central

    Costantino, H R; Brown, S H; Kelly, R M

    1990-01-01

    Pyrococcus furiosus is a strictly anaerobic hyperthermophilic archaebacterium with an optimal growth temperature of about 100 degrees C. When this organism was grown in the presence of certain complex carbohydrates, the production of several amylolytic enzymes was noted. These enzymes included an alpha-glucosidase that was located in the cell cytoplasm. This alpha-glucosidase has been purified 310-fold and corresponded to a protein band of 125 kilodaltons as resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited optimum activity at pH 5.0 to 6.0 and over a temperature range of 105 to 115 degrees C. Kinetic analysis conducted at 108 degrees C revealed hydrolysis of the substrates p-nitrophenyl-alpha-D-glucopyranoside (PNPG), methyl-alpha-D-glucopyranoside, maltose, and isomaltose. Trace activity was detected towards p-nitrophenyl-beta-D-glucopyranoside, and no activity could be detected towards starch or sucrose. Inhibition studies conducted at 108 degrees C with PNPG as the substrate and maltose as the inhibitor yielded a Ki for maltose of 14.3 mM. Preincubation for 30 min at 98 degrees C in 100 mM dithiothreitol and 1.0 M urea had little effect on enzyme activity, whereas preincubation in 1.0% sodium dodecyl sulfate and 1.0 M guanidine hydrochloride resulted in significant loss of enzyme activity. Purified alpha-glucosidase from P. furiosus exhibited remarkable thermostability; incubation of the enzyme at 98 degrees C resulted in a half life of nearly 48 h. Images PMID:2163383

  16. Observation of terahertz vibrations in Pyrococcus furiosus rubredoxin via impulsive coherent vibrational spectroscopy and nuclear resonance vibrational spectroscopy--interpretation by molecular mechanics.

    PubMed

    Tan, Ming-Liang; Bizzarri, Anna Rita; Xiao, Yuming; Cannistraro, Salvatore; Ichiye, Toshiko; Manzoni, Cristian; Cerullo, Giulio; Adams, Michael W W; Jenney, Francis E; Cramer, Stephen P

    2007-03-01

    We have used impulsive coherent vibrational spectroscopy (ICVS) to study the Fe(S-Cys)(4) site in oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). In this experiment, a 15 fs visible laser pulse is used to coherently pump the sample to an excited electronic state, and a second <10 fs pulse is used to probe the change in transmission as a function of the time delay. PfRd was observed to relax to the ground state by a single exponential decay with time constants of approximately 255-275 fs. Superimposed on this relaxation are oscillations caused by coherent excitation of vibrational modes in both excited and ground electronic states. Fourier transformation reveals the frequencies of these modes. The strongest ICV mode with 570 nm excitation is the symmetric Fe-S stretching mode near 310 cm(-1), compared to 313 cm(-1) in the low temperature resonance Raman. If the rubredoxin is pumped at 520 nm, a set of strong bands occurs between 20 and 110 cm(-1). Finally, there is a mode at approximately 500 cm(-1) which is similar to features near 508 cm(-1) in blue Cu proteins that have been attributed to excited state vibrations. Normal mode analysis using 488 protein atoms and 558 waters gave calculated spectra that are in good agreement with previous nuclear resonance vibrational spectra (NRVS) results. The lowest frequency normal modes are identified as collective motions of the entire protein or large segments of polypeptide. Motion in these modes may affect the polar environment of the redox site and thus tune the electron transfer functions in rubredoxins. PMID:17204331

  17. Hydrogenase of the hyperthermophile Pyrococcus furiosus is an elemental sulfur reductase or sulfhydrogenase: Evidence for a sulfur-reducing hydrogenase ancestor

    SciTech Connect

    Ma, K.; Adams, M.W.W. ); Schicho, R.N. ); Kelly, R.M. )

    1993-06-01

    Microorganisms growing near and above 100[degrees]C have recently been discovered near shallow and deep sea hydrothermal vents. Most are obligately dependent upon the reduction of elemental sulfur (S[sup 0]) to hydrogen sulfide (H[sub 2]S) for optimal growth, even though S[sup 0] reduction readily occurs abiotically at their growth temperatures. The sulfur reductase activity of the anaerobic archaeon Pyrococcus furiosus, which grows optimally at 100[degrees]C by a metabolism that produces H[sub 2]S if S[sup 0] is present, was found in the cytoplasm. It was purified anaerobically and was shown to be identical to the hydrogenase that had been previously purified from this organism. Both S[sup 0] and polysulfide served as substrates for H[sub 2]S production, and the S[sub 0] reduction activity but not the H[sub 2]-oxidation activity was enhanced by the redox protein rubredoxin. The H[sub 2]-oxidizing and S[sup 0]-reduction activities of the enzyme also showed different responses to pH, temperature, and inhibitors. This bifunctional [open quotes]sulfhydrogenase[close quotes] enzyme can, therefore, dispose of the excess reductant generated during fermentation using either protons or polysulfides as the electron acceptor. In addition, purified hydrogenases from both hyperthermophilic and mesophilic representatives of the archaeal and bacterial domains were shown to reduce S[sup 0] to H[sub 2]S. It is suggested that the function of some form of ancestral hydrogenase was S[sup 0] reduction rather than, or in addition, to the reduction of protons. 33 refs., 4 figs., 2 tabs.

  18. Insights into the hyperthermostability and unusual region-specificity of archaeal Pyrococcus abyssi tRNA m1A57/58 methyltransferase

    PubMed Central

    Guelorget, Amandine; Roovers, Martine; Guérineau, Vincent; Barbey, Carole; Li, Xuan; Golinelli-Pimpaneau, Béatrice

    2010-01-01

    The S-adenosyl-l-methionine dependent methylation of adenine 58 in the T-loop of tRNAs is essential for cell growth in yeast or for adaptation to high temperatures in thermophilic organisms. In contrast to bacterial and eukaryotic tRNA m1A58 methyltransferases that are site-specific, the homologous archaeal enzyme from Pyrococcus abyssi catalyzes the formation of m1A also at the adjacent position 57, m1A57 being a precursor of 1-methylinosine. We report here the crystal structure of P. abyssi tRNA m1A57/58 methyltransferase (PabTrmI), in complex with S-adenosyl-l-methionine or S-adenosyl-l-homocysteine in three different space groups. The fold of the monomer and the tetrameric architecture are similar to those of the bacterial enzymes. However, the inter-monomer contacts exhibit unique features. In particular, four disulfide bonds contribute to the hyperthermostability of the archaeal enzyme since their mutation lowers the melting temperature by 16.5°C. His78 in conserved motif X, which is present only in TrmIs from the Thermococcocales order, lies near the active site and displays two alternative conformations. Mutagenesis indicates His78 is important for catalytic efficiency of PabTrmI. When A59 is absent in tRNAAsp, only A57 is modified. Identification of the methylated positions in tRNAAsp by mass spectrometry confirms that PabTrmI methylates the first adenine of an AA sequence. PMID:20483913

  19. Hydrolysis of flavanone glycosides by β-glucosidase from Pyrococcus furiosus and its application to the production of flavanone aglycones from citrus extracts.

    PubMed

    Shin, Kyung-Chul; Nam, Hyun-Koo; Oh, Deok-Kun

    2013-11-27

    The hydrolytic activity of the recombinant β-glucosidase from Pyrococcus furiosus for the flavanone glycoside hesperidin was optimal at pH 5.5 and 95 °C in the presence of 0.5% (v/v) dimethyl sulfoxide (DMSO) and 0.1% (w/v) Tween 40 with a half-life of 88 h, a Km of 1.6 mM, and a kcat of 68.4 1/s. The specific activity of the enzyme for flavonoid glycosides followed the order hesperidin > neohesperidin > naringin > narirutin > poncirin > diosmin > neoponcirin > rutin. The specific activity for flavanone was higher than that for flavone or flavonol. DMSO at 10% (v/v) was used to increase the solubility of flavanone glycosides as substrates. The enzyme completely converted flavanone glycosides (1 g/L) to flavanone aglycones and disaccharides via one-step reaction. The major flavanone in grapefruit peel, grapefruit pulp, or orange peel extract was naringin (47.5 mg/g), naringin (16.6 mg/g), or hesperidin (18.2 mg/g), respectively. β-Glucosidase from P. furiosus completely converted naringin and narirutin in 100% (w/v) grapefruit peel extract to 22.5 g/L naringenin after 12 h, with a productivity of 1.88 g L(-1) h(-1); naringin and narirutin in 100% (w/v) grapefruit pulp extract to 8.1 g/L naringenin after 9 h, with a productivity of 0.90 g L(-1) h(-1); and hesperidin in 100% (w/v) orange peel extract to 9.0 g/L hesperetin after 9 h, with a productivity of 1.00 g L(-1) h(-1). The conversion yields, concentrations, and productivities of flavanone aglycones in this study are the highest among those obtained from citrus extracts. Thus, this enzyme may be useful for the industrial hydrolysis of flavanone glycosides in citrus extracts. PMID:24188428

  20. Enhancing Heat Tolerance of the Little Dogwood Cornus canadensis L. f. with Introduction of a Superoxide Reductase Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus

    PubMed Central

    Geng, Xing-Min; Liu, Xiang; Ji, Mikyoung; Hoffmann, William A.; Grunden, Amy; Xiang, Qiu-Yun J.

    2016-01-01

    Production of reactive oxygen species (ROS) can be accelerated under various biotic and abiotic stresses causing lipid peroxidation, protein degradation, enzyme inactivation, and DNA damage. Superoxide reductase (SOR) is a novel antioxidant enzyme from Pyrococcus furiosus and is employed by this anaerobic hyperthermophilic archaeon for efficient detoxification of ROS. In this study, SOR was introduced into a flowering plant Cornus canadensis to enhance its heat tolerance and reduce heat induced damage. A fusion construct of the SOR gene and Green Fluorescent Protein gene (GFP) was introduced into C. canadensis using Agrobacterium-mediated transformation. Heat tolerance of the GFP-SOR expressing transgenic plants was investigated by observing morphological symptoms of heat injury and by examining changes in photosynthesis, malondialdehyde (MDA), and proline levels in the plants. Our results indicate that the expression of the P. furiosus SOR gene in the transgenic plants alleviated lipid peroxidation of cell membranes and photoinhibition of PS II, and decreased the accumulation of proline at 40°C. After a series of exposures to increasing temperatures, the SOR transgenic plants remained healthy and green whereas most of the non-transgenic plants dried up and were unable to recover. While it had previously been reported that expression of SOR in Arabidopsis enhanced heat tolerance, this is the first report of the successful demonstration of improved heat tolerance in a non-model plant resulting from the introduction of P. furiosus SOR. The study demonstrates the potential of SOR for crop improvement and that inherent limitations of plant heat tolerance can be ameliorated with P. furiosus SOR. PMID:26858741

  1. DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction

    PubMed Central

    Elshawadfy, Ashraf M.; Keith, Brian J.; Ee Ooi, H'Ng; Kinsman, Thomas; Heslop, Pauline; Connolly, Bernard A.

    2014-01-01

    The polymerase chain reaction (PCR) is widely applied across the biosciences, with archaeal Family-B DNA polymerases being preferred, due to their high thermostability and fidelity. The enzyme from Pyrococcus furiosus (Pfu-Pol) is more frequently used than the similar protein from Thermococcus kodakarensis (Tkod-Pol), despite the latter having better PCR performance. Here the two polymerases have been comprehensively compared, confirming that Tkod-Pol: (1) extends primer-templates more rapidly; (2) has higher processivity; (3) demonstrates superior performance in normal and real time PCR. However, Tkod-Pol is less thermostable than Pfu-Pol and both enzymes have equal fidelities. To understand the favorable properties of Tkod-Pol, hybrid proteins have been prepared. Single, double and triple mutations were used to site arginines, present at the “forked-point” (the junction of the exonuclease and polymerase channels) of Tkod-Pol, at the corresponding locations in Pfu-Pol, slightly improving PCR performance. The Pfu-Pol thumb domain, responsible for double-stranded DNA binding, has been entirely replaced with that from Tkod-Pol, again giving better PCR properties. Combining the “forked-point” and thumb swap mutations resulted in a marked increase in PCR capability, maintenance of high fidelity and retention of the superior thermostability associated with Pfu-Pol. However, even the arginine/thumb swap mutant falls short of Tkod-Pol in PCR, suggesting further improvement within the Pfu-Pol framework is attainable. The significance of this work is the observation that improvements in PCR performance are easily attainable by blending elements from closely related archaeal polymerases, an approach that may, in future, be extended by using more polymerases from these organisms. PMID:24904539

  2. Transport mechanism of a glutamate transporter homologue GltPh.

    PubMed

    Ji, Yurui; Postis, Vincent L G; Wang, Yingying; Bartlam, Mark; Goldman, Adrian

    2016-06-15

    Glutamate transporters are responsible for uptake of the neurotransmitter glutamate in mammalian central nervous systems. Their archaeal homologue GltPh, an aspartate transporter isolated from Pyrococcus horikoshii, has been the focus of extensive studies through crystallography, MD simulations and single-molecule FRET (smFRET). Here, we summarize the recent research progress on GltPh, in the hope of gaining some insights into the transport mechanism of this aspartate transporter. PMID:27284058

  3. Transport mechanism of a glutamate transporter homologue GltPh

    PubMed Central

    Ji, Yurui; Postis, Vincent L.G.; Wang, Yingying; Bartlam, Mark; Goldman, Adrian

    2016-01-01

    Glutamate transporters are responsible for uptake of the neurotransmitter glutamate in mammalian central nervous systems. Their archaeal homologue GltPh, an aspartate transporter isolated from Pyrococcus horikoshii, has been the focus of extensive studies through crystallography, MD simulations and single-molecule FRET (smFRET). Here, we summarize the recent research progress on GltPh, in the hope of gaining some insights into the transport mechanism of this aspartate transporter. PMID:27284058

  4. Dynamics of the [4Fe-4S] Cluster in Pyrococcus furiosus D14C Ferredoxin via Nuclear Resonance Vibrational and Resonance Raman Spectroscopies, Force Field Simulations, and Density Functional Theory Calculations

    PubMed Central

    Mitra, Devrani; Pelmenschikov, Vladimir; Guo, Yisong; Case, David A.; Wang, Hongxin; Dong, Weibing; Tan, Ming-Liang; Ichiye, Toshiko; Jenney, Francis E.; Adams, Michael W. W.; Yoda, Yoshitaka; Zhao, Jiyong; Cramer, Stephen P.

    2011-01-01

    We have used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from Pyrococcus furiosus (Pf D14C Fd). To assist the normal mode assignments, we recorded the NRVS of D14C ferredoxin samples with 36S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains: (Ph4P)2[Fe4S4Cl4]. Several distinct regions of NRVS intensity are identified, ranging from `protein' and torsional modes below 100 cm−1, through bending and breathing modes near 150 cm−1, to strong bands from Fe-S stretching modes between 250 cm−1 and ~400 cm−1. The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra. The 57Fe partial vibrational densities of states (PVDOS) for the oxidized samples were interpreted by normal mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low frequency modes that may be relevant to electron transfer with Pf Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe4S4]2+/1+ redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins. PMID:21500788

  5. Dimeric 3-phosphoglycerate kinases from hyperthermophilic Archaea. Cloning, sequencing and expression of the 3-phosphoglycerate kinase gene of Pyrococcus woesei in Escherichia coli and characterization of the protein. Structural and functional comparison with the 3-phosphoglycerate kinase of Methanothermus fervidus.

    PubMed

    Hess, D; Krüger, K; Knappik, A; Palm, P; Hensel, R

    1995-10-01

    The gene coding for the 3-phosphoglycerate kinase (EC 2.7.2.3) of Pyrococcus woesei was cloned and sequenced. The gene sequence comprises 1230 bp coding for a polypeptide with the theoretical M(r) of 46,195. The deduced protein sequence exhibits a high similarity (46.1% and 46.6% identity) to the other known archaeal 3-phosphoglycerate kinases of Methanobacterium bryantii and Methanothermus fervidus [Fabry, S., Heppner, P., Dietmaier, W. & Hensel, R. (1990) Gene 91, 19-25]. By comparing the 3-phosphoglycerate kinase sequences of the mesophilic and the two thermophilic Archaea, trends in thermoadaptation were confirmed that could be deduced from comparisons of glyceraldehyde-3-phosphate dehydrogenase sequences from the same organisms [Zwickl, P., Fabry, S., Bogedain, C., Haas, A. & Hensel, R. (1990) J. Bacteriol. 172, 4329-4338]. With increasing temperature the average hydrophobicity and the portion of aromatic residues increases, whereas the chain flexibility as well as the content in chemically labile residues (Asn, Cys) decreases. To study the phenotypic properties of the 3-phosphoglycerate kinases from thermophilic Archaea in more detail, the 3-phosphoglycerate kinase genes from P. woesei and M. fervidus were expressed in Escherichia coli. Comparisons of kinetic and molecular properties of the enzymes from the original organisms and from E. coli indicate that the proteins expressed in the mesophilic host are folded correctly. Besides their higher thermostability according to their origin from hyperthermophilic organisms, both enzymes differ from their bacterial and eucaryotic homologues mainly in two respects. (a) The 3-phosphoglycerate kinases from P. woesei and M. fervidus are homomeric dimers in their native state contrary to all other known 3-phosphoglycerate kinases, which are monomers including the enzyme from the mesophilic Archaeum M. bryantii. (b) Monovalent cations are essential for the activity of both archaeal enzymes with K+ being significantly more

  6. Hyper-thermostability of CutA1 protein, with a denaturation temperature of nearly 150 degrees C.

    PubMed

    Tanaka, Tomoyuki; Sawano, Masahide; Ogasahara, Kyoko; Sakaguchi, Yasushi; Bagautdinov, Bagautdin; Katoh, Etsuko; Kuroishi, Chizu; Shinkai, Akeo; Yokoyama, Shigeyuki; Yutani, Katsuhide

    2006-07-24

    We found that the CutA1 protein, from Pyrococcus horikoshii (PhCutA1), has an extremely high denaturation temperature (T(d)) of nearly 150 degrees C, which exceeds the highest record determined by DSC by about 30 degrees C. To elucidate the mechanism of the ultra-high stability of PhCutA1, we analyzed the crystal structures of CutA1 proteins from three different sources, P. horikoshii, Thermus thermophilus, and Escherichia coli, with different growth temperatures (98, 75, and 37 degrees C). This analysis revealed that the remarkably increased number of ion pairs in the monomeric structure contributes to the stabilization of the trimeric structure and plays an important role in enhancing the T(d), up to 150 degrees C, for PhCutA1. PMID:16831434

  7. Solution Structure of an Archaeal RNase P Binary Protein Complex. Formation of the 30-kDa Complex Between Pyrococcus furiosus RPP21 and RPP29 is Accompanied by Coupled Protein Folding, and Highlights Critical Features for Protein-Protein and Protein-RNA Interactions

    PubMed Central

    Xu, Yiren; Amero, Carlos D.; Pulukkunat, Dileep K.; Gopalan, Venkat; Foster, Mark P.

    2009-01-01

    RNase P is a ribonucleoprotein (RNP) enzyme that catalyzes the Mg2+-dependent 5’ maturation of precursor tRNAs. In all domains of life, it is a ribozyme: the RNase P RNA (RPR) component has been demonstrated to be responsible for catalysis. However, the number of RNase P protein subunits (RPPs) varies from one in bacteria to nine or ten in eukarya. The archaeal RPR is associated with at least four RPPs, which function in pairs (RPP21–RPP29 and RPP30-POP5). We used solution NMR spectroscopy to determine the three-dimensional structure of the protein-protein complex comprising Pyrococcus furiosus (Pfu) RPP21 and RPP29. We found that the protein-protein interaction is characterized by coupled folding of secondary structural elements that participate in interface formation. In addition to detailing the intermolecular contacts that stabilize this 30-kDa binary complex, the structure identifies surfaces rich in conserved basic residues likely vital for recognition of the RPR and/or precursor tRNA. Furthermore, enzymatic footprinting experiments allowed us to localize the RPP21–RPP29 complex to the specificity domain of the RPR. These findings provide valuable new insights into mechanisms of RNP assembly and serve as important steps towards a three-dimensional model of this ancient RNP enzyme. PMID:19733182

  8. Functional reconstitution and characterization of Pyrococcus furiosus RNase P

    PubMed Central

    Tsai, Hsin-Yue; Pulukkunat, Dileep K.; Woznick, Walter K.; Gopalan, Venkat

    2006-01-01

    RNase P, which catalyzes the magnesium-dependent 5′-end maturation of tRNAs in all three domains of life, is composed of one essential RNA and a varying number of protein subunits depending on the source: at least one in bacteria, four in archaea, and nine in eukarya. To address why multiple protein subunits are needed for archaeal/eukaryal RNase P catalysis, in contrast to their bacterial relative, in vitro reconstitution of these holoenzymes is a prerequisite. Using recombinant subunits, we have reconstituted in vitro the RNase P holoenzyme from the thermophilic archaeon Pyroccocus furiosus (Pfu) and furthered our understanding regarding its functional organization and assembly pathway(s). Whereas Pfu RNase P RNA (RPR) alone is capable of multiple turnover, addition of all four RNase P protein (Rpp) subunits to Pfu RPR results in a 25-fold increase in its kcat and a 170-fold decrease in Km. In fact, even in the presence of only one of two specific pairs of Rpps, the RPR displays activity at lower substrate and magnesium concentrations. Moreover, a pared-down, mini-Pfu RNase P was identified with an RPR deletion mutant. Results from our kinetic and footprinting studies on Pfu RNase P, together with insights from recent structures of bacterial RPRs, provide a framework for appreciating the role of multiple Rpps in archaeal RNase P. PMID:17053064

  9. Crystal Structures of the Iron-Sulfur Cluster-Dependent Quinolinate Synthase in Complex with Dihydroxyacetone Phosphate, Iminoaspartate Analogues, and Quinolinate.

    PubMed

    Fenwick, Michael K; Ealick, Steven E

    2016-08-01

    The quinolinate synthase of prokaryotes and photosynthetic eukaryotes, NadA, contains a [4Fe-4S] cluster with unknown function. We report crystal structures of Pyrococcus horikoshii NadA in complex with dihydroxyacetone phosphate (DHAP), iminoaspartate analogues, and quinolinate. DHAP adopts a nearly planar conformation and chelates the [4Fe-4S] cluster via its keto and hydroxyl groups. The active site architecture suggests that the cluster acts as a Lewis acid in enediolate formation, like zinc in class II aldolases. The DHAP and putative iminoaspartate structures suggest a model for a condensed intermediate. The ensemble of structures suggests a two-state system, which may be exploited in early steps. PMID:27404889

  10. Transmembrane structure predictions with hydropathy index/charge two-dimensional trajectories of stochastic dynamical systems.

    PubMed

    Kaburagi, Takashi; Muramatsu, Daigo; Matsumoto, Takashi

    2007-06-01

    A novel algorithm is proposed for predicting transmembrane protein secondary structure from two-dimensional vector trajectories consisting of a hydropathy index and formal charge of a test amino acid sequence using stochastic dynamical system models. Two prediction problems are discussed. One is the prediction of transmembrane region counts; another is that of transmembrane regions, i.e. predicting whether or not each amino acid belongs to a transmembrane region. The prediction accuracies, using a collection of well-characterized transmembrane protein sequences and benchmarking sequences, suggest that the proposed algorithm performs reasonably well. An experiment was performed with a glutamate transporter homologue from Pyrococcus horikoshii. The predicted transmembrane regions of the five human glutamate transporter sequences and observations based on the computed likelihood are reported. PMID:17688311

  11. Transport Mechanism of a Bacterial Homologue of Glutamate Transporters

    SciTech Connect

    Reyes, N.; Ginter, C; Boudker, O

    2009-01-01

    Glutamate transporters are integral membrane proteins that catalyse a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glia and neuronal cells by harnessing the energy of pre-existing electrochemical gradients of ions. Crucial to the reaction is the conformational transition of the transporters between outward and inward facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm, respectively. Here we describe the crystal structure of a double cysteine mutant of a glutamate transporter homologue from Pyrococcus horikoshii, GltPh, which is trapped in the inward facing state by cysteine crosslinking. Together with the previously determined crystal structures of Glt{sub Ph} in the outward facing state, the structure of the crosslinked mutant allows us to propose a molecular mechanism by which Glt{sub Ph} and, by analogy, mammalian glutamate transporters mediate sodium-coupled substrate uptake.

  12. Crystal structure of stable protein CutA1 from psychrotrophic bacterium Shewanella sp. SIB1

    PubMed Central

    Sato, Aya; Yokotani, Sonoko; Tadokoro, Takashi; Tanaka, Shun-ichi; Angkawidjaja, Clement; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2011-01-01

    CutA1 is widely found in bacteria, plants and animals, including humans. The functions of CutA1, however, have not been well clarified. It is known that CutA1s from Pyrococcus horikoshii, Thermus thermophilus and Oryza sativa unfold at temperatures remarkably higher than the growth temperatures of the host organisms. In this work the crystal structure of CutA1 from the psychrotrophic bacterium Shewanella sp. SIB1 (SIB1–CutA1) in a trimeric form was determined at 2.7 Å resolution. This is the first crystal structure of a psychrotrophic CutA1. The overall structure of SIB1–CutA1 is similar to those of CutA1 from Homo sapiens, Escherichia coli, Pyrococcus horikoshii, Thermus thermophilus, Termotoga maritima, Oryza sativa and Rattus norvergicus. A peculiarity is observed in the β2 strand. The β2 strand is divided into two short β strands, β2a and β2b, in SIB1–CutA1. A thermal denaturation experiment revealed that SIB1–CutA1 does not unfold completely at 363 K at pH 7.0, although Shewanella sp. SIB1 cannot grow at temperatures exceeding 303 K. These results indicate that the trimeric structural motif of CutA1 is the critical factor in its unusually high stability and suggest that CutA1 needs to maintain its high stability in order to function, even in psychrotrophs. PMID:21169681

  13. Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme

    SciTech Connect

    Zhang, Yang; Zhu, Xuling; Torelli, Andrew T; Lee, Michael; Dzikovski, Boris; Koralewski, Rachel M; Wang, Eileen; Freed, Jack; Krebs, Carsten; Ealick, Steve E; Lin, Hening

    2010-08-30

    Archaeal and eukaryotic translation elongation factor 2 contain a unique post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide was proposed to involve three steps, with the first being the formation of a C-C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosyl-l-methionine (SAM). However, further details of the biosynthesis remain unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron-sulphur-cluster enzyme, Dph2. Dph2 is a homodimer and each of its monomers can bind a [4Fe-4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5'-deoxyadenosyl radical. Instead, it breaks the Cγ,Met-S bond of SAM and generates a 3-amino-3-carboxypropyl radical. Our results suggest that P. horikoshii Dph2 represents a previously unknown, SAM-dependent, [4Fe-4S]-containing enzyme that catalyses unprecedented chemistry.

  14. Bacterial start site prediction.

    PubMed

    Hannenhalli, S S; Hayes, W S; Hatzigeorgiou, A G; Fickett, J W

    1999-09-01

    With the growing number of completely sequenced bacterial genes, accurate gene prediction in bacterial genomes remains an important problem. Although the existing tools predict genes in bacterial genomes with high overall accuracy, their ability to pinpoint the translation start site remains unsatisfactory. In this paper, we present a novel approach to bacterial start site prediction that takes into account multiple features of a potential start site, viz., ribosome binding site (RBS) binding energy, distance of the RBS from the start codon, distance from the beginning of the maximal ORF to the start codon, the start codon itself and the coding/non-coding potential around the start site. Mixed integer programing was used to optimize the discriminatory system. The accuracy of this approach is up to 90%, compared to 70%, using the most common tools in fully automated mode (that is, without expert human post-processing of results). The approach is evaluated using Bacillus subtilis, Escherichia coli and Pyrococcus furiosus. These three genomes cover a broad spectrum of bacterial genomes, since B.subtilis is a Gram-positive bacterium, E.coli is a Gram-negative bacterium and P. furiosus is an archaebacterium. A significant problem is generating a set of 'true' start sites for algorithm training, in the absence of experimental work. We found that sequence conservation between P. furiosus and the related Pyrococcus horikoshii clearly delimited the gene start in many cases, providing a sufficient training set. PMID:10446249

  15. Comparative analysis of the catalytic components in the archaeal dye-linked L-proline dehydrogenase complexes.

    PubMed

    Kawakami, Ryushi; Noguchi, Chiaki; Higashi, Marie; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2013-04-01

    Two types of hetero-oligomeric dye-linked L-proline dehydrogenases (α4β4 and αβγδ types) are expressed in the hyperthermophilic archaea belonging to Thermococcales. In both enzymes, the β subunit (PDHβ) is responsible for catalyzing L-proline dehydrogenation. The genes encoding the two enzyme types form respective clusters that are completely conserved among Pyrococcus and Thermococcus strains. To compare the enzymatic properties of PDHβs from α4β4- and αβγδ-type enzyme complexes, eight PDHβs (four of each type) from Pyrococcus furiosus DSM3638, Pyrococcus horikoshii OT-3, Thermococcus kodakaraensis KOD1 JCM12380 and Thermococcus profundus DSM9503 were expressed in Escherichia coli cells and purified to homogeneity using one-step Ni-chelating chromatography. The α4β4-type PDHβs showed greater thermostability than most of the αβγδ-type PDHβs: the former retained more than 80 % of their activity after heating at 70 °C for 20 min, while the latter showed different thermostabilities under the same conditions. In addition, the α4β4-type PDHβs utilized ferricyanide as the most preferable electron acceptor, whereas αβγδ-type PDHβs preferred 2, 6-dichloroindophenol, with one exception. These results indicate that the differences in the enzymatic properties of the PDHβs likely reflect whether they were from an αβγδ- or α4β4-type complex, though the wider divergence observed within αβγδ-type PDHβs based on the phylogenetic analysis may also be responsible for their inconsistent enzymatic properties. By contrast, differences in the kinetic parameters among the PDHβs did not reflect the complex type. Interestingly, the k cat value for free α4β4-type PDHβ from P. horikoshii was much larger than the value for the same subunit within the α4β4-complex. This indicates that the isolated PDHβ could be a useful element for an electrochemical system for detection of L-proline. PMID:22752365

  16. Characterization of three putative Lon proteases of Thermus thermophilus HB27 and use of their defective mutants as hosts for production of heterologous proteins.

    PubMed

    Maehara, Tomoko; Hoshino, Takayuki; Nakamura, Akira

    2008-03-01

    In the genome of a thermophilic bacterium, Thermus thermophilus HB27, three genes, TTC0418, TTC0746 and TTC1975, were annotated as ATP-dependent protease La (Lon). Sequence comparisons indicated that TTC0418 and TTC0746 showed significant similarities to bacterial LonA-type proteases, such as Escherichia coli Lon protease, especially in regions corresponding to domains for ATP-binding and hydrolysis, and for proteolysis, but TTC1975 exhibited a similarity only at the C-terminal proteolytic domain. The enzymatic analyses, using purified recombinant proteins produced by E. coli, revealed that TTC0418 and TTC0746 exhibited peptidase and protease activities against two synthetic peptides and casein, respectively, in an ATP-dependent manner, and at the same time, both the enzymes had significant ATPase activities in the presence of substrates. On the other hand, TTC1975 possessed a protease activity against casein, but addition of ATP did not enhance this activity. Moreover, a T. thermophilus mutant deficient in both TTC0418 and TTC0746 showed a similar growth characteristic to an E. coli lon mutant, i.e., a growth defect lag after a nutritional downshift. These results indicate that TTC0418 and TTC0746 are actually members of bacterial LonA-type proteases with different substrate specificities, whereas TTC1975 should not be classified as a Lon protease. Finally, the effects of mutations deficient in these proteases were assessed on production of several heterologous gene products from Pyrococcus horikoshii and Geobacillus stearothermophilus. It was shown that TTC0746 mutation was more effective in improving production than the other two mutations, especially for production of P. horikoshii alpha-mannosidase and G. stearothermophilus alpha-amylase, indicating that the TTC0746 mutant of T. thermophilus HB27 may be useful for production of heterologous proteins from thermophiles and hyperthermophiles. PMID:18157502

  17. Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase.

    PubMed

    Sasaki, Hiroshi M; Sekine, Shun-ichi; Sengoku, Toru; Fukunaga, Ryuya; Hattori, Motoyuki; Utsunomiya, Yukiko; Kuroishi, Chizu; Kuramitsu, Seiki; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2006-10-01

    To achieve accurate aminoacylation of tRNAs with their cognate amino acids, errors in aminoacylation are corrected by the "editing" mechanism in several aminoacyl-tRNA synthetases. Phenylalanyl-tRNA synthetase (PheRS) hydrolyzes, or edits, misformed tyrosyl-tRNA with its editing domain in the beta subunit. We report the crystal structure of an N-terminal fragment of the PheRS beta subunit (PheRS-beta(N)) from the archaeon, Pyrococcus horikoshii, at 1.94-A resolution. PheRS-beta(N) includes the editing domain B3/4, which has archaea/eukarya-specific insertions/deletions and adopts a different orientation relative to other domains, as compared with that of bacterial PheRS. Surprisingly, most residues constituting the editing active-site pocket were substituted between the archaeal/eukaryal and bacterial PheRSs. We prepared Ala-substituted mutants of P. horikoshii PheRS for 16 editing-pocket residues, of which 12 are archaea/eukarya-specific and four are more widely conserved. On the basis of their activities, Tyr-adenosine was modeled on the B3/4-domain structure. First, the mutations of Leu-202, Ser-211, Asp-234, and Thr-236 made the PheRS incorrectly hydrolyze the cognate Phe-tRNA(Phe), indicating that these residues participate in the Tyr hydroxy group recognition and are responsible for discrimination against Phe. Second, the mutations of Leu-168 and Arg-223, which could interact with the tRNA 3'-terminal adenosine, reduced Tyr-tRNA(Phe) deacylation activity. Third, the mutations of archaea/eukarya-specific Gln-126, Glu-127, Arg-137, and Asn-217, which are proximal to the ester bond to be cleaved, also reduced Tyr-tRNA(Phe) deacylation activity. In particular, the replacement of Asn-217 abolished the activity, revealing its absolute requirement for the catalysis. PMID:17003130

  18. Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase

    PubMed Central

    Sasaki, Hiroshi M.; Sekine, Shun-ichi; Sengoku, Toru; Fukunaga, Ryuya; Hattori, Motoyuki; Utsunomiya, Yukiko; Kuroishi, Chizu; Kuramitsu, Seiki; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2006-01-01

    To achieve accurate aminoacylation of tRNAs with their cognate amino acids, errors in aminoacylation are corrected by the “editing” mechanism in several aminoacyl-tRNA synthetases. Phenylalanyl-tRNA synthetase (PheRS) hydrolyzes, or edits, misformed tyrosyl-tRNA with its editing domain in the β subunit. We report the crystal structure of an N-terminal fragment of the PheRS β subunit (PheRS-βN) from the archaeon, Pyrococcus horikoshii, at 1.94-Å resolution. PheRS-βN includes the editing domain B3/4, which has archaea/eukarya-specific insertions/deletions and adopts a different orientation relative to other domains, as compared with that of bacterial PheRS. Surprisingly, most residues constituting the editing active-site pocket were substituted between the archaeal/eukaryal and bacterial PheRSs. We prepared Ala-substituted mutants of P. horikoshii PheRS for 16 editing-pocket residues, of which 12 are archaea/eukarya-specific and four are more widely conserved. On the basis of their activities, Tyr-adenosine was modeled on the B3/4-domain structure. First, the mutations of Leu-202, Ser-211, Asp-234, and Thr-236 made the PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe, indicating that these residues participate in the Tyr hydroxy group recognition and are responsible for discrimination against Phe. Second, the mutations of Leu-168 and Arg-223, which could interact with the tRNA 3′-terminal adenosine, reduced Tyr-tRNAPhe deacylation activity. Third, the mutations of archaea/eukarya-specific Gln-126, Glu-127, Arg-137, and Asn-217, which are proximal to the ester bond to be cleaved, also reduced Tyr-tRNAPhe deacylation activity. In particular, the replacement of Asn-217 abolished the activity, revealing its absolute requirement for the catalysis. PMID:17003130

  19. Maltose Metabolism in the Hyperthermophilic Archaeon Thermococcus litoralis: Purification and Characterization of Key Enzymes

    PubMed Central

    Xavier, Karina B.; Peist, Ralf; Kossmann, Marina; Boos, Winfried; Santos, Helena

    1999-01-01

    Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in T. litoralis cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, a p-nitrophenyl-α-d-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. A Km of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the T. litoralis

  20. Cloning, expression, and purification of the general stress protein YhbO from Escherichia coli.

    PubMed

    Abdallah, Jad; Kern, Renee; Malki, Abderrahim; Eckey, Viola; Richarme, Gilbert

    2006-06-01

    We cloned, expressed, and purified the Escherichia coli yhbO gene product, which is an amino acid sequence homolog to the Bacillus subtilis general stress protein 18 (the yfkM gene product), the Pyrococcus furiosus intracellular protease PfpI, and the human Parkinson disease protein DJ-1. The gene coding for YhbO was generated by amplifying the yhbO gene from E. coli by polymerase chain reaction. It was inserted into the expression plasmid pET-21a, under the transcriptional control of the bacteriophage T7 promoter and lac operator. A BL21 (DE3) E. coli strain transformed with the YhbO-expression vector, pET-21a-yhbO, accumulates large amounts of a soluble protein with a molecular mass of 20 kDa in SDS-PAGE that matches the expected YhbO molecular weight. YhbO was purified to homogeneity by ion exchange chromatography and hydroxyapatite chromatography, and its identity was confirmed by N-terminal sequencing and mass spectrometry analysis. The native protein exists in monomeric, trimeric, and hexameric forms. We also report a strong sequence homology between YhbO and the general stress protein YfkM (64% identities), which suggests that YhbO is a stress protein, and a strong structural homology between YhbO and the Pyrococcus horikoshii intracellular protease PhpI. We could not, however, detect any proteolytic or peptidolytic activity of YhbO, using classical biochemical substrates. PMID:16380269

  1. Gene Transfer & Hybridization Studies in Hyperthermophilic Species

    SciTech Connect

    Nelson, Karen E.

    2005-10-14

    A. ABSTRACT The importance of lateral gene transfer (LGT) in the evolution of microbial species has become increasingly evident with each completed microbial genome sequence. Most significantly, the genome of Thermotoga maritima MSB8, a hyperthermophilic bacterium isolated by Karl Stetter and workers from Vulcano Italy in 1986, and sequenced at The Institute for Genomic Research (TIGR) in Rockville Maryland in 1999, revealed extensive LGT between % . this bacterium and members of the archaeal domain (in particular Archaeoglobus fulgidus, and Pyracoccus frcriosus species). Based on whole genome comparisons, it was estimated that 24% of the genetic information in this organism was acquired by genetic exchange with archaeal species, Independent analyses including periodicity analysis of the T. maritimu genomic DNA sequence, phylogenetic reconstruction based on genes that appear archaeal-like, and codon and amino acid usage, have provided additional evidence for LGT between T. maritima and the archaea. More recently, DiRuggiero and workers have identified a very recent LGT event between two genera of hyperthermophilic archaea, where a nearly identical DNA fragment of 16 kb in length flanked by insertion sequence (IS) elements, exists. Undoubtedly, additional examples of LGT will be identified as more microbial genomes are completed. For the present moment however, the genome sequence of T. maritima and other hyperthermophiles including P. furiosus, Pyrococcus horikoshii, Pyrococcus abyssi, A. fulgidus, and Aquifex aeolicus, have significantly increased out awareness of evolution being a web of life rather than a tree of life, as suggested by single gene phylogenies. In this proposal, we will aim to determine the extent of LGT across the hyperthemophiles, employing iY maritima as the model organism. A variety of biochemical techniques and phylogenetic reconstructions will allow for a detailed and thorough characterization of the extent of LGT in this species. The

  2. Evaluation of sulfur-reducing microorganisms for organic desulfurization. [Pyrococcus furiosus

    SciTech Connect

    Miller, K.W.

    1991-01-01

    Because of substantial portion of the sulfur in Illinois coal is organic, microbial desulfurization of sulfidic and thiophenic functionalities could hold great potential for completing pyritic sulfur removal. We are testing the hypothesis that organic sulfur can be reductively removed as H{sub 2}S through the activities of anaerobic microorganisms. Our objectives for this year include the following: (1) To obtain cultures that will reductively desulfurize thiophenic model compounds. In addition to crude oil enrichments begun last year, we sampled municipal sewage sludge. (2) To continue to work toward optimizing the activity of the DBDS-reducing cultures obtained during the previous year. (3) To expand coal desulfurization work to include other coals including Illinois Basin Coal 101 and a North Dakota lignite, which might be more susceptible to the dibenzyldisulfide reducing cultures due to its lower rank. (4) To address the problem of sulfide sorption, by investigating the sorption capacity of coals in addition to Illinois Basin Coal 108.

  3. Features for instantaneous emissions of low-level infrared signals of glucokinase enzyme from Pyrococcus furiosus.

    PubMed

    Torres, Sergio; Mella, Héctor; Reyes, Claudio; Meza, Pablo; Gallardo, Maria J; Staforelli, Juan P

    2015-03-10

    A noncontact infrared (IR) imaging-based methodology and signal recovery tools are applied on an enzyme reaction as a test target. The method is implemented by a long-wave (8-12 μm) IR microbolometer imaging array and a germanium-based IR optical vision. The reaction is carried out by the glucokinase, which produces a rapid exothermal release of energy that is weak, and, even worse, the IR video captured by the uncooled microbolometer detector is affected by spatial and temporal noise with specific complexities. Hitherto, IR-based signal recovery tools have worked with a standard acquisition frequency, which is clearly beyond the time scale of a real scenario. The implications of this (and similar) rapid reactions motivate the designs of a signal recovery method using prior information of the processes to extract and quantify the spontaneity of the enzymatic reaction in a three-dimensional (space and time) single and noncontact online measurement. PMID:25968383

  4. Post-translational environmental switch of RadA activity by extein–intein interactions in protein splicing

    PubMed Central

    Topilina, Natalya I.; Novikova, Olga; Stanger, Matthew; Banavali, Nilesh K.; Belfort, Marlene

    2015-01-01

    Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control. PMID:26101259

  5. Crystal structures of type III{sub H} NAD-dependent D-3-phosphoglycerate dehydrogenase from two thermophiles

    SciTech Connect

    Kumar, S.M.; Pampa, K.J.; Manjula, M.; Hemantha Kumar, G.; Kunishima, Naoki; Lokanath, N.K.

    2014-08-15

    Highlights: • Determined the crystal structures of PGDH from two thermophiles. • Monomer is composed of nucleotide binding domain and substrate binding domain. • Crystal structures of type III{sub H} PGDH. - Abstract: In the L-Serine biosynthesis, D-3-phosphoglycerate dehydrogenase (PGDH) catalyzes the inter-conversion of D-3-phosphoglycerate to phosphohydroxypyruvate. PGDH belongs to 2-hydroxyacid dehydrogenases family. We have determined the crystal structures of PGDH from Sulfolobus tokodaii (StPGDH) and Pyrococcus horikoshii (PhPGDH) using X-ray diffraction to resolution of 1.77 Å and 1.95 Å, respectively. The PGDH protomer from both species exhibits identical structures, consisting of substrate binding domain and nucleotide binding domain. The residues and water molecules interacting with the NAD are identified. The catalytic triad residues Glu-His-Arg are highly conserved. The residues involved in the dimer interface and the structural features responsible for thermostability are evaluated. Overall, structures of PGDHs with two domains and histidine at the active site are categorized as type III{sub H} and such PGDHs structures having this type are reported for the first time.

  6. Structure of RNA 3'-phosphate cyclase bound to substrate RNA.

    PubMed

    Desai, Kevin K; Bingman, Craig A; Cheng, Chin L; Phillips, George N; Raines, Ronald T

    2014-10-01

    RNA 3'-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3'-phosphate to form a 2',3'-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA-AMP and RNA(3')pp(5')A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme. PMID:25161314

  7. Induction of apoptotic effects of antiproliferative protein from the seeds of Borreria hispida on lung cancer (A549) and cervical cancer (HeLa) cell lines.

    PubMed

    Rupachandra, S; Sarada, D V L

    2014-01-01

    A 35 KDa protein referred to as F3 was purified from the seeds of Borreria hispida by precipitation with 80% ammonium sulphate and gel filtration on Sephadex G-100 column. RP-HPLC analysis of protein fraction (F3) on an analytical C-18 column produced a single peak, detected at 220 nm. F3 showed an apparent molecular weight of 35 KDa by SDS PAGE and MALDI-TOF-MS analyses. Peptide mass fingerprinting analysis of F3 showed the closest homology with the sequence of 1-aminocyclopropane-1-carboxylate deaminase of Pyrococcus horikoshii. The protein (F3) exhibited significant cytotoxic activity against lung (A549) and cervical (HeLa) cancer cells in a dose-dependent manner at concentrations ranging from 10 µg to 1000 µg/mL, as revealed by the MTT assay. Cell cycle analysis revealed the increased growth of sub-G0 population in both cell lines exposed to a concentration of 1000 µg/mL of protein fraction F3 as examined from flow cytometry. This is the first report of a protein from the seeds of Borreria hispida with antiproliferative and apoptotic activity in lung (A549) and cervical (HeLa) cancer cells. PMID:24605320

  8. Tuning the ion selectivity of glutamate transporter-associated uncoupled conductances.

    PubMed

    Cater, Rosemary J; Vandenberg, Robert J; Ryan, Renae M

    2016-07-01

    The concentration of glutamate within a glutamatergic synapse is tightly regulated by excitatory amino acid transporters (EAATs). In addition to their primary role in clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl(-) conductance. This conductance is activated by the binding of substrate and Na(+), but the direction of Cl(-) flux is independent of the rate or direction of substrate transport; thus, the two processes are thermodynamically uncoupled. A recent molecular dynamics study of the archaeal EAAT homologue GltPh (an aspartate transporter from Pyrococcus horikoshii) identified an aqueous pore at the interface of the transport and trimerization domains, through which anions could permeate, and it was suggested that an arginine residue at the most restricted part of this pathway might play a role in determining anion selectivity. In this study, we mutate this arginine to a histidine in the human glutamate transporter EAAT1 and investigate the role of the protonation state of this residue on anion selectivity and transporter function. Our results demonstrate that a positive charge at this position is crucial for determining anion versus cation selectivity of the uncoupled conductance of EAAT1. In addition, because the nature of this residue influences the turnover rate of EAAT1, we reveal an intrinsic link between the elevator movement of the transport domain and the Cl(-) channel. PMID:27296367

  9. Structural basis for substrate recognition and processive cleavage mechanisms of the trimeric exonuclease PhoExo I

    PubMed Central

    Miyazono, Ken-ichi; Ishino, Sonoko; Tsutsumi, Kanae; Ito, Tomoko; Ishino, Yoshizumi; Tanokura, Masaru

    2015-01-01

    Nucleases play important roles in nucleic acid processes, such as replication, repair and recombination. Recently, we identified a novel single-strand specific 3′-5′ exonuclease, PfuExo I, from the hyperthermophilic archaeon Pyrococcus furiosus, which may be involved in the Thermococcales-specific DNA repair system. PfuExo I forms a trimer and cleaves single-stranded DNA at every two nucleotides. Here, we report the structural basis for the cleavage mechanism of this novel exonuclease family. A structural analysis of PhoExo I, the homologous enzyme from P. horikoshii OT3, showed that PhoExo I utilizes an RNase H-like active site and possesses a 3′-OH recognition site ∼9 Å away from the active site, which enables cleavage at every two nucleotides. Analyses of the heterotrimeric and monomeric PhoExo I activities showed that trimerization is indispensable for its processive cleavage mechanism, but only one active site of the trimer is required. PMID:26138487

  10. Transport domain unlocking sets the uptake rate of an aspartate transporter.

    PubMed

    Akyuz, Nurunisa; Georgieva, Elka R; Zhou, Zhou; Stolzenberg, Sebastian; Cuendet, Michel A; Khelashvili, George; Altman, Roger B; Terry, Daniel S; Freed, Jack H; Weinstein, Harel; Boudker, Olga; Blanchard, Scott C

    2015-02-01

    Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the 'humanizing' mutations favour structurally 'unlocked' intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold. PMID:25652997

  11. Comparison of Three Ionic Liquid-Tolerant Cellulases by Molecular Dynamics

    PubMed Central

    Jaeger, Vance; Burney, Patrick; Pfaendtner, Jim

    2015-01-01

    We have employed molecular dynamics to investigate the differences in ionic liquid tolerance among three distinct family 5 cellulases from Trichoderma viride, Thermogata maritima, and Pyrococcus horikoshii. Simulations of the three cellulases were conducted at a range of temperatures in various binary mixtures of the ionic liquid 1-ethyl-3-methyl-imidazolium acetate with water. Our analysis demonstrates that the effects of ionic liquids on the enzymes vary in each individual case from local structural disturbances to loss of much of one of the enzyme’s secondary structure. Enzymes with more negatively charged surfaces tend to resist destabilization by ionic liquids. Specific and unique structural changes in the enzymes are induced by the presence of ionic liquids. Disruption of the secondary structure, changes in dynamical motion, and local changes in the binding pocket are observed in less tolerant enzymes. Ionic-liquid-induced denaturation of one of the enzymes is indicated over the 500 ns timescale. In contrast, the most tolerant cellulase behaves similarly in water and in ionic-liquid-containing mixtures. Unlike the heuristic approaches that attempt to predict enzyme stability using macroscopic properties, molecular dynamics allows us to predict specific atomic-level structural and dynamical changes in an enzyme’s behavior induced by ionic liquids and other mixed solvents. Using these insights, we propose specific experimentally testable hypotheses regarding the origin of activity loss for each of the systems investigated in this study. PMID:25692593

  12. Induction of Apoptotic Effects of Antiproliferative Protein from the Seeds of Borreria hispida on Lung Cancer (A549) and Cervical Cancer (HeLa) Cell Lines

    PubMed Central

    Rupachandra, S.; Sarada, D. V. L.

    2014-01-01

    A 35 KDa protein referred to as F3 was purified from the seeds of Borreria hispida by precipitation with 80% ammonium sulphate and gel filtration on Sephadex G-100 column. RP-HPLC analysis of protein fraction (F3) on an analytical C-18 column produced a single peak, detected at 220 nm. F3 showed an apparent molecular weight of 35 KDa by SDS PAGE and MALDI-TOF-MS analyses. Peptide mass fingerprinting analysis of F3 showed the closest homology with the sequence of 1-aminocyclopropane-1-carboxylate deaminase of Pyrococcus horikoshii. The protein (F3) exhibited significant cytotoxic activity against lung (A549) and cervical (HeLa) cancer cells in a dose-dependent manner at concentrations ranging from 10 µg to 1000 µg/mL, as revealed by the MTT assay. Cell cycle analysis revealed the increased growth of sub-G0 population in both cell lines exposed to a concentration of 1000 µg/mL of protein fraction F3 as examined from flow cytometry. This is the first report of a protein from the seeds of Borreria hispida with antiproliferative and apoptotic activity in lung (A549) and cervical (HeLa) cancer cells. PMID:24605320

  13. Multisite-specific archaeosine tRNA-guanine transglycosylase (ArcTGT) from Thermoplasma acidophilum, a thermo-acidophilic archaeon

    PubMed Central

    Kawamura, Takuya; Hirata, Akira; Ohno, Satoshi; Nomura, Yuichiro; Nagano, Tomoko; Nameki, Nobukazu; Yokogawa, Takashi; Hori, Hiroyuki

    2016-01-01

    Archaeosine (G+), which is found only at position 15 in many archaeal tRNA, is formed by two steps, the replacement of the guanine base with preQ0 by archaeosine tRNA-guanine transglycosylase (ArcTGT) and the subsequent modification of preQ0 to G+ by archaeosine synthase. However, tRNALeu from Thermoplasma acidophilum, a thermo-acidophilic archaeon, exceptionally has two G+13 and G+15 modifications. In this study, we focused on the biosynthesis mechanism of G+13 and G+15 modifications in this tRNALeu. Purified ArcTGT from Pyrococcus horikoshii, for which the tRNA recognition mechanism and structure were previously characterized, exchanged only the G15 base in a tRNALeu transcript with 14C-guanine. In contrast, T. acidophilum cell extract exchanged both G13 and G15 bases. Because T. acidophilum ArcTGT could not be expressed as a soluble protein in Escherichia coli, we employed an expression system using another thermophilic archaeon, Thermococcus kodakarensis. The arcTGT gene in T. kodakarensis was disrupted, complemented with the T. acidophilum arcTGT gene, and tRNALeu variants were expressed. Mass spectrometry analysis of purified tRNALeu variants revealed the modifications of G+13 and G+15 in the wild-type tRNALeu. Thus, T. acidophilum ArcTGT has a multisite specificity and is responsible for the formation of both G+13 and G+15 modifications. PMID:26721388

  14. The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex

    SciTech Connect

    Wang, Ruiying; Zheng, Han; Preamplume, Gan; Shao, Yaming; Li, Hong

    2012-03-15

    The repeat-associated mysterious proteins (RAMPs) comprise the most abundant family of proteins involved in prokaryotic immunity against invading genetic elements conferred by the clustered regularly interspaced short palindromic repeat (CRISPR) system. Cas6 is one of the first characterized RAMP proteins and is a key enzyme required for CRISPR RNA maturation. Despite a strong structural homology with other RAMP proteins that bind hairpin RNA, Cas6 distinctly recognizes single-stranded RNA. Previous structural and biochemical studies show that Cas6 captures the 5' end while cleaving the 3' end of the CRISPR RNA. Here, we describe three structures and complementary biochemical analysis of a noncatalytic Cas6 homolog from Pyrococcus horikoshii bound to CRISPR repeat RNA of different sequences. Our study confirms the specificity of the Cas6 protein for single-stranded RNA and further reveals the importance of the bases at Positions 5-7 in Cas6-RNA interactions. Substitutions of these bases result in structural changes in the protein-RNA complex including its oligomerization state.

  15. Clustering of OB-fold domains of the partner protease complexed with trimeric stomatin from Thermococcales.

    PubMed

    Yokoyama, Hideshi; Matsui, Eriko; Hiramoto, Kana; Forterre, Patrick; Matsui, Ikuo

    2013-07-01

    The C-terminal soluble domain of stomatin operon partner protein (STOPP) of the hyperthermophilic archaeon Pyrococcus horikoshii has an oligonucleotide binding-fold (OB-fold). STOPP lacks the conserved surface residues necessary for binding to DNA/RNA. A tryptophan (W) residue is conserved instead at the molecular surface. Solvent-accessible W residues are often found at interfaces of protein-protein complexes, which suggested the possibility of self-assembling of STOPP. Protein-protein interactions among the C-terminal soluble domains of STOPP PH1510 (1510-C) were then analyzed by chemical linking and blue native polyacrylamide gel electrophoresis (BN-PAGE) methods. These results suggest that the soluble domains of STOPP could assemble into homo-oligomers. Since hexameric subcomplex I from archaeal proteasome consists of coiled-coil segments and OB-fold domains, molecular modeling of 1510-C was performed using hexameric subcomplex I as a template. Although 1510-C is a comparatively small polypeptide consisting of approximately 60 residues, numerous salt bridges and hydrophobic interactions were observed in the predicted hexamer of 1510-C, suggesting the stability of the homo-oligomeric structure. This oligomeric property of STOPP may be favorable for triplicate proteolysis of the trimer of prokaryotic stomatin. PMID:23587725

  16. Active-site models for complexes of quinolinate synthase with substrates and intermediates

    SciTech Connect

    Soriano, Erika V.; Zhang, Yang; Colabroy, Keri L.; Sanders, Jennie M.; Settembre, Ethan C.; Dorrestein, Pieter C.; Begley, Tadhg P.; Ealick, Steven E.

    2013-09-01

    Structural studies of quinolinate synthase suggest a model for the enzyme–substrate complex and an enzyme–intermediate complex with a [4Fe–4S] cluster. Quinolinate synthase (QS) catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8 Å resolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four-stranded parallel β-sheet flanked by four α-helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a [4Fe–4S] cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii [Sakuraba et al. (2005 ▶), J. Biol. Chem.280, 26645–26648]. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the [4Fe–4S] cluster, prior to cluster assembly.

  17. Characterization of a novel acylaminoacyl peptidase with hexameric structure and endopeptidase activity.

    PubMed

    Szeltner, Zoltán; Kiss, András L; Domokos, Klarissza; Harmat, Veronika; Náray-Szabó, Gábor; Polgár, László

    2009-08-01

    We have overexpressed in E. coli, purified and investigated the kinetic, thermodynamic and biophysical properties of an acylaminoacyl peptidase (AAP), from the thermophile Pyrococcus horikoshii (PhAAP). It was shown that the electrostatic environment of the catalytic site of PhAAP substantially influenced the pH dependence of the specificity rate constant (k(cat)/K(m)). However, 0.3 M NaCl, which depressed the electrostatic effects, simplified the complex pH-rate profile. The rate of formation of the enzyme-substrate complex (k(1)) was obtained from a non-linear Arrhenius plot. The lack of substrate leaving group effects indicated that k(1) is the rate determining step in the catalysis. DSC and CD measurements demonstrated that PhAAP displayed a stable structure in the catalytically competent pH range. It was shown that PhAAP is not just an acylaminoacyl peptidase, but it also has an endopeptidase activity and so differs from the mammalian AAPs. Size exclusion chromatography with PhAAP revealed a hexameric structure, which is unique among the known members of the prolyl oligopeptidase family that includes AAPs and suggests that its cellular function may be different from that of the dimeric AAP also found in the same organism. PMID:19303951

  18. Organometallic Complex Formed by an Unconventional Radical S-Adenosylmethionine Enzyme.

    PubMed

    Dong, Min; Horitani, Masaki; Dzikovski, Boris; Pandelia, Maria-Eirini; Krebs, Carsten; Freed, Jack H; Hoffman, Brian M; Lin, Hening

    2016-08-10

    Pyrococcus horikoshii Dph2 (PhDph2) is an unusual radical S-adenosylmethionine (SAM) enzyme involved in the first step of diphthamide biosynthesis. It catalyzes the reaction by cleaving SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. To probe the reaction mechanism, we synthesized a SAM analogue (SAMCA), in which the ACP group of SAM is replaced with a 3-carboxyallyl group. SAMCA is cleaved by PhDph2, yielding a paramagnetic (S = 1/2) species, which is assigned to a complex formed between the reaction product, α-sulfinyl-3-butenoic acid, and the [4Fe-4S] cluster. Electron-nuclear double resonance (ENDOR) measurements with (13)C and (2)H isotopically labeled SAMCA support a π-complex between the C═C double bond of α-sulfinyl-3-butenoic acid and the unique iron of the [4Fe-4S] cluster. This is the first example of a radical SAM-related [4Fe-4S](+) cluster forming an organometallic complex with an alkene, shedding additional light on the mechanism of PhDph2 and expanding our current notions for the reactivity of [4Fe-4S] clusters in radical SAM enzymes. PMID:27465315

  19. Structural basis for recognition of a kink-turn motif by an archaeal homologue of human RNase P protein Rpp38.

    PubMed

    Oshima, Kosuke; Kakiuchi, Yosuke; Tanaka, Yoshikazu; Ueda, Toshifumi; Nakashima, Takashi; Kimura, Makoto; Yao, Min

    2016-06-01

    PhoRpp38 in the hyperthermophilic archaeon Pyrococcus horikoshii, a homologue of human ribonuclease P (RNase P) protein Rpp38, belongs to the ribosomal protein L7Ae family that specifically recognizes a kink-turn (K-turn) motif. A previous biochemical study showed that PhoRpp38 specifically binds to two stem-loops, SL12 and SL16, containing helices P12.1/12.2 and P15/16 respectively, in P. horikoshii RNase P RNA (PhopRNA). In order to gain insight into the PhoRpp38 binding mode to PhopRNA, we determined the crystal structure of PhoRpp38 in complex with the SL12 mutant (SL12M) at a resolution of 3.4 Å. The structure revealed that Lys35 on the β-strand (β1) and Asn38, Glu39, and Lys42 on the α-helix (α2) in PhoRpp38 interact with characteristic G•A and A•G pairs in SL12M, where Ile93, Glu94, and Val95, on a loop between α4 and β4 in PhoRpp38, interact with the 3-nucleotide bulge (G-G-U) in the SL12M. The structure demonstrates the previously proposed secondary structure of SL12, including helix P12.2. Structure-based mutational analysis indicated that amino acid residues involved in the binding to SL12 are also responsible for the binding to SL16. This result suggested that each PhoRpp38 binds to the K-turns in SL12 and SL16 in PhopRNA. A pull-down assay further suggested the presence of a second K-turn in SL12. Based on the present results, together with available data, we discuss a structural basis for recognition of K-turn motifs in PhopRNA by PhoRpp38. PMID:27114305

  20. TrmBL2 from Pyrococcus furiosus Interacts Both with Double-Stranded and Single-Stranded DNA

    PubMed Central

    Wierer, Sebastian; Daldrop, Peter; Ud Din Ahmad, Misbha; Boos, Winfried; Drescher, Malte; Welte, Wolfram; Seidel, Ralf

    2016-01-01

    In many hyperthermophilic archaea the DNA binding protein TrmBL2 or one of its homologues is abundantly expressed. TrmBL2 is thought to play a significant role in modulating the chromatin architecture in combination with the archaeal histone proteins and Alba. However, its precise physiological role is poorly understood. It has been previously shown that upon binding TrmBL2 covers double-stranded DNA, which leads to the formation of a thick and fibrous filament. Here we investigated the filament formation process as well as the stabilization of DNA by TrmBL2 from Pyroccocus furiosus in detail. We used magnetic tweezers that allow to monitor changes of the DNA mechanical properties upon TrmBL2 binding on the single-molecule level. Extended filaments formed in a cooperative manner and were considerably stiffer than bare double-stranded DNA. Unlike Alba, TrmBL2 did not form DNA cross-bridges. The protein was found to bind double- and single-stranded DNA with similar affinities. In mechanical disruption experiments of DNA hairpins this led to stabilization of both, the double- (before disruption) and the single-stranded (after disruption) DNA forms. Combined, these findings suggest that the biological function of TrmBL2 is not limited to modulating genome architecture and acting as a global repressor but that the protein acts additionally as a stabilizer of DNA secondary structure. PMID:27214207

  1. Crystallization and preliminary X-ray characterization of a ferritin from the hyperthermophilic archaeon and anaerobe Pyrococcus furiosus

    SciTech Connect

    Matias, Pedro M.; Tatur, Jana; Carrondo, Maria Arménia; Hagen, Wilfred R.

    2005-05-01

    Ferritin from P. furiosus crystallizes in space group C222{sub 1}, with unit-cell parameters a = 258.1, b = 340.1, c = 266.5 Å and 36 monomers in the asymmetric unit, corresponding to one and a half 24-mers. Crystals of the title protein have been produced and preliminary structural analysis has been carried out. The crystals belong to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 258.1, b = 340.1, c = 266.5 Å. The protein forms a 24-mer of 20 kDa subunits, which assemble with 432 non-crystallographic symmetry. A total of 36 monomers are found in the asymmetric unit, corresponding to one and a half 24-mers.

  2. Small-angle neutron scattering reveals the assembly mode and oligomeric architecture of TET, a large, dodecameric aminopeptidase

    SciTech Connect

    Appolaire, Alexandre; Girard, Eric; Colombo, Matteo; Durá, M. Asunción; Moulin, Martine; Härtlein, Michael; Franzetti, Bruno; Gabel, Frank

    2014-11-01

    The present work illustrates that small-angle neutron scattering, deuteration and contrast variation, combined with in vitro particle reconstruction, constitutes a very efficient approach to determine subunit architectures in large, symmetric protein complexes. In the case of the 468 kDa heterododecameric TET peptidase machine, it was demonstrated that the assembly of the 12 subunits is a highly controlled process and represents a way to optimize the catalytic efficiency of the enzyme. The specific self-association of proteins into oligomeric complexes is a common phenomenon in biological systems to optimize and regulate their function. However, de novo structure determination of these important complexes is often very challenging for atomic-resolution techniques. Furthermore, in the case of homo-oligomeric complexes, or complexes with very similar building blocks, the respective positions of subunits and their assembly pathways are difficult to determine using many structural biology techniques. Here, an elegant and powerful approach based on small-angle neutron scattering is applied, in combination with deuterium labelling and contrast variation, to elucidate the oligomeric organization of the quaternary structure and the assembly pathways of 468 kDa, hetero-oligomeric and symmetric Pyrococcus horikoshii TET2–TET3 aminopeptidase complexes. The results reveal that the topology of the PhTET2 and PhTET3 dimeric building blocks within the complexes is not casual but rather suggests that their quaternary arrangement optimizes the catalytic efficiency towards peptide substrates. This approach bears important potential for the determination of quaternary structures and assembly pathways of large oligomeric and symmetric complexes in biological systems.

  3. Recyclable thermoresponsive polymer-cellulase bioconjugates for biomass depolymerization.

    PubMed

    Mackenzie, Katherine J; Francis, Matthew B

    2013-01-01

    Here we report the construction and characterization of a recoverable, thermoresponsive polymer-endoglucanase bioconjugate that matches the activity of unmodified enzymes on insoluble cellulose substrates. Two copolymers exhibiting a thermoresponsive lower critical solution temperature (LCST) were created through the copolymerization of an aminooxy-bearing methacrylamide with N-isopropylacrylamide (NIPAm) or N-isopropylmethacrylamide (NIPMa). The aminooxy group provided a handle through which the LCST was adjusted through small-molecule quenching. This allowed materials with LCSTs ranging from 20.9 to 60.5 °C to be readily obtained after polymerization. The thermostable endoglucanase EGPh from the hypothermophilic Pyrococcus horikoshii was transaminated with pyridoxal-5'-phosphate to produce a ketone-bearing protein, which was then site-selectively modified through oxime linkage with benzylalkoxyamine or 5 kDa-poly(ethylene glycol)-alkoxyamine. These modified proteins showed activity comparable to the controls when assayed on an insoluble cellulosic substrate. Two polymer bioconjugates were then constructed using transaminated EGPh and the aminooxy-bearing copolymers. After 12 h, both bioconjugates produced an equivalent amount of free reducing sugars as the unmodified control using insoluble cellulose as a substrate. The recycling ability of the NIPAm copolymer-EGPh conjugate was determined through three rounds of activity, maintaining over 60% activity after two cycles of reuse and affording significantly more soluble carbohydrates than unmodified enzyme alone. When assayed on acid-pretreated Miscanthus, this bioconjugate increased the amount of reducing sugars by 2.8-fold over three rounds of activity. The synthetic strategy of this bioconjugate allows the LCST of the material to be changed readily from a common stock of copolymer and the method of attachment is applicable to a variety of proteins, enabling the same approach to be amenable to thermophile

  4. Recyclable Thermoresponsive Polymer-Cellulase Bioconjugates for Biomass Depolymerization

    PubMed Central

    Mackenzie, Katherine J.; Francis, Matthew B.

    2013-01-01

    Here we report the construction and characterization of a recoverable, thermoresponsive polymer-endoglucanase bioconjugate that matches the activity of unmodified enzymes on insoluble cellulose substrates. Two copolymers exhibiting a thermoresponsive lower critical solution temperature (LCST) were created through the copolymerization of an aminooxy-bearing methacrylamide with N-isopropylacrylamide (NIPAm) or N-isopropylmethacrylamide (NIPMa). The aminooxy group provided a handle through which the LCST was adjusted through small-molecule quenching. This allowed materials with LCSTs ranging from 20.9 °C to 60.5 °C to be readily obtained after polymerization. The thermostable endoglucanase EGPh from the hypothermophilic Pyrococcus horikoshii was transaminated with pyridoxal-5’-phosphate to produce a ketone-bearing protein, which was then site-selectively modified through oxime linkage with benzylalkoxyamine or 5 kDa-poly(ethylene glycol)-alkoxyamine. These modified proteins showed activity comparable to the controls when assayed on an insoluble cellulosic substrate. Two polymer bioconjugates were then constructed using transaminated EGPh and the aminooxy-bearing copolymers. After twelve hours, both bioconjugates produced an equivalent amount of free reducing sugars as the unmodified control using insoluble cellulose as a substrate. The recycling ability of the NIPAm copolymer-EGPh conjugate was determined through three rounds of activity, maintaining over 60% activity after two cycles of reuse and affording significantly more soluble carbohydrates than unmodified enzyme alone. When assayed on acid-pretreated Miscanthus, this bioconjugate increased the amount of reducing sugars by 2.8-fold over three rounds of activity. The synthetic strategy of this bioconjugate allows the LCST of the material to be changed readily from a common stock of copolymer and the method of attachment is applicable to a variety of proteins, enabling the same approach to be amenable to

  5. Functional implication of archaeal homologues of human RNase P protein pair Pop5 and Rpp30.

    PubMed

    Hamasaki, Masato; Hazeyama, Kohsuke; Iwasaki, Fumihiko; Ueda, Toshifumi; Nakashima, Takashi; Kakuta, Yoshimitsu; Kimura, Makoto

    2016-01-01

    PhoPop5 and PhoRpp30 in the hyperthermophilic archaeon Pyrococcus horikoshii, homologues of human ribonuclease P (RNase P) proteins hPop5 and Rpp30, respectively, fold into a heterotetramer [PhoRpp30-(PhoPop5)2-PhoRpp30], which plays a crucial role in the activation of RNase P RNA (PhopRNA). Here, we examined the functional implication of PhoPop5 and PhoRpp30 in the tetramer. Surface plasmon resonance (SPR) analysis revealed that the tetramer strongly interacts with an oligonucleotide including the nucleotide sequence of a stem-loop SL3 in PhopRNA. In contrast, PhoPop5 had markedly reduced affinity to SL3, whereas PhoRpp30 had little affinity to SL3. SPR studies of PhoPop5 mutants further revealed that the C-terminal helix (α4) in PhoPop5 functions as a molecular recognition element for SL3. Moreover, gel filtration indicated that PhoRpp30 exists as a monomer, whereas PhoPop5 is an oligomer in solution, suggesting that PhoRpp30 assists PhoPop5 in attaining a functionally active conformation by shielding hydrophobic surfaces of PhoPop5. These results, together with available data, allow us to generate a structural and mechanistic model for the PhopRNA activation by PhoPop5 and PhoRpp30, in which the two C-terminal helices (α4) of PhoPop5 in the tetramer whose formation is assisted by PhoRpp30 act as binding elements and bridge SL3 and SL16 in PhopRNA. PMID:26152732

  6. Transport Rates of a Glutamate Transporter Homologue Are Influenced by the Lipid Bilayer*

    PubMed Central

    McIlwain, Benjamin C.; Vandenberg, Robert J.; Ryan, Renae M.

    2015-01-01

    The aspartate transporter from Pyrococcus horikoshii (GltPh) is a model for the structure of the SLC1 family of amino acid transporters. Crystal structures of GltPh provide insight into mechanisms of ion coupling and substrate transport; however, structures have been solved in the absence of a lipid bilayer so they provide limited information regarding interactions that occur between the protein and lipids of the membrane. Here, we investigated the effect of the lipid environment on aspartate transport by reconstituting GltPh into liposomes of defined lipid composition where the primary lipid is phosphatidylethanolamine (PE) or its methyl derivatives. We showed that the rate of aspartate transport and the transmembrane orientation of GltPh were influenced by the primary lipid in the liposomes. In PE liposomes, we observed the highest transport rate and showed that 85% of the transporters were orientated right-side out, whereas in trimethyl PE liposomes, 50% of transporters were right-side out, and we observed a 4-fold reduction in transport rate. Differences in orientation can only partially explain the lipid composition effect on transport rate. Crystal structures of GltPh revealed a tyrosine residue (Tyr-33) that we propose interacts with lipid headgroups during the transport cycle. Based on site-directed mutagenesis, we propose that a cation-π interaction between Tyr-33 and the lipid headgroups can influence conformational flexibility of the trimerization domain and thus the rate of transport. These results provide a specific example of how interactions between membrane lipids and membrane-bound proteins can influence function and highlight the importance of the role of the membrane in transporter function. PMID:25713135

  7. Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold

    PubMed Central

    Lomonosova, Anna V.; Ovchinnikova, Elena V.; Kazakov, Alexei S.; Denesyuk, Alexander I.; Sofin, Alexander D.; Mikhailov, Roman V.; Ulitin, Andrei B.; Mirzabekov, Tajib A.; Permyakov, Eugene A.; Permyakov, Sergei E.

    2015-01-01

    Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C) and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M). Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X) was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M) and is more prone to oligomerization. This investigation of an extremophile protein’s scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds. PMID:26247602

  8. The solution structure of the C-terminal domain of NfeD reveals a novel membrane-anchored OB-fold.

    PubMed

    Kuwahara, Yohta; Ohno, Ayako; Morii, Taichi; Yokoyama, Hideshi; Matsui, Ikuo; Tochio, Hidehito; Shirakawa, Masahiro; Hiroaki, Hidekazu

    2008-11-01

    Nodulation formation efficiency D (NfeD) is a member of a class of membrane-anchored ClpP-class proteases. There is a second class of NfeD homologs that lack the ClpP domain. The genes of both NfeD classes usually are part of an operon that also contains a gene for a prokaryotic homolog of stomatin. (Stomatin is a major integral-membrane protein of mammalian erythrocytes.) Such NfeD/stomatin homolog gene pairs are present in more than 290 bacterial and archaeal genomes, and their protein products may be part of the machinery used for quality control of membrane proteins. Herein, we report the structure of the isolated C-terminal domain of PH0471, a Pyrococcus horikoshii NfeD homolog, which lacks the ClpP domain. This C-terminal domain (termed NfeDC) contains a five-strand beta-barrel, which is structurally very similar to the OB-fold (oligosaccharide/oligonucleotide-binding fold) domain. However, there is little sequence similarity between it and previously characterized OB-fold domains. The NfeDC domain lacks the conserved surface residues that are necessary for the binding of an OB-fold domain to DNA/RNA, an ion. Instead, its surface is composed of residues that are uniquely conserved in NfeD homologs and that form the structurally conserved surface turns and beta-bulges. There is also a conserved tryptophan present on the surface. We propose that, in general, NfeDC domains may interact with other spatially proximal membrane proteins and thereby regulate their activities. PMID:18687870

  9. Thermodynamic analysis of unusually thermostable CutA1 protein from human brain and its protease susceptibility.

    PubMed

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Yamamoto, Hitoshi; Sawano, Masahide; Ogasahara, Kyoko; Takehira, Michiyo; Kunishima, Naoki; Katoh, Etsuko; Yutani, Katsuhide

    2015-03-01

    Unusually stable proteins are a disadvantage for the metabolic turnover of proteins in cells. The CutA1 proteins from Pyrococcus horikoshii and from Oryza sativa (OsCutA1) have unusually high denaturation temperatures (Td) of nearly 150 and 100 °C, respectively, at pH 7.0. It seemed that the CutA1 protein from the human brain (HsCutA1) also has a remarkably high stability. Therefore, the thermodynamic stabilities of HsCutA1 and its protease susceptibility were examined. The Td was remarkably high, being over 95 °C at pH 7.0. The unfolding Gibbs energy (ΔG(0)H2O) was 174 kJ/mol at 37 °C from the denaturant denaturation. The thermodynamic analysis showed that the unfolding enthalpy and entropy values of HsCutA1 were considerably lower than those of OsCutA1 with a similar stability to HsCutA1, which should be related to flexibility of the unstructured properties in both N- and C-terminals of HsCutA1. HsCutA1 was almost completely digested after 1-day incubation at 37 °C by subtilisin, although OsCutA1 was hardly digested at the same conditions. These results indicate that easily available fragmentation of HsCutA1 with remarkably high thermodynamic stability at the body temperature should be important for its protein catabolism in the human cells. PMID:25344844

  10. Remarkable improvement in the heat stability of CutA1 from Escherichia coli by rational protein design.

    PubMed

    Matsuura, Yoshinori; Ota, Motonori; Tanaka, Tomoyuki; Takehira, Michiyo; Ogasahara, Kyoko; Bagautdinov, Bagautdin; Kunishima, Naoki; Yutani, Katsuhide

    2010-10-01

    To enhance the heat stability of the CutA1 protein from Escherichia coli (EcCutA1) so that it has comparable stability to CutA1 from Pyrococcus horikoshii with a denaturation temperature (T(d)) of 150°C, we used the Stability Profile of Mutant Protein (SPMP) to examine the structure-sequence (3D-1D) compatibility between the conformation of EcCutA1 and its native sequence [J. Mol. Biol., 248, 733-738, (1995)]. We identified seven residues in EcCutA1 that were incompatible in terms of dihedral angles and hydrophobicity. These residues were replaced with appropriate amino acids, and the mutant proteins were evaluated for changes in stability by DSC and denaturant denaturation. The mutations that were introduced at five out of the seven positions improved the stability of EcCutA1. The T(d) values of single (S11A) and triple (S11V/E61V/Q73V) mutants improved by 16.5 and 26.6°C, respectively, compared to that of the wild-type protein (89.9°C). These analyses showed that (1) the stability of EcCutA1 is remarkably improved by slight substitutions, even though the stability of the wild-type protein is considerably high, (2) remarkable improvements in the stability can be quantitatively explained based on the newly solved native structure, and (3) SPMP is a powerful tool to examine substitutions that improve protein stability. PMID:20639520

  11. Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold.

    PubMed

    Lomonosova, Anna V; Ovchinnikova, Elena V; Kazakov, Alexei S; Denesyuk, Alexander I; Sofin, Alexander D; Mikhailov, Roman V; Ulitin, Andrei B; Mirzabekov, Tajib A; Permyakov, Eugene A; Permyakov, Sergei E

    2015-01-01

    Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C) and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M). Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X) was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M) and is more prone to oligomerization. This investigation of an extremophile protein's scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds. PMID:26247602

  12. Altering the Enantioselectivity of Tyrosyl-tRNA Synthetase by Insertion of a Stereospecific Editing Domain.

    PubMed

    Richardson, Charles J; First, Eric A

    2016-03-15

    Translation of mRNAs by the ribosome is stereospecific, with only l-amino acids being incorporated into the nascent polypeptide chain. This stereospecificity results from the exclusion of d-amino acids at three steps during protein synthesis: (1) the aminoacylation of tRNA by aminoacyl-tRNA synthetases, (2) binding of aminoacyl-tRNAs to EF-Tu, and (3) recognition of aminoacyl-tRNAs by the ribosome. As a first step toward incorporating d-amino acids during protein synthesis, we have altered the enantioselectivity of tyrosyl-tRNA synthetase. This enzyme is unusual among aminoacyl-tRNA synthetases, as it can aminoacylate tRNA with d-tyrosine (albeit at a reduced rate compared to l-tyrosine). To change the enantioselectivity of tyrosyl-tRNA synthetase, we introduced the post-transfer editing domain from Pyrococcus horikoshii phenylalanyl-tRNA synthetase into the connective polypeptide 1 (CP1) domain of Geobacillus stearothermophilus tyrosyl-tRNA synthetase (henceforth designated TyrRS-FRSed). We show that the phenylalanyl-tRNA synthetase editing domain is stereospecific, hydrolyzing l-Tyr-tRNA(Tyr), but not d-Tyr-tRNA(Tyr). We further show that inserting the phenylalanyl-tRNA synthetase editing domain into the CP1 domain of tyrosyl-tRNA synthetase decreases the activity of the synthetic site in tyrosyl-tRNA synthetase. This decrease in activity is critical, as it prevents the rate of synthesis from overwhelming the ability of the editing domain to hydrolyze the l-Tyr-tRNA(Tyr) product. Overall, inserting the phenylalanyl-tRNA synthetase editing domain results in a 2-fold shift in the enantioselectivity of tyrosyl-tRNA synthetase toward the d-Tyr-tRNA(Tyr) product. When a 4-fold excess of d-tyrosine is used, approximately 40% of the tRNA(Tyr) is aminoacylated with d-tyrosine. PMID:26890980

  13. A sampling of molecular dynamics

    NASA Astrophysics Data System (ADS)

    Sindhikara, Daniel Jon

    regulatory protein whose binding to free Ni++ increases its binding affinity for a nickel transporter gene. Three forms of the Pyrococcus Horikoshii species of NikR were simulated including two apo-forms and one nickel-bound form. A quantum-mechanics-based force field parameterization was required to accurately represent the four nickel-centers in the holo-form. Extensive analysis of the three 100-ns-long trajectories was performed.

  14. 4-Demethylwyosine Synthase from Pyrococcus abyssi Is a Radical-S-adenosyl-l-methionine Enzyme with an Additional [4Fe-4S]+2 Cluster That Interacts with the Pyruvate Co-substrate*

    PubMed Central

    Perche-Letuvée, Phanélie; Kathirvelu, Velavan; Berggren, Gustav; Clemancey, Martin; Latour, Jean-Marc; Maurel, Vincent; Douki, Thierry; Armengaud, Jean; Mulliez, Etienne; Fontecave, Marc; Garcia-Serres, Ricardo; Gambarelli, Serge; Atta, Mohamed

    2012-01-01

    Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called “Radical-SAM.” These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX3CX2C motif, and S-adenosyl-l-methionine (SAM) to generate a 5′-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed. PMID:23043105

  15. Revisiting the structure/function relationships of H/ACA(-like) RNAs: a unified model for Euryarchaea and Crenarchaea

    PubMed Central

    Toffano-Nioche, Claire; Gautheret, Daniel; Leclerc, Fabrice

    2015-01-01

    A structural and functional classification of H/ACA and H/ACA-like motifs is obtained from the analysis of the H/ACA guide RNAs which have been identified previously in the genomes of Euryarchaea (Pyrococcus) and Crenarchaea (Pyrobaculum). A unified structure/function model is proposed based on the common structural determinants shared by H/ACA and H/ACA-like motifs in both Euryarchaea and Crenarchaea. Using a computational approach, structural and energetic rules for the guide:target RNA-RNA interactions are derived from structural and functional data on the H/ACA RNP particles. H/ACA(-like) motifs found in Pyrococcus are evaluated through the classification and their biological relevance is discussed. Extra-ribosomal targets found in both Pyrococcus and Pyrobaculum might support the hypothesis of a gene regulation mediated by H/ACA(-like) guide RNAs in archaea. PMID:26240384

  16. Nuclear resonance vibrational spectroscopy (NRVS) of rubredoxin and MoFe protein crystals

    PubMed Central

    Guo, Yisong; Brecht, Eric; Aznavour, Kristen; Nix, Jay C.; Xiao, Yuming; Wang, Hongxin; George, Simon J.; Bau, Robert; Keable, Stephen; Peters, John W.; Adams, Michael W.W.; Jenney, Francis; Sturhahn, Wolfgang; Alp, Ercan E.; Zhao, Jiyong; Yoda, Yoshitaka; Cramer, Stephen P.

    2014-01-01

    We have applied 57Fe nuclear resonance vibrational spectroscopy (NRVS) for the first time to study the dynamics of Fe centers in Fe-S protein crystals, including oxidized wild type rubredoxin crystals from Pyrococcus furiosus, and the MoFe protein of nitrogenase from Azotobacter vinelandii. Thanks to the NRVS selection rule, selectively probed vibrational modes have been observed in both oriented rubredoxin and MoFe protein crystals. The NRVS work was complemented by extended X-ray absorption fine structure spectroscopy (EXAFS) measurements on oxidized wild type rubredoxin crystals from Pyrococcus furiosus. The EXAFS spectra revealed the Fe-S bond length difference in oxidized Pf Rd protein, which is qualitatively consistent with the X-ray crystal structure. PMID:26052177

  17. Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon.

    PubMed

    Myllykallio, H; Lopez, P; López-García, P; Heilig, R; Saurin, W; Zivanovic, Y; Philippe, H; Forterre, P

    2000-06-23

    Despite a rapid increase in the amount of available archaeal sequence information, little is known about the duplication of genetic material in the third domain of life. We identified a single origin of bidirectional replication in Pyrococcus abyssi by means of in silico analyses of cumulative oligomer skew and the identification of an early replicating chromosomal segment. The replication origin in three Pyrococcus species was found to be highly conserved, and several eukaryotic-like DNA replication genes were clustered around it. As in Bacteria, the chromosomal region containing the replication terminus was a hot spot of genome shuffling. Thus, although bacterial and archaeal replication proteins differ profoundly, they are used to replicate chromosomes in a similar manner in both prokaryotic domains. PMID:10864870

  18. Nuclear resonance vibrational spectroscopy (NRVS) of rubredoxin and MoFe protein crystals

    NASA Astrophysics Data System (ADS)

    Guo, Yisong; Brecht, Eric; Aznavour, Kristen; Nix, Jay C.; Xiao, Yuming; Wang, Hongxin; George, Simon J.; Bau, Robert; Keable, Stephen; Peters, John W.; Adams, Michael W. W.; , Francis E. Jenney, Jr.; Sturhahn, Wolfgang; Alp, Ercan E.; Zhao, Jiyong; Yoda, Yoshitaka; Cramer, Stephen P.

    2013-12-01

    We have applied 57Fe nuclear resonance vibrational spectroscopy (NRVS) for the first time to study the dynamics of Fe centers in Iron-sulfur protein crystals, including oxidized wild type rubredoxin crystals from Pyrococcus furiosus, and the MoFe protein of nitrogenase from Azotobacter vinelandii. Thanks to the NRVS selection rule, selectively probed vibrational modes have been observed in both oriented rubredoxin and MoFe protein crystals. The NRVS work was complemented by extended X-ray absorption fine structure spectroscopy (EXAFS) measurements on oxidized wild type rubredoxin crystals from Pyrococcus furiosus. The EXAFS spectra revealed the Fe-S bond length difference in oxidized Pf Rd protein, which is qualitatively consistent with the crystal structure.

  19. Non-discriminating and discriminating aspartyl-tRNA synthetases differ in the anticodon-binding domain

    PubMed Central

    Charron, Christophe; Roy, Hervé; Blaise, Mickael; Giegé, Richard; Kern, Daniel

    2003-01-01

    In most organisms, tRNA aminoacylation is ensured by 20 aminoacyl-tRNA synthetases (aaRSs). In eubacteria, however, synthetases can be duplicated as in Thermus thermophilus, which contains two distinct AspRSs. While AspRS-1 is specific, AspRS-2 is non-discriminating and aspartylates tRNAAsp and tRNAAsn. The structure at 2.3 Å resolution of AspRS-2, the first of a non-discriminating synthetase, was solved. It differs from that of AspRS-1 but has resemblance to that of discriminating and archaeal AspRS from Pyrococcus kodakaraensis. The protein presents non-conventional features in its OB-fold anticodon-binding domain, namely the absence of a helix inserted between two β-strands of this fold and a peculiar L1 loop differing from the large loops known to interact with tRNAAsp identity determinant C36 in conventional AspRSs. In AspRS-2, this loop is small and structurally homologous to that in AsnRSs, including conservation of a proline. In discriminating Pyrococcus AspRS, the L1 loop, although small, lacks this proline and is not superimposable with that of AspRS-2 or AsnRS. Its particular status is demonstrated by a loop-exchange experiment that renders the Pyrococcus AspRS non-discriminating. PMID:12660169

  20. Biogeography and evolution of Thermococcus isolates from hydrothermal vent systems of the Pacific.

    PubMed

    Price, Mark T; Fullerton, Heather; Moyer, Craig L

    2015-01-01

    Thermococcus is a genus of hyperthermophilic archaea that is ubiquitous in marine hydrothermal environments growing in anaerobic subsurface habitats but able to survive in cold oxygenated seawater. DNA analyses of Thermococcus isolates were applied to determine the relationship between geographic distribution and relatedness focusing primarily on isolates from the Juan de Fuca Ridge and South East Pacific Rise. Amplified fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) were used to resolve genomic differences in 90 isolates of Thermococcus, making biogeographic patterns and evolutionary relationships apparent. Isolates were differentiated into regionally endemic populations however there was also evidence in some lineages of cosmopolitan distribution. The biodiversity identified in Thermococcus isolates and presence of distinct lineages within the same vent site suggests the utilization of varying ecological niches in this genus. In addition to resolving biogeographic patterns in Thermococcus, this study has raised new questions about the closely related Pyrococcus genus. The phylogenetic placement of Pyrococcus type strains shows the close relationship between Thermococcus and Pyrococcus and the unresolved divergence of these two genera. PMID:26441901

  1. Biogeography and evolution of Thermococcus isolates from hydrothermal vent systems of the Pacific

    PubMed Central

    Price, Mark T.; Fullerton, Heather; Moyer, Craig L.

    2015-01-01

    Thermococcus is a genus of hyperthermophilic archaea that is ubiquitous in marine hydrothermal environments growing in anaerobic subsurface habitats but able to survive in cold oxygenated seawater. DNA analyses of Thermococcus isolates were applied to determine the relationship between geographic distribution and relatedness focusing primarily on isolates from the Juan de Fuca Ridge and South East Pacific Rise. Amplified fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) were used to resolve genomic differences in 90 isolates of Thermococcus, making biogeographic patterns and evolutionary relationships apparent. Isolates were differentiated into regionally endemic populations however there was also evidence in some lineages of cosmopolitan distribution. The biodiversity identified in Thermococcus isolates and presence of distinct lineages within the same vent site suggests the utilization of varying ecological niches in this genus. In addition to resolving biogeographic patterns in Thermococcus, this study has raised new questions about the closely related Pyrococcus genus. The phylogenetic placement of Pyrococcus type strains shows the close relationship between Thermococcus and Pyrococcus and the unresolved divergence of these two genera. PMID:26441901

  2. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    SciTech Connect

    Woodward, J.

    1996-10-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based upon the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with continuous cofactor recycle. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value commodity chemical.

  3. Sulfide Ameliorates Metal Toxicity for Deep-Sea Hydrothermal Vent Archaea†

    PubMed Central

    Edgcomb, Virginia P.; Molyneaux, Stephen J.; Saito, Mak A.; Lloyd, Karen; Böer, Simone; Wirsen, Carl O.; Atkins, Michael S.; Teske, Andreas

    2004-01-01

    The chemical stress factors for microbial life at deep-sea hydrothermal vents include high concentrations of heavy metals and sulfide. Three hyperthermophilic vent archaea, the sulfur-reducing heterotrophs Thermococcus fumicolans and Pyrococcus strain GB-D and the chemolithoautotrophic methanogen Methanocaldococcus jannaschii, were tested for survival tolerance to heavy metals (Zn, Co, and Cu) and sulfide. The sulfide addition consistently ameliorated the high toxicity of free metal cations by the formation of dissolved metal-sulfide complexes as well as solid precipitates. Thus, chemical speciation of heavy metals with sulfide allows hydrothermal vent archaea to tolerate otherwise toxic metal concentrations in their natural environment. PMID:15066859

  4. NMR studies on mechanism of isomerisation of fructose 6-phosphate to glucose 6-phosphate catalysed by phosphoglucose isomerase from Thermococcus kodakarensis.

    PubMed

    Abbas, Shahzada Nadeem; Mok, Kenneth Hun; Rashid, Naeem; Xie, Yongjing; Ruether, Manuel; O'Brien, John; Akhtar, Muhammad

    2016-06-01

    The fate of hydrogen atoms at C-2 of glucose 6-phosphate (G6P) and C-1 of fructose 6-phosphate (F6P) was studied in the reaction catalysed by phosphoglucose isomerase from Thermococcus kodakarensis (TkPGI) through 1D and 2D NMR methods. When the reaction was performed in (2)H2O the hydrogen atoms in the aforementioned positions were exchanged with deuterons indicating that the isomerization occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. These features are similar to those described for phosphoglucose isomerases from rabbit muscle and Pyrococcus furiosus. PMID:27014866

  5. Overview of thermostable DNA polymerases for classical PCR applications: from molecular and biochemical fundamentals to commercial systems.

    PubMed

    Terpe, Kay

    2013-12-01

    During the genomics era, the use of thermostable DNA polymerases increased greatly. Many were identified and described-mainly of the genera Thermus, Thermococcus and Pyrococcus. Each polymerase has different features, resulting from origin and genetic modification. However, the rational choice of the adequate polymerase depends on the application itself. This review gives an overview of the most commonly used DNA polymerases used for PCR application: KOD, Pab (Isis™), Pfu, Pst (Deep Vent™), Pwo, Taq, Tbr, Tca, Tfi, Tfl, Tfu, Tgo, Tli (Vent™), Tma (UITma™), Tne, Tth and others. PMID:24177730

  6. Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea.

    PubMed

    Ishino, Sonoko; Nishi, Yuki; Oda, Soichiro; Uemori, Takashi; Sagara, Takehiro; Takatsu, Nariaki; Yamagami, Takeshi; Shirai, Tsuyoshi; Ishino, Yoshizumi

    2016-04-20

    The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeonPyrococcus furiosus The corresponding gene revealed that the activity originates from PF0012, and we named this enzyme Endonuclease MS (EndoMS) as themismatch-specific Endonuclease. The sequence similarity suggested that EndoMS is the ortholog of NucS isolated fromPyrococcus abyssi, published previously. Biochemical characterizations of the EndoMS homolog fromThermococcus kodakarensisclearly showed that EndoMS specifically cleaves both strands of double-stranded DNA into 5'-protruding forms, with the mismatched base pair in the central position. EndoMS cleaves G/T, G/G, T/T, T/C and A/G mismatches, with a more preference for G/T, G/G and T/T, but has very little or no effect on C/C, A/C and A/A mismatches. The discovery of this endonuclease suggests the existence of a novel mismatch repair process, initiated by the double-strand break generated by the EndoMS endonuclease, in Archaea and some Bacteria. PMID:27001046

  7. Redox-Promoting Protein Motions in Rubredoxin

    SciTech Connect

    Myles, Dean A A; He, Junhong; Meilleur, Flora; Weiss, Kevin L; Agarwal, Pratul K; Borreguero Calvo, Jose M; Barthes, Mariette; Brown, Craig; Herwig, Kenneth W

    2011-01-01

    Proteins are dynamic objects, constantly undergoing conformational fluctuations, yet the linkage between internal protein motion and function is widely debated. This manuscript reports on the characterization of temperature-activated collective and individual atomic motions of oxidized rubredoxin, a small 53 residue protein from thermophilic Pyrococcus furiosus (RdPf), by neutron scattering and computational simulations. The changes in motion have been explored in connection to their role in promoting reduction of the Fe+3 ion which is responsible for the electron transfer function of RdPf. Just above the dynamical transition temperature of 220 K which marks the onset of significant anharmonic motions of the protein, the computer simulations show both a significant reorientation of the average electrostatic force experienced by the Fe+3 ion and a dramatic rise in its strength. At higher temperatures, additional anharmonic modes become activated which dominate the electrostatic fluctuations experienced by the ion. At 360 K, close to the optimal growth temperature of Pyrococcus furiosus, computer simulations show that three anharmonic modes involving two conserved residues located at the protein active site (Ile7 and Ile40) give rise to the majority of the electrostatic fluctuations experienced by the Fe+3 ion and include displacements which allow solvent access to the ion. The low-frequency, high amplitude motions of these residues at low temperatures may be precursors of the high temperature, anharmonic motions necessary for protein function.

  8. Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea

    PubMed Central

    Ishino, Sonoko; Nishi, Yuki; Oda, Soichiro; Uemori, Takashi; Sagara, Takehiro; Takatsu, Nariaki; Yamagami, Takeshi; Shirai, Tsuyoshi; Ishino, Yoshizumi

    2016-01-01

    The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus. The corresponding gene revealed that the activity originates from PF0012, and we named this enzyme Endonuclease MS (EndoMS) as the mismatch-specific Endonuclease. The sequence similarity suggested that EndoMS is the ortholog of NucS isolated from Pyrococcus abyssi, published previously. Biochemical characterizations of the EndoMS homolog from Thermococcus kodakarensis clearly showed that EndoMS specifically cleaves both strands of double-stranded DNA into 5′-protruding forms, with the mismatched base pair in the central position. EndoMS cleaves G/T, G/G, T/T, T/C and A/G mismatches, with a more preference for G/T, G/G and T/T, but has very little or no effect on C/C, A/C and A/A mismatches. The discovery of this endonuclease suggests the existence of a novel mismatch repair process, initiated by the double-strand break generated by the EndoMS endonuclease, in Archaea and some Bacteria. PMID:27001046

  9. Box C/D RNA guides for the ribose methylation of archaeal tRNAs. The tRNATrp intron guides the formation of two ribose-methylated nucleosides in the mature tRNATrp

    PubMed Central

    d’Orval, Béatrice Clouet; Bortolin, Marie-Line; Gaspin, Christine; Bachellerie, Jean-Pierre

    2001-01-01

    Following a search of the Pyrococcus genomes for homologs of eukaryotic methylation guide small nucleolar RNAs, we have experimentally identified in Pyrococcus abyssi four novel box C/D small RNAs predicted to direct 2′-O-ribose methylations onto the first position of the anticodon in tRNALeu(CAA), tRNALeu(UAA), elongator tRNAMet and tRNATrp, respectively. Remarkably, one of them corresponds to the intron of its presumptive target, pre-tRNATrp. This intron is predicted to direct in cis two distinct ribose methylations within the unspliced tRNA precursor, not only onto the first position of the anticodon in the 5′ exon but also onto position 39 (universal tRNA numbering) in the 3′ exon. The two intramolecular RNA duplexes expected to direct methylation, which both span an exon–intron junction in pre-tRNATrp, are phylogenetically conserved in euryarchaeotes. We have experimentally confirmed the predicted guide function of the box C/D intron in halophile Haloferax volcanii by mutagenesis analysis, using an in vitro splicing/RNA modification assay in which the two cognate ribose methylations of pre-tRNATrp are faithfully reproduced. Euryarchaeal pre-tRNATrp should provide a unique system to further investigate the molecular mechanisms of RNA-guided ribose methylation and gain new insights into the origin and evolution of the complex family of archaeal and eukaryotic box C/D small RNAs. PMID:11713301

  10. Identification of potential inhibitors for AIRS from de novo purine biosynthesis pathway through molecular modeling studies - a computational approach.

    PubMed

    Rao, R Guru Raj; Biswal, Jayashree; Dhamodharan, Prabhu; Kanagarajan, Surekha; Jeyaraman, Jeyakanthan

    2016-10-01

    In cancer, de novo pathway plays an important role in cell proliferation by supplying huge demand of purine nucleotides. Aminoimidazole ribonucleotide synthetase (AIRS) catalyzes the fifth step of de novo purine biosynthesis facilitating in the conversion of formylglycinamidine ribonucleotide to aminoimidazole ribonucleotide. Hence, inhibiting AIRS is crucial due to its involvement in the regulation of uncontrollable cancer cell proliferation. In this study, the three-dimensional structure of AIRS from P. horikoshii OT3 was constructed based on the crystal structure from E. coli and the modeled protein is verified for stability using molecular dynamics for a time frame of 100 ns. Virtual screening and induced fit docking were performed to identify the best antagonists based on their binding mode and affinity. Through mutational studies, the residues necessary for catalytic activity of AIRS were identified and among which the following residues Lys35, Asp103, Glu137, and Thr138 are important in determination of AIRS function. The mutational studies help to understand the structural and energetic characteristics of the specified residues. In addition to Molecular Dynamics, ADME properties, binding free-energy, and density functional theory calculations of the compounds were carried out to find the best lead molecule. Based on these analyses, the compound from the NCI database, NCI_121957 was adjudged as the best molecule and could be suggested as the suitable inhibitor of AIRS. In future studies, experimental validation of these ligands as AIRS inhibitors will be carried out. PMID:26524231

  11. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.

    PubMed

    Hopfner, K P; Karcher, A; Shin, D S; Craig, L; Arthur, L M; Carney, J P; Tainer, J A

    2000-06-23

    To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins. PMID:10892749

  12. Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation.

    PubMed

    Buller, Andrew R; Brinkmann-Chen, Sabine; Romney, David K; Herger, Michael; Murciano-Calles, Javier; Arnold, Frances H

    2015-11-24

    Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope. PMID:26553994

  13. Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

    SciTech Connect

    Hura, Greg L.; Menon, Angeli L.; Hammel, Michal; Rambo, Robert P.; Poole II, Farris L.; Tsutakawa, Susan E.; Jenney Jr, Francis E.; Classen, Scott; Frankel, Kenneth A.; Hopkins, Robert C.; Yang, Sungjae; Scott, Joseph W.; Dillard, Bret D.; Adams, Michael W. W.; Tainer, John A.

    2009-07-20

    We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

  14. A variable-temperature direct electrochemical study of metalloproteins from hyperthermophilic microorganisms involves in hydrogen production from pyruvate

    SciTech Connect

    Smith, E.T.; Blamey, J.; Zhou, Z.Z.; Adams, M.W.W.

    1995-05-30

    The hyperthomophilic bacterium Thermotoga maritima and the hyperthermolic archaeon Pyrococcus furiosus grow optimally at 80{degrees} and 100{degrees}C, respectively, by the fermentation of carbohydrates to organic acids, CO{sub 2}, and H{sub 2}. Pyruvate is a major source of reductant for H{sub 2} production during fermentation, and pyruvate ferredoxin oxidoreductase (POR), a 4Fe-type ferredoxin, and hydrogenase have been previously purified from both species. P. furiosus utilizes copper-iron-containing POR and a nickel-iron-containing hydrogenase, whereas the POR of T. maritima lacks copper and its hydrogenase lacks nickel. For all four enzymes and for the two ferredoxins, we have determined their reproduction potentials (E{degrees}` and, where possible, thermodynamic parameters associated with electron transfer {Delta}S{degrees} and {Delta}H{degrees}), using differential pulse voltammetry at temperatures ranging from 25 to 95{degrees}C. 55 refs., 7 fig., 2 tabs.

  15. Microbial metalloproteomes explored using MIRAGE.

    PubMed

    Sevcenco, Ana-Maria; Hagen, Wilfred R; Hagedoorn, Peter-Leon

    2012-09-01

    Metalloproteomics is a rapidly developing field of science that involves the comprehensive analysis of all metal-containing or metal-binding proteins in a biological sample. The purpose of this review is to offer an overview of the research involving Metal Isotope native RadioAutography in Gel Electrophoresis (MIRAGE), a powerful new method to visualize and study the proteome of a particular metal ion. MIRAGE involves four steps: i) labelling of target proteins with a radioisotope; ii) separation of intact holo-proteins using native isoelectric focusing (1D) combined with Blue Native PAGE (2D); iii) spot visualization and quantification using autoradiography; and iv) protein identification by tandem mass spectrometry. MIRAGE Investigations of the soluble Cu, Zn, and Fe metalloproteomes of Escherichia coli, and of the soluble Mo and W proteomes of the hyperthermophilic archaeon Pyrococcus furiosus are reviewed. PMID:22976984

  16. POLYPEPTIDE AND POLYSACCHARIDE PROCESSING IN HYPERTHERMOPHILIC MICROORGANISMS

    SciTech Connect

    KELLY, ROBERT M.

    2008-12-22

    This project focused on the microbial physiology and biochemistry of heterotrophic hyperthermophiles with respect to mechanisms by which these organisms process polypeptides and polysaccharides under normal and stressed conditions. Emphasis is on two model organisms, for which completed genome sequences are available: Pyrococcus furiosus (growth Topt of 98°C), an archaeon, and Thermotoga maritima (growth Topt of 80°C), a bacterium. Both organisms are obligately anaerobic heterotrophs that reduce sulfur facultatively. Whole genome cDNA spotted microarrays were used to follow transcriptional response to a variety of environmental conditions in order to identify genes encoding proteins involved in the acquisition, synthesis, processing and utilization of polypeptides and polysaccharides. This project provided new insights into the physiological aspects of hyperthermophiles as these relate to microbial biochemistry and biological function in high temperature habitats. The capacity of these microorganisms to produce biohydrogen from renewable feedstocks makes them important for future efforts to develop biofuels.

  17. Synthesis of β-Branched Tryptophan Analogues Using an Engineered Subunit of Tryptophan Synthase.

    PubMed

    Herger, Michael; van Roye, Paul; Romney, David K; Brinkmann-Chen, Sabine; Buller, Andrew R; Arnold, Frances H

    2016-07-13

    We report that l-threonine may substitute for l-serine in the β-substitution reaction of an engineered subunit of tryptophan synthase from Pyrococcus furiosus, yielding (2S,3S)-β-methyltryptophan (β-MeTrp) in a single step. The trace activity of the wild-type β-subunit on this substrate was enhanced more than 1000-fold by directed evolution. Structural and spectroscopic data indicate that this increase is correlated with stabilization of the electrophilic aminoacrylate intermediate. The engineered biocatalyst also reacts with a variety of indole analogues and thiophenol for diastereoselective C-C, C-N, and C-S bond-forming reactions. This new activity circumvents the 3-enzyme pathway that produces β-MeTrp in nature and offers a simple and expandable route to preparing derivatives of this valuable building block. PMID:27355405

  18. The Mode of Cell Wall Growth in Selected Archaea Is Similar to the General Mode of Cell Wall Growth in Bacteria as Revealed by Fluorescent Dye Analysis ▿ †

    PubMed Central

    Wirth, Reinhard; Bellack, Annett; Bertl, Markus; Bilek, Yvonne; Heimerl, Thomas; Herzog, Bastian; Leisner, Madeleine; Probst, Alexander; Rachel, Reinhard; Sarbu, Christina; Schopf, Simone; Wanner, Gerhard

    2011-01-01

    The surfaces of 8 bacterial and 23 archaeal species, including many hyperthermophilic Archaea, could be stained using succinimidyl esters of fluorescent dyes. This allowed us for the first time to analyze the mode of cell wall growth in Archaea by subculturing stained cells. The data obtained show that incorporation of new cell wall material in Archaea follows the pattern observed for Bacteria: in the coccoid species Pyrococcus furiosus incorporation was in the region of septum formation while for the rod-shaped species Methanopyrus kandleri and Methanothermus sociabilis, a diffuse incorporation of cell wall material over the cell length was observed. Cell surface appendages like fimbriae/pili, fibers, or flagella were detectable by fluorescence staining only in a very few cases although their presence was proven by electron microscopy. Our data in addition prove that Alexa Fluor dyes can be used for in situ analyses at temperatures up to 100°C. PMID:21169435

  19. Proteolysis in hyperthermophilic microorganisms

    DOE PAGESBeta

    Ward, Donald E.; Shockley, Keith R.; Chang, Lara S.; Levy, Ryan D.; Michel, Joshua K.; Conners, Shannon B.; Kelly, Robert M.

    2002-01-01

    Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus , the crenarchaeote Sulfolobus solfataricus , and the bacterium Thermotoga maritima . An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putativemore » proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.« less

  20. Diversity of CRISPR systems in the euryarchaeal Pyrococcales

    PubMed Central

    Norais, Cédric; Moisan, Annick; Gaspin, Christine; Clouet-d'Orval, Béatrice

    2013-01-01

    Pyrococcales are members of the order Thermococcales, a group of hyperthermophilic euryarchaea that are frequently found in deep sea hydrothermal vents. Infectious genetic elements, such as plasmids and viruses, remain a threat even in this remote environment and these microorganisms have developed several ways to fight their genetic invaders. Among these are the recently discovered CRISPR systems. In this review, we have combined and condensed available information on genetic elements infecting the Thermococcales and on the multiple CRISPR systems found in the Pyrococcales to fight them. Their organization and mode of action will be presented with emphasis on the Type III-B system that is the only CRISPR system known to target RNA molecules in a process reminiscent of RNA interference. The intriguing case of Pyrococcus abyssi, which is among the rare strains to present a CRISPR system devoid of the universal cas1 and cas2 genes, is also discussed. PMID:23422322

  1. Monomer structure of a hyperthermophilic β-glucosidase mutant forming a dodecameric structure in the crystal form

    PubMed Central

    Nakabayashi, Makoto; Kataoka, Misumi; Watanabe, Masahiro; Ishikawa, Kazuhiko

    2014-01-01

    One of the β-glucosidases from Pyrococcus furiosus (BGLPf) is found to be a hyperthermophilic tetrameric enzyme that can degrade cellooligosaccharides. Recently, the crystal structures of the tetrameric and dimeric forms were solved. Here, a new monomeric form of BGLPf was constructed by removing the C-terminal region of the enzyme and its crystal structure was solved at a resolution of 2.8 Å in space group P1. It was discovered that the mutant enzyme forms a unique dodecameric structure consisting of two hexameric rings in the asymmetric unit of the crystal. Under biological conditions, the mutant enzyme forms a monomer. This result helps explain how BGLPf has attained its oligomeric structure and thermostability. PMID:25005077

  2. Phylogenetic and Comparative Sequence Analysis of Thermostable Alpha Amylases of kingdom Archea, Prokaryotes and Eukaryotes.

    PubMed

    Huma, Tayyaba; Maryam, Arooma; Rehman, Shahid Ur; Qamar, Muhammad Tahir Ul; Shaheen, Tayyaba; Haque, Asma; Shaheen, Bushra

    2014-01-01

    Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase α-(1, 4) or α-(1, 6) glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family, nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis (70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm. Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and productive for industry. PMID:25187685

  3. Characterization of two members among the five ADP-forming acyl coenzyme A (Acyl-CoA) synthetases reveals the presence of a 2-(Imidazol-4-yl)acetyl-CoA synthetase in Thermococcus kodakarensis.

    PubMed

    Awano, Tomotsugu; Wilming, Anja; Tomita, Hiroya; Yokooji, Yuusuke; Fukui, Toshiaki; Imanaka, Tadayuki; Atomi, Haruyuki

    2014-01-01

    The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α2β2 structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales. PMID:24163338

  4. Bacillus mesophilus sp. nov., an alginate-degrading bacterium isolated from a soil sample collected from an abandoned marine solar saltern.

    PubMed

    Zhou, Yan-Xia; Liu, Guo-Hong; Liu, Bo; Chen, Guan-Jun; Du, Zong-Jun

    2016-07-01

    A novel Gram-stain positive, endospore-forming bacterium, designated SA4(T), was isolated from a soil sample collected from an abandoned marine solar saltern at Wendeng, Shandong Province, PR China. Cells were observed to be rod shaped, alginase positive, catalase positive and motile. The strain was found to grow at temperatures ranging from 15 to 40 °C (optimum 35 °C), and pH 5.0-11.0 (optimum pH 8.0) with 0-7.0 % (w/v) NaCl concentration (optimum NaCl 3.0 %). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SA4(T) belongs to the genus Bacillus and exhibits 16S rRNA gene sequence similarities of 96.6, 96.5, 96.3 and 96.2 % with Bacillus horikoshii DSM 8719(T), Bacillus acidicola 105-2(T), Bacillus shackletonii LMG 18435(T) and Bacillus pocheonensis Gsoil 420(T), respectively. The menaquinone was identified as MK-7 and the major polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids detected were anteiso-C15:0 (22.3 %), iso-C15:0 (22.6 %), iso-C16:0 (14.8 %) and iso-C14:0 (14.7 %). The DNA G+C content was determined to be 42.4 mol %. Phenotypic, chemotaxonomic and genotypic properties clearly indicated that isolate SA4(T) represents a novel species within the genus Bacillus, for which the name Bacillus mesophius sp. nov. is proposed. The type strain is SA4(T) (=DSM 101000(T)=CCTCC AB 2015209(T)). PMID:27084709

  5. Engineering a Hyperthermophilic Archaeon for Temperature-Dependent Product Formation

    SciTech Connect

    Basen, M; Sun, JS; Adams, MWW

    2012-02-24

    Microorganisms growing near the boiling point have enormous biotechnological potential but only recently have molecular engineering tools become available for them. We have engineered the hyperthermophilic archaeon Pyrococcus furiosus, which grows optimally at 100 degrees C, to switch its end products of fermentation in a temperature-controlled fashion without the need for chemical inducers. The recombinant strain (LAC) expresses a gene (ldh) encoding lactate dehydrogenase from the moderately thermophilic Caldicellulosiruptor bescii (optimal growth temperature [T-opt] of 78 degrees C) controlled by a "cold shock" promoter that is upregulated when cells are transferred from 98 degrees C to 72 degrees C. At 98 degrees C, the LAC strain fermented sugar to produce acetate and hydrogen as end products, and lactate was not detected. When the LAC strain was grown at 72 degrees C, up to 3 mM lactate was produced instead. Expression of a gene from a moderately thermophilic bacterium in a hyperthermophilic archaeon at temperatures at which the hyperthermophile has low metabolic activity provides a new perspective to engineering microorganisms for bioproduct and biofuel formation. IMPORTANCE Extremely thermostable enzymes from microorganisms that grow near or above the boiling point of water are already used in biotechnology. However, the use of hyperthermophilic microorganisms themselves for biotechnological applications has been limited by the lack of their genetic accessibility. Recently, a genetic system for Pyrococcus furiosus, which grows optimally near 100 degrees C, was developed in our laboratory. In this study, we present the first heterologous protein expression system for a microorganism that grows optimally at 100 degrees C, a first step towards the potential expression of genes involved in biomass degradation or biofuel production in hyperthermophiles. Moreover, we developed the first system for specific gene induction in P. furiosus. As the cold shock promoter

  6. A high-performance thioredoxin-based scaffold for peptide immunogen construction: proof-of-concept testing with a human papillomavirus epitope

    PubMed Central

    Canali, Elena; Bolchi, Angelo; Spagnoli, Gloria; Seitz, Hanna; Rubio, Ivonne; Pertinhez, Thelma A.; Müller, Martin; Ottonello, Simone

    2014-01-01

    Escherichia coli thioredoxin has been previously exploited as a scaffold for the presentation/stabilization of peptide aptamers as well as to confer immunogenicity to peptide epitopes. Here we focused on other key features of thioredoxin that are of general interest for the production of safer and more effective peptide immunogens, such as a high thermal stability, lack of cross-reactivity and a low-cost of production. We identified thioredoxin from the archaebacterium Pyrococcus furiosus (PfTrx) as a novel scaffold meeting all the above criteria. PfTrx is a highly thermostable and protease-resistant scaffold with a strong (poly)peptide solubilisation capacity. Anti-PfTrx antibodies did not cross-react with mouse, nor human thioredoxin. Untagged PfTrx bearing a previously identified HPV16-L2 peptide epitope was obtained in a >90% pure form with a one-step thermal purification procedure and effectively elicited the production of neutralizing anti-HPV antibodies. We thus propose PfTrx as a superior, general-purpose scaffold for the construction of safe, stable, and low-cost peptide immunogens. PMID:24751665

  7. Organic Solutes in Hyperthermophilic Archaea

    PubMed Central

    Martins, L. O.; Huber, R.; Huber, H.; Stetter, K. O.; Da Costa, M. S.; Santos, H.

    1997-01-01

    We examined the accumulation of organic solutes under optimum growth conditions in 12 species of thermophilic and hyperthermophilic Archaea belonging to the Crenarchaeota and Euryarchaeota. Pyrobaculum aerophilum, Thermoproteus tenax, Thermoplasma acidophilum, and members of the order Sulfolobales accumulated trehalose. Pyrococcus furiosus accumulated di-myo-inositol-1,1(prm1)(3,3(prm1))-phosphate and (beta)-mannosylglycerate, Methanothermus fervidus accumulated cyclic-2,3-bisphosphoglycerate and (beta)-mannosylglycerate, while the only solute detected in Pyrodictium occultum was di-myo-inositol-1,1(prm1)(3,3(prm1))-phosphate. Methanopyrus kandleri accumulated large concentrations of cyclic-2,3-bisphosphoglycerate. On the other hand, Archaeoglobus fulgidus accumulated three phosphorylated solutes; prominent among them was a compound identified as di-glycerol-phosphate. This solute increased in concentration as the salinity of the medium and the growth temperature were raised, suggesting that this compound serves as a general stress solute. Di-myo-inositol-1,1(prm1)(3,3(prm1))-phosphate accumulated at supraoptimal temperature only. The relationship between the accumulation of unusual solutes and high temperatures is also discussed. PMID:16535556

  8. The Achilles' Heel of "Ultrastable" Hyperthermophile Proteins: Submillimolar Concentrations of SDS Stimulate Rapid Conformational Change, Aggregation, and Amyloid Formation in Proteins Carrying Overall Positive Charge.

    PubMed

    Khan, Javed M; Sharma, Prerna; Arora, Kanika; Kishor, Nitin; Kaila, Pallavi; Guptasarma, Purnananda

    2016-07-19

    Low concentrations (<3.0 mM) of the anionic surfactant sodium dodecyl sulfate (SDS) have been shown to induce the formation of amyloid fibers in more than 20 different mesophile-derived proteins in the cationic state. It is not known whether SDS has similar effects on hyperthermophile-derived proteins, which are otherwise thought to be "ultrastable" and inordinately resistant to structural perturbations at room temperature. Here, we show that low (<4.5 mM) concentrations of SDS rapidly induce the formation of aggregates and amyloid fibers in five different ultrastable Pyrococcus furiosus proteins in the cationic state. We also show that amyloid formation is accompanied by the development of a characteristic, negative circular dichroism band at ∼230 nm. These effects are not seen if the proteins have a net negative charge or when higher concentrations of SDS are used (which induce helix formation instead). Our results appear to reveal a potential weakness or "Achilles' heel" in ultrastable proteins from hyperthermophiles. They also provide very strong support for the view that SDS initially interacts with proteins through electrostatic interactions, and not hydrophobic interactions, eliciting similar effects entirely regardless of protein molecular weight, or structural features such as quaternary structure or tertiary structural stability. PMID:27331826

  9. Cloning and Characterization of an Alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

    NASA Technical Reports Server (NTRS)

    Bernhardsdotter, Eva C. M. J.; Pusey, Marc L.; Ng, Joseph D.; Garriott, Owen K.

    2004-01-01

    The gene encoding an extracellular a-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

  10. Cloning and Characterization of an alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

    NASA Technical Reports Server (NTRS)

    Bernhardsdotter, Eva C. M. J.; Pusey, Mark L.; Ng, Joseph D.; Garriott, Owen K.

    2004-01-01

    The gene encoding an extracellular alpha-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

  11. An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics

    PubMed Central

    Vandemoortele, Giel; Staes, An; Gonnelli, Giulia; Samyn, Noortje; De Sutter, Delphine; Vandermarliere, Elien; Timmerman, Evy; Gevaert, Kris; Martens, Lennart; Eyckerman, Sven

    2016-01-01

    The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these ’Proteotypic peptides for Quantification by SRM’ (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins. PMID:27264994

  12. On the mineral core of ferritin-like proteins: structural and magnetic characterization.

    PubMed

    García-Prieto, A; Alonso, J; Muñoz, D; Marcano, L; Abad Díaz de Cerio, A; Fernández de Luis, R; Orue, I; Mathon, O; Muela, A; Fdez-Gubieda, M L

    2016-01-14

    It is generally accepted that the mineral core synthesized by ferritin-like proteins consists of a ferric oxy-hydroxide mineral similar to ferrihydrite in the case of horse spleen ferritin (HoSF) and an oxy-hydroxide-phosphate phase in plant and prokaryotic ferritins. The structure reflects a dynamic process of deposition and dissolution, influenced by different biological, chemical and physical variables. In this work we shed light on this matter by combining a structural (High Resolution Transmission Electron Microscopy (HRTEM) and Fe K-edge X-ray Absorption Spectroscopy (XAS)) and a magnetic study of the mineral core biomineralized by horse spleen ferritin (HoSF) and three prokaryotic ferritin-like proteins: bacterial ferritin (FtnA) and bacterioferritin (Bfr) from Escherichia coli and archaeal ferritin (PfFtn) from Pyrococcus furiosus. The prokaryotic ferritin-like proteins have been studied under native conditions and inside the cells for the sake of preserving their natural attributes. They share with HoSF a nanocrystalline structure rather than an amorphous one as has been frequently reported. However, the presence of phosphorus changes drastically the short-range order and magnetic response of the prokaryotic cores with respect to HoSF. The superparamagnetism observed in HoSF is absent in the prokaryotic proteins, which show a pure atomic-like paramagnetic behaviour attributed to phosphorus breaking the Fe-Fe exchange interaction. PMID:26666195

  13. A new crystal form of a hyperthermophilic endocellulase

    SciTech Connect

    Kataoka, Misumi; Ishikawa, Kazuhiko

    2014-06-18

    The hyperthermostable endocellulase from P. furiosus was crystallized at pH 5.5. The new crystal form has symmetry consistent with space group C2 and exhibits a structure different from that of the protein crystallized at pH 9.0. The hyperthermophilic glycoside hydrolase family endocellulase 12 from the archaeon Pyrococcus furiosus (EGPf; Gene ID PF0854; EC 3.2.1.4) catalyzes the hydrolytic cleavage of the β-1,4-glucosidic linkage in β-glucan in lignocellulose biomass. A crystal of EGPf was previously prepared at pH 9.0 and its structure was determined at an atomic resolution of 1.07 Å. This article reports the crystallization of EGPf at the more physiologically relevant pH of 5.5. Structure determination showed that this new crystal form has the symmetry of space group C2. Two molecules of the enzyme are observed in the asymmetric unit. Crystal packing is weak at pH 5.5 owing to two flexible interfaces between symmetry-related molecules. Comparison of the EGPf structures obtained at pH 9.0 and pH 5.5 reveals a significant conformational difference at the active centre and in the surface loops. The interfaces in the vicinity of the flexible surface loops impact the quality of the EGPf crystal.

  14. Backbone Solution Structures of Proteins Using Residual Dipolar Couplings: Application to a Novel Structural Genomics Target

    PubMed Central

    Valafar, H.; Mayer, K. L.; Bougault, C. M.; LeBlond, P. D.; Jenney, F. E.; Brereton, P. S.; Adams, M.W.W.; Prestegard, J.H.

    2006-01-01

    Structural genomics (or proteomics) activities are critically dependent on the availability of high-throughput structure determination methodology. Development of such methodology has been a particular challenge for NMR based structure determination because of the demands for isotopic labeling of proteins and the requirements for very long data acquisition times. We present here a methodology that gains efficiency from a focus on determination of backbone structures of proteins as opposed to full structures with all side chains in place. This focus is appropriate given the presumption that many protein structures in the future will be built using computational methods that start from representative fold family structures and replace as many as 70% of the side chains in the course of structure determination. The methodology we present is based primarily on residual dipolar couplings (RDCs), readily accessible NMR observables that constrain the orientation of backbone fragments irrespective of separation in space. A new software tool is described for the assembly of backbone fragments under RDC constraints and an application to a structural genomics target is presented. The target is an 8.7 kDa protein from Pyrococcus furiosus, PF1061, that was previously not well annotated, and had a nearest structurally characterized neighbor with only 33% sequence identity. The structure produced shows structural similarity to this sequence homologue, but also shows similarity to other proteins that suggests a functional role in sulfur transfer. Given the backbone structure and a possible functional link this should be an ideal target for development of modeling methods. PMID:15704012

  15. A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria.

    PubMed

    Scott, Israel M; Rubinstein, Gabe M; Lipscomb, Gina L; Basen, Mirko; Schut, Gerrit J; Rhaesa, Amanda M; Lancaster, W Andrew; Poole, Farris L; Kelly, Robert M; Adams, Michael W W

    2015-10-01

    Caldicellulosiruptor bescii grows optimally at 78°C and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C5 and C6 sugars primarily to hydrogen gas, lactate, acetate, and CO2 and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) toward highly reduced products such as biofuels. During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus. Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, here termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii, XOR represents ca. 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary redox metabolism of this cellulolytic microorganism. PMID:26276113

  16. PCNA is involved in the EndoQ-mediated DNA repair process in Thermococcales

    PubMed Central

    Shiraishi, Miyako; Ishino, Sonoko; Yoshida, Kotaro; Yamagami, Takeshi; Cann, Isaac; Ishino, Yoshizumi

    2016-01-01

    To maintain genome integrity for transfer to their offspring, and to maintain order in cellular processes, all living organisms have DNA repair systems. Besides the well-conserved DNA repair machineries, organisms thriving in extreme environments are expected to have developed efficient repair systems. We recently discovered a novel endonuclease, which cleaves the 5′ side of deoxyinosine, from the hyperthermophilic archaeon, Pyrococcus furiosus. The novel endonuclease, designated as Endonulcease Q (EndoQ), recognizes uracil, abasic site and xanthine, as well as hypoxanthine, and cuts the phosphodiester bond at their 5′ sides. To understand the functional process involving EndoQ, we searched for interacting partners of EndoQ and identified Proliferating Cell Nuclear Angigen (PCNA). The EndoQ activity was clearly enhanced by addition of PCNA in vitro. The physical interaction between the two proteins through a PIP-motif of EndoQ and the toroidal structure of PCNA are critical for the stimulation of the endonuclease activity. These findings provide us a clue to elucidate a unique DNA repair system in Archaea. PMID:27150116

  17. STRUCTURAL FOLD, CONSERVATION AND FE(II) BINDING OF THE INTRACELLULAR DOMAIN OF PROKARYOTE FEOB

    PubMed Central

    Hung, Kuo-Wei; Chang, Yi-Wei; Eng, Edward T.; Chen, Jai-Hui; Chen, Yi-Chung; Sun, Yuh-Ju; Hsiao, Chwan-Deng; Dong, Gang; Spasov, Krasimir A.; Unger, Vinzenz M.; Huang, Tai-huang

    2010-01-01

    FeoB is a G-protein coupled membrane protein essential for Fe(II) uptake in prokaryotes. Here, we report the crystal structures of the intracellular domain of FeoB (NFeoB) from Klebsiella pneumoniae (KpNFeoB) and Pyrococcus furiosus (PfNFeoB) with and without bound ligands. In the structures, a canonical G-protein domain (G domain) is followed by a helical bundle domain (S-domain), which despite its lack of sequence similarity between species is structurally conserved. In the nucleotide-free state, the G-domain’s two switch regions point away from the binding site. This gives rise to an open binding pocket whose shallowness is likely to be responsible for the low nucleotide binding affinity. Nucleotide binding induced significant conformational changes in the G5 motif which in the case of GMPPNP binding was accompanied by destabilization of the switch I region. In addition to the structural data, we demonstrate that Fe(II)-induced foot printing cleaves the protein close to a putative Fe(II)-binding site at the tip of switch I, and we identify functionally important regions within the S-domain. Moreover, we show that NFeoB exists as a monomer in solution, and that its two constituent domains can undergo large conformational changes. The data show that the S-domain plays important roles in FeoB function. PMID:20123128

  18. Molecular basis of transcription initiation in Archaea.

    PubMed

    De Carlo, Sacha; Lin, Shih-Chieh; Taatjes, Dylan J; Hoenger, Andreas

    2010-01-01

    Compared with eukaryotes, the archaeal transcription initiation machinery-commonly known as the Pre-Initiation Complex-is relatively simple. The archaeal PIC consists of the TFIIB ortholog TFB, TBP, and an 11-subunit RNA polymerase (RNAP). The relatively small size of the entire archaeal PIC makes it amenable to structural analysis. Using purified RNAP, TFB, and TBP from the thermophile Pyrococcus furiosus, we assembled the biochemically active PIC at 65ºC. The intact archaeal PIC was isolated by implementing a cross-linking technique followed by size-exclusion chromatography, and the structure of this 440 kDa assembly was determined using electron microscopy and single-particle reconstruction techniques. Combining difference maps with crystal structure docking of various sub-domains, TBP and TFB were localized within the macromolecular PIC. TBP/TFB assemble near the large RpoB subunit and the RpoD/L "foot" domain behind the RNAP central cleft. This location mimics that of yeast TBP and TFIIB in complex with yeast RNAP II. Collectively, these results define the structural organization of the archaeal transcription machinery and suggest a conserved core PIC architecture. PMID:21326901

  19. Novel protein fold discovered in the PabI family of restriction enzymes

    PubMed Central

    Miyazono, Ken-ichi; Watanabe, Miki; Kosinski, Jan; Ishikawa, Ken; Kamo, Masayuki; Sawasaki, Tatsuya; Nagata, Koji; Bujnicki, Janusz M.; Endo, Yaeta; Tanokura, Masaru; Kobayashi, Ichizo

    2007-01-01

    Although structures of many DNA-binding proteins have been solved, they fall into a limited number of folds. Here, we describe an approach that led to the finding of a novel DNA-binding fold. Based on the behavior of Type II restriction–modification gene complexes as mobile elements, our earlier work identified a restriction enzyme, R.PabI, and its cognate modification enzyme in Pyrococcus abyssi through comparison of closely related genomes. While the modification methyltransferase was easily recognized, R.PabI was predicted to have a novel 3D structure. We expressed cytotoxic R.PabI in a wheat-germ-based cell-free translation system and determined its crystal structure. R.PabI turned out to adopt a novel protein fold. Homodimeric R.PabI has a curved anti-parallel β-sheet that forms a ‘half pipe’. Mutational and in silico DNA-binding analyses have assigned it as the double-strand DNA-binding site. Unlike most restriction enzymes analyzed, R.PabI is able to cleave DNA in the absence of Mg2+. These results demonstrate the value of genome comparison and the wheat-germ-based system in finding a novel DNA-binding motif in mobile DNases and, in general, a novel protein fold in horizontally transferred genes. PMID:17332011

  20. Solution Structure of Pfu RPP21, a Component of the Archaeal RNase P Holoenzyme, and Interactions with its RPP29 Protein Partner

    PubMed Central

    Amero, Carlos D; Boomershine, William P; Xu, Yiren; Foster, Mark

    2009-01-01

    RNase P is the ubiquitous ribonucleoprotein metalloenzyme responsible for cleaving the 5′-leader sequence of precursor tRNAs during their maturation. While the RNA subunit is catalytically active on its own at high monovalent and divalent ion concentration, four proteins subunits are associated with archaeal RNase P activity in vivo: RPP21, RPP29, RPP30 and POP5. These proteins have been shown to function in pairs: RPP21-RPP29 and POP5-RPP30. We have determined the solution structure of RPP21 from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) using conventional and paramagnetic NMR techniques. Pfu RPP21 in solution consists of an unstructured N-terminus, two alpha helices, a zinc binding motif, and an unstructured C-terminus. Moreover, we have used chemical shift perturbations to characterize the interaction of RPP21 with Pfu RPP29. The data show that the primary contact with RPP29 is localized to the two helices of RPP21. This information represents a fundamental step towards understanding structure-function relationships of the archaeal RNase P holoenzyme. PMID:18922021

  1. Tuned by metals: the TET peptidase activity is controlled by 3 metal binding sites

    PubMed Central

    Colombo, Matteo; Girard, Eric; Franzetti, Bruno

    2016-01-01

    TET aminopeptidases are dodecameric particles shared in the three life domains involved in various biological processes, from carbon source provider in archaea to eye-pressure regulation in humans. Each subunit contains a dinuclear metal site (M1 and M2) responsible for the enzyme catalytic activity. However, the role of each metal ion is still uncharacterized. Noteworthy, while mesophilic TETs are activated by Mn2+, hyperthermophilic TETs prefers Co2+. Here, by means of anomalous x-ray crystallography and enzyme kinetics measurements of the TET3 aminopeptidase from the hyperthermophilic organism Pyrococcus furiosus (PfTET3), we show that M2 hosts the catalytic activity of the enzyme, while M1 stabilizes the TET3 quaternary structure and controls the active site flexibility in a temperature dependent manner. A new third metal site (M3) was found in the substrate binding pocket, modulating the PfTET3 substrate preferences. These data show that TET activity is tuned by the molecular interplay among three metal sites. PMID:26853450

  2. Crystal Structure and Function of 5-Formaminoimidazole-4-carboxamide Ribonucleotide Synthetase from Methanocaldococcus jannaschii

    SciTech Connect

    Zhang, Yang; White, Robert H.; Ealick, Steven E.

    2008-08-06

    Purine biosynthesis requires 10 enzymatic steps in higher organisms, while prokaryotes require an additional enzyme for step 6. In most organisms steps 9 and 10 are catalyzed by the purH gene product, a bifunctional enzyme with both 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) synthase and inosine monophosphate (IMP) cyclohydrolase activity. Recently it was discovered that Archaea utilize different enzymes to catalyze steps 9 and 10. An ATP-dependent FAICAR synthetase is encoded by the purP gene, and IMP cyclohydrolase is encoded by the purO gene. We have determined the X-ray crystal structures of FAICAR synthetase from Methanocaldococcus jannaschii complexed with various ligands, including the tertiary substrate complex and product complex. The enzyme belongs to the ATP grasp superfamily and is predicted to use a formyl phosphate intermediate formed by an ATP-dependent phosphorylation. In addition, we have determined the structures of a PurP orthologue from Pyrococcus furiosus, which is functionally unclassified, in three crystal forms. With approximately 50% sequence identity, P. furiosus PurP is structurally homologous to M. jannaschii PurP. A phylogenetic analysis was performed to explore the possible role of this functionally unclassified PurP.

  3. Structural fold, conservation and Fe(II) binding of the intracellular domain of prokaryote FeoB

    SciTech Connect

    Hung, Kuo-Wei; Chang, Yi-Wei; Eng, Edward T.; Chen, Jai-Hui; Chen, Yi-Chung; Sun, Yuh-Ju; Hsiao, Chwan-Deng; Dong, Gang; Spasov, Krasimir A.; Unger, Vinzenz M.; Huang, Tai-huang

    2010-09-17

    FeoB is a G-protein coupled membrane protein essential for Fe(II) uptake in prokaryotes. Here, we report the crystal structures of the intracellular domain of FeoB (NFeoB) from Klebsiella pneumoniae (KpNFeoB) and Pyrococcus furiosus (PfNFeoB) with and without bound ligands. In the structures, a canonical G-protein domain (G domain) is followed by a helical bundle domain (S-domain), which despite its lack of sequence similarity between species is structurally conserved. In the nucleotide-free state, the G-domain's two switch regions point away from the binding site. This gives rise to an open binding pocket whose shallowness is likely to be responsible for the low nucleotide-binding affinity. Nucleotide binding induced significant conformational changes in the G5 motif which in the case of GMPPNP binding was accompanied by destabilization of the switch I region. In addition to the structural data, we demonstrate that Fe(II)-induced foot printing cleaves the protein close to a putative Fe(II)-binding site at the tip of switch I, and we identify functionally important regions within the S-domain. Moreover, we show that NFeoB exists as a monomer in solution, and that its two constituent domains can undergo large conformational changes. The data show that the S-domain plays important roles in FeoB function.

  4. Hierarchically ordered supramolecular protein-polymer composites with thermoresponsive properties.

    PubMed

    Välimäki, Salla; Mikkilä, Joona; Liljeström, Ville; Rosilo, Henna; Ora, Ari; Kostiainen, Mauri A

    2015-01-01

    Synthetic macromolecules that can bind and co-assemble with proteins are important for the future development of biohybrid materials. Active systems are further required to create materials that can respond and change their behavior in response to external stimuli. Here we report that stimuli-responsive linear-branched diblock copolymers consisting of a cationic multivalent dendron with a linear thermoresponsive polymer tail at the focal point, can bind and complex Pyrococcus furiosus ferritin protein cages into crystalline arrays. The multivalent dendron structure utilizes cationic spermine units to bind electrostatically on the surface of the negatively charged ferritin cage and the in situ polymerized poly(di(ethylene glycol) methyl ether methacrylate) linear block enables control with temperature. Cloud point of the final product was determined with dynamic light scattering (DLS), and it was shown to be approximately 31 °C at a concentration of 150 mg/L. Complexation of the polymer binder and apoferritin was studied with DLS, small-angle X-ray scattering, and transmission electron microscopy, which showed the presence of crystalline arrays of ferritin cages with a face-centered cubic (fcc, Fm3m)) Bravais lattice where lattice parameter a=18.6 nm. The complexation process was not temperature dependent but the final complexes had thermoresponsive characteristics with negative thermal expansion. PMID:25950765

  5. Comparative structural biology of eubacterial and archaeal oligosaccharyltransferases.

    PubMed

    Maita, Nobuo; Nyirenda, James; Igura, Mayumi; Kamishikiryo, Jun; Kohda, Daisuke

    2010-02-12

    Oligosaccharyltransferase (OST) catalyzes the transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. In the bacterium Campylobacter jejuni, a single-subunit membrane protein, PglB, catalyzes N-glycosylation. We report the 2.8 A resolution crystal structure of the C-terminal globular domain of PglB and its comparison with the previously determined structure from the archaeon Pyrococcus AglB. The two distantly related oligosaccharyltransferases share unexpected structural similarity beyond that expected from the sequence comparison. The common architecture of the putative catalytic sites revealed a new catalytic motif in PglB. Site-directed mutagenesis analyses confirmed the contribution of this motif to the catalytic function. Bacterial PglB and archaeal AglB constitute a protein family of the catalytic subunit of OST along with STT3 from eukaryotes. A structure-aided multiple sequence alignment of the STT3/PglB/AglB protein family revealed three types of OST catalytic centers. This novel classification will provide a useful framework for understanding the enzymatic properties of the OST enzymes from Eukarya, Archaea, and Bacteria. PMID:20007322

  6. Structure of the Class IV Adenylyl Cyclase Reveals a Novel Fold

    SciTech Connect

    Gallagher,D.; Smith, N.; Kim, S.; Heroux, A.; Robinson, H.; Reddy, P.

    2006-01-01

    The crystal structure of the class IV adenylyl cyclase (AC) from Yersinia pestis (Yp) is reported at 1.9 {angstrom} resolution. The class IV AC fold is distinct from the previously described folds for class II and class III ACs. The dimeric AC-IV folds into an antiparallel eight-stranded barrel whose connectivity has been seen in only three previous structures: yeast RNA triphosphatase and two proteins of unknown function from Pyrococcus furiosus and Vibrio parahaemolyticus. Eight highly conserved ionic residues E10, E12, K14, R63, K76, K111, D126, and E136 lie in the barrel core and form the likely binding sites for substrate and divalent cations. A phosphate ion is observed bound to R63, K76, K111, and R113 near the center of the conserved cluster. Unlike the AC-II and AC-III active sites that utilize two-Asp motifs for cation binding, the AC-IV active site is relatively enriched in glutamate and features an ExE motif as its most conserved element. Homologs of Y. pestis AC-IV, including human thiamine triphosphatase, span the three kingdoms of life and delineate an ancient family of phosphonucleotide processing enzymes.

  7. An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA*

    PubMed Central

    Fischer, Susan; Maier, Lisa-Katharina; Stoll, Britta; Brendel, Jutta; Fischer, Eike; Pfeiffer, Friedhelm; Dyall-Smith, Mike; Marchfelder, Anita

    2012-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum. PMID:22767603

  8. Prediction of transcription regulatory sites in Archaea by a comparative genomic approach.

    PubMed

    Gelfand, M S; Koonin, E V; Mironov, A A

    2000-02-01

    Intragenomic and intergenomic comparisons of upstream nucleotide sequences of archaeal genes were performed with the goal of predicting transcription regulatory sites (operators) and identifying likely regulons. Learning sets for the detection of regulatory sites were constructed using the available experimental data on archaeal transcription regulation or by analogy with known bacterial regulons, and further analysis was performed using iterative profile searches. The information content of the candidate signals detected by this method is insufficient for reliable predictions to be made. Therefore, this approach has to be complemented by examination of evolutionary conservation in different archaeal genomes. This combined strategy resulted in the prediction of a conserved heat shock regulon in all euryarchaea, a nitrogen fixation regulon in the methanogens Methanococcus jannaschii and Methanobacterium thermoautotrophicum and an aromatic amino acid regulon in M.thermoautotrophicum. Unexpectedly, the heat shock regulatory site was detected not only for genes that encode known chaperone proteins but also for archaeal histone genes. This suggests a possible function for archaeal histones in stress-related changes in DNA condensation. In addition, comparative analysis of the genomes of three Pyrococcus species resulted in the prediction of their purine metabolism and transport regulon. The results demonstrate the feasibility of prediction of at least some transcription regulatory sites by comparing poorly characterized prokaryotic genomes, particularly when several closely related genome sequences are available. PMID:10637320

  9. Insights into Dynamics of Mobile Genetic Elements in Hyperthermophilic Environments from Five New Thermococcus Plasmids

    PubMed Central

    Krupovic, Mart; Gonnet, Mathieu; Hania, Wajdi Ben; Forterre, Patrick; Erauso, Gaël

    2013-01-01

    Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1), with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA) systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles. PMID:23326305

  10. MCM ring hexamerization is a prerequisite for DNA-binding

    DOE PAGESBeta

    Froelich, Clifford A.; Nourse, Amanda; Enemark, Eric J.

    2015-09-13

    The hexameric Minichromosome Maintenance (MCM) protein complex forms a ring that unwinds DNA at the replication fork in eukaryotes and archaea. Our recent crystal structure of an archaeal MCM N-terminal domain bound to single-stranded DNA (ssDNA) revealed ssDNA associating across tight subunit interfaces but not at the loose interfaces, indicating that DNA-binding is governed not only by the DNA-binding residues of the subunits (MCM ssDNA-binding motif, MSSB) but also by the relative orientation of the subunits. We now extend these findings to show that DNA-binding by the MCM N-terminal domain of the archaeal organism Pyrococcus furiosus occurs specifically in themore » hexameric oligomeric form. We show that mutants defective for hexamerization are defective in binding ssDNA despite retaining all the residues observed to interact with ssDNA in the crystal structure. One mutation that exhibits severely defective hexamerization and ssDNA-binding is at a conserved phenylalanine that aligns with the mouse Mcm4(Chaos3) mutation associated with chromosomal instability, cancer, and decreased intersubunit association.« less

  11. Parallel evolution of transcriptome architecture during genome reorganization.

    PubMed

    Yoon, Sung Ho; Reiss, David J; Bare, J Christopher; Tenenbaum, Dan; Pan, Min; Slagel, Joseph; Moritz, Robert L; Lim, Sujung; Hackett, Murray; Menon, Angeli Lal; Adams, Michael W W; Barnebey, Adam; Yannone, Steven M; Leigh, John A; Baliga, Nitin S

    2011-11-01

    Assembly of genes into operons is generally viewed as an important process during the continual adaptation of microbes to changing environmental challenges. However, the genome reorganization events that drive this process are also the roots of instability for existing operons. We have determined that there exists a statistically significant trend that correlates the proportion of genes encoded in operons in archaea to their phylogenetic lineage. We have further characterized how microbes deal with operon instability by mapping and comparing transcriptome architectures of four phylogenetically diverse extremophiles that span the range of operon stabilities observed across archaeal lineages: a photoheterotrophic halophile (Halobacterium salinarum NRC-1), a hydrogenotrophic methanogen (Methanococcus maripaludis S2), an acidophilic and aerobic thermophile (Sulfolobus solfataricus P2), and an anaerobic hyperthermophile (Pyrococcus furiosus DSM 3638). We demonstrate how the evolution of transcriptional elements (promoters and terminators) generates new operons, restores the coordinated regulation of translocated, inverted, and newly acquired genes, and introduces completely novel regulation for even some of the most conserved operonic genes such as those encoding subunits of the ribosome. The inverse correlation (r=-0.92) between the proportion of operons with such internally located transcriptional elements and the fraction of conserved operons in each of the four archaea reveals an unprecedented view into varying stages of operon evolution. Importantly, our integrated analysis has revealed that organisms adapted to higher growth temperatures have lower tolerance for genome reorganization events that disrupt operon structures. PMID:21750103

  12. Role of Mn2+ and compatible solutes in the radiation resistance of thermophilic bacteria and archaea.

    PubMed

    Webb, Kimberly M; DiRuggiero, Jocelyne

    2012-01-01

    Radiation-resistant bacteria have garnered a great deal of attention from scientists seeking to expose the mechanisms underlying their incredible survival abilities. Recent analyses showed that the resistance to ionizing radiation (IR) in the archaeon Halobacterium salinarum is dependent upon Mn-antioxidant complexes responsible for the scavenging of reactive oxygen species (ROS) generated by radiation. Here we examined the role of the compatible solutes trehalose, mannosylglycerate, and di-myo-inositol phosphate in the radiation resistance of aerobic and anaerobic thermophiles. We found that the IR resistance of the thermophilic bacteria Rubrobacter xylanophilus and Rubrobacter radiotolerans was highly correlated to the accumulation of high intracellular concentration of trehalose in association with Mn, supporting the model of Mn(2+)-dependent ROS scavenging in the aerobes. In contrast, the hyperthermophilic archaea Thermococcus gammatolerans and Pyrococcus furiosus did not contain significant amounts of intracellular Mn, and we found no significant antioxidant activity from mannosylglycerate and di-myo-inositol phosphate in vitro. We therefore propose that the low levels of IR-generated ROS under anaerobic conditions combined with highly constitutively expressed detoxification systems in these anaerobes are key to their radiation resistance and circumvent the need for the accumulation of Mn-antioxidant complexes in the cell. PMID:23209374

  13. A conserved MCM single-stranded DNA binding element is essential for replication initiation

    PubMed Central

    Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

    2014-01-01

    The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001 PMID:24692448

  14. Bioenergetic and physiological studies of hyperthermophilic archaea. Final report

    SciTech Connect

    Kelly, R.M.

    1999-03-01

    This project focuses on physiological and bioenergetic characteristics of two representative hyperthermophilic archaea: Thermococcus litoralis (T{sub opt} 88 C) and Pyrococcus furiosus (T{sub opt} 98 C). Both are obligately anaerobic heterotrophs which grow in the presence or absence of reducible sulfur compounds. T. litoralis was studied in relation to information previously developed for P. furiosus: effect of sulfur reduction on bioenergetics, preferred fermentation patterns, tungsten requirement, etc. A defined medium was developed for T. litoralis consisting of amino acids, vitamins and nucleotides. This serves as the basis for continuous culture studies probing metabolic response to media changes. P. furiosus and T. litoralis have also been found to produce a polysaccharide in the presence of maltose and yeast extract. The composition and chemical structure of this polysaccharide was investigated as well as the metabolic motivation for its production. A novel and, perhaps, primitive intracellular proteolytic complex (previously designated as protease S66) in P. furiosus was isolated and the gene encoding the subunit of the complex was cloned, sequenced and the protease expressed in active form in Eschericia coli. Among other issues, the role of this complex in protein turnover and stress response was examined in the context of this organism in addition to comparing it to other complexes in eubacterial and eukaryotic cells. Biochemical characteristics of the protease have been measured in addition to examining other proteolytic species in P. furiosus.

  15. Structural Determinant for Switching between the Polymerase and Exonuclease Modes in the PCNA-Replicative DNA Polymerase Complex

    NASA Astrophysics Data System (ADS)

    Nishida, Hirokazu; Mayanagi, Kouta; Ishino, Yoshizumi; Morikawa, Kosuke

    Proliferating cell nuclear antigen (PCNA) is responsible for the processivity of DNA polymerase. We determined the crystal structure of Pyrococcus furiosus DNA polymerase (PfuPol) complexed with a cognate monomeric PCNA, which allowed us to construct a convincing model of the polymerase-PCNA ring interaction. Electron microscopy analyses confirmed that this complex structure exists among the multiple functional configurations in solution. Together with data from mutational analyses, this structural study indicated that the novel interaction between a stretched loop of PCNA and the PfuPol Thumb domain is quite important, in addition to the authentic PCNA-polymerase recognition site (PIP box). A comparison of the present structures with the previously reported structures of polymerases complexed with DNA suggested that the second interaction site plays a crucial role in switching between the polymerase and exonuclease modes, by stabilizing only the polymerase mode. This proposed mechanism of fidelity control of replicative DNA polymerases was supported by experiments, in which a mutation within the second interaction site caused an enhancement in the exonuclease activity in the presence of PCNA.

  16. PCNA is involved in the EndoQ-mediated DNA repair process in Thermococcales.

    PubMed

    Shiraishi, Miyako; Ishino, Sonoko; Yoshida, Kotaro; Yamagami, Takeshi; Cann, Isaac; Ishino, Yoshizumi

    2016-01-01

    To maintain genome integrity for transfer to their offspring, and to maintain order in cellular processes, all living organisms have DNA repair systems. Besides the well-conserved DNA repair machineries, organisms thriving in extreme environments are expected to have developed efficient repair systems. We recently discovered a novel endonuclease, which cleaves the 5' side of deoxyinosine, from the hyperthermophilic archaeon, Pyrococcus furiosus. The novel endonuclease, designated as Endonulcease Q (EndoQ), recognizes uracil, abasic site and xanthine, as well as hypoxanthine, and cuts the phosphodiester bond at their 5' sides. To understand the functional process involving EndoQ, we searched for interacting partners of EndoQ and identified Proliferating Cell Nuclear Angigen (PCNA). The EndoQ activity was clearly enhanced by addition of PCNA in vitro. The physical interaction between the two proteins through a PIP-motif of EndoQ and the toroidal structure of PCNA are critical for the stimulation of the endonuclease activity. These findings provide us a clue to elucidate a unique DNA repair system in Archaea. PMID:27150116

  17. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    SciTech Connect

    Basen, M; Schut, GJ; Nguyen, DM; Lipscomb, GL; Benn, RA; Prybol, CJ; Vaccaro, BJ; Poole, FL; Kelly, RM; Adams, MWW

    2014-12-09

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 degrees C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.

  18. Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology

    PubMed Central

    Chambers, Cecilia R.; Patrick, Wayne M.

    2015-01-01

    With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and biotechnology. Currently, the nucleic acid ligases from bacteriophage T4 are used extensively in these protocols. In this review, we argue that the nucleic acid ligases from Archaea represent a largely untapped pool of enzymes with diverse and potentially favourable properties for new and emerging biotechnological applications. We summarise the current state of knowledge on archaeal DNA and RNA ligases, which makes apparent the relative scarcity of information on in vitro activities that are of most relevance to biotechnologists (such as the ability to join blunt- or cohesive-ended, double-stranded DNA fragments). We highlight the existing biotechnological applications of archaeal DNA ligases and RNA ligases. Finally, we draw attention to recent experiments in which protein engineering was used to modify the activities of the DNA ligase from Pyrococcus furiosus and the RNA ligase from Methanothermobacter thermautotrophicus, thus demonstrating the potential for further work in this area. PMID:26494982

  19. Accurate placement of substrate RNA by Gar1 in H/ACA RNA-guided pseudouridylation

    PubMed Central

    Wang, Peng; Yang, Lijiang; Gao, Yi Qin; Zhao, Xin Sheng

    2015-01-01

    H/ACA RNA-guided ribonucleoprotein particle (RNP), the most complicated RNA pseudouridylase so far known, uses H/ACA guide RNA for substrate capture and four proteins (Cbf5, Nop10, L7Ae and Gar1) for pseudouridylation. Although it was shown that Gar1 not only facilitates the product release, but also enhances the catalytic activity, the chemical role that Gar1 plays in this complicated machinery is largely unknown. Kinetics measurement on Pyrococcus furiosus RNPs at different temperatures making use of fluorescence anisotropy showed that Gar1 reduces the catalytic barrier through affecting the activation entropy instead of enthalpy. Site-directed mutagenesis combined with molecular dynamics simulations demonstrated that V149 in the thumb loop of Cbf5 is critical in placing the target uridine to the right position toward catalytic D85 of Cbf5. The enzyme elegantly aligns the position of uridine in the catalytic site with the help of Gar1. In addition, conversion of uridine to pseudouridine results in a rigid syn configuration of the target nucleotide in the active site and causes Gar1 to pull out the thumb. Both factors guarantee the efficient release of the product. PMID:26206671

  20. Co-expression of chaperones from P. furiosus enhanced the soluble expression of the recombinant hyperthermophilic α-amylase in E. coli.

    PubMed

    Peng, Shuaiying; Chu, Zhongmei; Lu, Jianfeng; Li, Dongxiao; Wang, Yonghong; Yang, Shengli; Zhang, Yi

    2016-05-01

    The extracellular α-amylase from the hyperthermophilic archaeum Pyrococcus furiosus (PFA) is extremely thermostable and of an industrial importance and interest. PFA aggregates and accumulates as insoluble inclusion bodies when expressed as a heterologous protein at a high level in Escherichia coli. In the present study, we investigated the roles of chaperones from P. furiosus in the soluble expression of recombinant PFA in E. coli. The results indicate that co-expression of PFA with the molecular chaperone prefoldin alone significantly increased the soluble expression of PFA. Although, co-expression of other main chaperone components from P. furiosus, such as the small heat shock protein (sHSP) or chaperonin (HSP60), was also able to improve the soluble expression of PFA to a certain extent. Co-expression of chaperonin or sHSP in addition to prefoldin did not further increase the soluble expression of PFA. This finding emphasizes the biotechnological potentials of the molecular chaperone prefoldin from P. furiosus, which may facilitate the production of recombinant PFA. PMID:26862080

  1. Anion binding in biological systems

    NASA Astrophysics Data System (ADS)

    Feiters, Martin C.; Meyer-Klaucke, Wolfram; Kostenko, Alexander V.; Soldatov, Alexander V.; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Küpper, Frithjof C.; Hollenstein, Kaspar; Locher, Kaspar P.; Bevers, Loes E.; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

    2009-11-01

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L3 (2p3/2) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  2. Hierarchically Ordered Supramolecular Protein-Polymer Composites with Thermoresponsive Properties

    PubMed Central

    Välimäki, Salla; Mikkilä, Joona; Liljeström, Ville; Rosilo, Henna; Ora, Ari; Kostiainen, Mauri A.

    2015-01-01

    Synthetic macromolecules that can bind and co-assemble with proteins are important for the future development of biohybrid materials. Active systems are further required to create materials that can respond and change their behavior in response to external stimuli. Here we report that stimuli-responsive linear-branched diblock copolymers consisting of a cationic multivalent dendron with a linear thermoresponsive polymer tail at the focal point, can bind and complex Pyrococcus furiosus ferritin protein cages into crystalline arrays. The multivalent dendron structure utilizes cationic spermine units to bind electrostatically on the surface of the negatively charged ferritin cage and the in situ polymerized poly(di(ethylene glycol) methyl ether methacrylate) linear block enables control with temperature. Cloud point of the final product was determined with dynamic light scattering (DLS), and it was shown to be approximately 31 °C at a concentration of 150 mg/L. Complexation of the polymer binder and apoferritin was studied with DLS, small-angle X-ray scattering, and transmission electron microscopy, which showed the presence of crystalline arrays of ferritin cages with a face-centered cubic (fcc Fm3¯m) Bravais lattice where lattice parameter a = 18.6 nm. The complexation process was not temperature dependent but the final complexes had thermoresponsive characteristics with negative thermal expansion. PMID:25950765

  3. Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex*

    PubMed Central

    Lipscomb, Gina L.; Schut, Gerrit J.; Thorgersen, Michael P.; Nixon, William J.; Kelly, Robert M.; Adams, Michael W. W.

    2014-01-01

    Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications. PMID:24318960

  4. A First Analysis of Metallome Biosignatures of Hyperthermophilic Archaea

    PubMed Central

    Cameron, Vyllinniskii; House, Christopher H.; Brantley, Susan L.

    2012-01-01

    To date, no experimental data has been reported for the metallome of hyperthermophilic microorganisms although their metal requirements for growth are known to be unique. Here, experiments were conducted to determine (i) cellular trace metal concentrations of the hyperthermophilic Archaea Methanococcus jannaschii and Pyrococcus furiosus, and (ii) a first estimate of the metallome for these hyperthermophilic species via ICP-MS. The metal contents of these cells were compared to parallel experiments using the mesophilic bacterium Escherichia coli grown under aerobic and anaerobic conditions. Fe and Zn were typically the most abundant metals in cells. Metal concentrations for E. coli grown aerobically decreased in the order Fe > Zn > Cu > Mo > Ni > W > Co. In contrast, M. jannaschii and P. furiosus show almost the reverse pattern with elevated Ni, Co, and W concentrations. Of the three organisms, a biosignature is potentially demonstrated for the methanogen M. jannaschii that may, in part, be related to the metallome requirements of methanogenesis. The bioavailability of trace metals more than likely has varied through time. If hyperthermophiles are very ancient, then the trace metal patterns observed here may begin to provide some insights regarding Earth's earliest cells and in turn, early Earth chemistry. PMID:23243390

  5. Anaerobic High-Throughput Cultivation Method for Isolation of Thermophiles Using Biomass-Derived Substrates

    SciTech Connect

    Hamilton-Brehm, Scott; Vishnivetskaya, Tatiana A; Allman, Steve L; Mielenz, Jonathan R; Elkins, James G

    2012-01-01

    Flow cytometry (FCM) techniques have been developed for sorting mesophilic organisms, but the difficulty increases if the target microbes are thermophilic anaerobes. We demonstrate a reliable, high-throughput method of screening thermophilic anaerobic organisms using FCM and 96-well plates for growth on biomass-relevant substrates. The method was tested using the cellulolytic thermophiles Clostridium ther- mocellum (Topt = 55 C), Caldicellulosiruptor obsidiansis (Topt = 78 C) and the fermentative hyperthermo- philes, Pyrococcus furiosus (Topt = 100 C) and Thermotoga maritima (Topt = 80 C). Multi-well plates were incubated at various temperatures for approximately 72 120 h and then tested for growth. Positive growth resulting from single cells sorted into individual wells containing an anaerobic medium was verified by OD600. Depending on the growth substrate, up to 80 % of the wells contained viable cultures, which could be transferred to fresh media. This method was used to isolate thermophilic microbes from Rabbit Creek, Yellowstone National Park (YNP), Wyoming. Substrates for enrichment cultures including crystalline cellulose (Avicel), xylan (from Birchwood), pretreated switchgrass and Populus were used to cultivate organisms that may be of interest to lignocellulosic biofuel production.

  6. Stabilization of Enzymes against Thermal Stress and Freeze-Drying by Mannosylglycerate

    PubMed Central

    Ramos, A.; Raven, N.; Sharp, R. J.; Bartolucci, S.; Rossi, M.; Cannio, R.; Lebbink, J.; Van Der Oost, J.; De Vos, W. M.; Santos, H.

    1997-01-01

    2-O-(beta)-Mannosylglycerate, a solute that accumulates in some (hyper)thermophilic organisms, was purified from Pyrococcus furiosus cells, and its effect on enzyme stabilization in vitro was assessed. Enzymes from hyperthermophilic, thermophilic, and mesophilic sources were examined. The thermostabilities of alcohol dehydrogenases from P. furiosus and Bacillus stearothermophilus and of glutamate dehydrogenases from Thermotoga maritima and Clostridium difficile were improved to a significant extent when enzyme solutions were incubated at supraoptimal temperatures in the presence of 2-O-(beta)-mannosylglycerate, but no effect on the thermostability of glutamate dehydrogenase from P. furiosus was detected. On the other hand, there was a remarkable effect on the thermal stabilities of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and bovine liver glutamate dehydrogenase, which were used as model systems to evaluate stabilization of enzymes of mesophilic origin. For all of the enzymes examined and at the highest temperatures tested, 2-O-(beta)-mannosylglycerate was a better thermoprotectant than trehalose. The stabilizing effect exerted by 2-O-(beta)-mannosylglycerate on enzymes suggests a role for this compound as a protein thermostabilizer under physiological conditions. 2-O-(beta)-Mannosylglycerate was also effective in the protection of enzymes against stress imposed by freeze-drying, with its protecting effect being similar to or better than that exerted by trehalose. The data show 2-O-(beta)-mannosylglycerate to be a potential enzyme stabilizer in biotechnological applications. PMID:16535713

  7. Crystal structure of Argonaute and its implications for RISC slicer activity.

    PubMed

    Song, Ji-Joon; Smith, Stephanie K; Hannon, Gregory J; Joshua-Tor, Leemor

    2004-09-01

    Argonaute proteins and small interfering RNAs (siRNAs) are the known signature components of the RNA interference effector complex RNA-induced silencing complex (RISC). However, the identity of "Slicer," the enzyme that cleaves the messenger RNA (mRNA) as directed by the siRNA, has not been resolved. Here, we report the crystal structure of the Argonaute protein from Pyrococcus furiosus at 2.25 angstrom resolution. The structure reveals a crescent-shaped base made up of the amino-terminal, middle, and PIWI domains. The Piwi Argonaute Zwille (PAZ) domain is held above the base by a "stalk"-like region. The PIWI domain (named for the protein piwi) is similar to ribonuclease H, with a conserved active site aspartate-aspartate-glutamate motif, strongly implicating Argonaute as "Slicer." The architecture of the molecule and the placement of the PAZ and PIWI domains define a groove for substrate binding and suggest a mechanism for siRNA-guided mRNA cleavage. PMID:15284453

  8. Crystal structure of tRNA m(1)A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-L-methionine.

    PubMed

    Kuratani, Mitsuo; Yanagisawa, Tatsuo; Ishii, Ryohei; Matsuno, Michiyo; Si, Shu-Yi; Katsura, Kazushige; Ushikoshi-Nakayama, Ryoko; Shibata, Rie; Shirouzu, Mikako; Bessho, Yoshitaka; Yokoyama, Shigeyuki

    2014-09-01

    The N (1)-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m(1)A58 methyltransferase TrmI, using S-adenosyl-L-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi. PMID:24894648

  9. Genomic Characterization of Methanomicrobiales Reveals Three Classes of Methanogens

    SciTech Connect

    Anderson, Iain; Ulrich, Luke; Lupa, Boguslaw; Susanti, Dwi; Porat, I.; Hooper, Sean; Lykidis, A; Sieprawska-Lupa, Magdalena; Dharmarajan, Lakshmi; Goltsman, Eugene; Lapidus, Alla L.; Saunders, Elizabeth H; Han, Cliff; Land, Miriam L; Lucas, Susan; Mukhopadhyay, Biswarup; Whitman, William; Woese, Carl; Bristow, James; Kyrpides, Nikos C

    2009-01-01

    Background Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens. Methodology/Principal Findings In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales. Conclusions/Significance Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).

  10. Experimental fossilisation of viruses from extremophilic Archaea

    NASA Astrophysics Data System (ADS)

    Orange, F.; Chabin, A.; Gorlas, A.; Lucas-Staat, S.; Geslin, C.; Le Romancer, M.; Prangishvili, D.; Forterre, P.; Westall, F.

    2011-06-01

    The role of viruses at different stages of the origin of life has recently been reconsidered. It appears that viruses may have accompanied the earliest forms of life, allowing the transition from an RNA to a DNA world and possibly being involved in the shaping of tree of life in the three domains that we know presently. In addition, a large variety of viruses has been recently identified in extreme environments, hosted by extremophilic microorganisms, in ecosystems considered as analogues to those of the early Earth. Traces of life on the early Earth were preserved by the precipitation of silica on the organic structures. We present the results of the first experimental fossilisation by silica of viruses from extremophilic Archaea (SIRV2 - Sulfolobus islandicus rod-shaped virus 2, TPV1 - Thermococcus prieurii virus 1, and PAV1 - Pyrococcus abyssi virus 1). Our results confirm that viruses can be fossilised, with silica precipitating on the different viral structures (proteins, envelope) over several months in a manner similar to that of other experimentally and naturally fossilised microorganisms. This study thus suggests that viral remains or traces could be preserved in the rock record although their identification may be challenging due to the small size of the viral particles.

  11. Experimental fossilisation of viruses from extremophilic Archaea

    NASA Astrophysics Data System (ADS)

    Orange, F.; Chabin, A.; Gorlas, A.; Lucas-Staat, S.; Geslin, C.; Le Romancer, M.; Prangishvili, D.; Forterre, P.; Westall, F.

    2011-03-01

    The role of viruses at different stages of the origin of life has recently been reconsidered. It appears that viruses may have accompanied the earliest forms of life, allowing the transition from an RNA to a DNA world and possibly being involved in the shaping of tree of life in the three domains that we know presently. In addition, a large variety of viruses has been recently identified in extreme environments, hosted by extremophilic microorganisms, in ecosystems considered as analogues to those of the early Earth. The earliest traces of life were preserved by the precipitation of silica on organic structures. The study of the in situ and experimental fossilisation of microorganisms allows better understanding of the fossilisation processes and helps identification of traces of life in ancient rocks. In a continuation of these studies, we present the results of the first experimental fossilisation by silica of viruses from extremophilic Archaea (SIRV2 - Sulfolobus islandicus Virus 2, TPV1 - Thermococcus prieurii virus 1, and PAV1 - Pyrococcus abyssi virus 1). Our results confirm that viruses can be fossilised, with silica precipitating on the different viral structures (proteins, envelope) over several months. However differences in the silicification process were noticed, depending on the viral structure and composition. The fossilisation mechanism is similar to that of the fossilisation of microorganisms. This study thus suggests that viral remains or traces could be preserved in the rock record although their identification may be challenging due to the small size of the viral particles.

  12. A New Thermophilic Nitrilase from an Antarctic Hyperthermophilic Microorganism

    PubMed Central

    Dennett, Geraldine V.; Blamey, Jenny M.

    2016-01-01

    Several environmental samples from Antarctica were collected and enriched to search for microorganisms with nitrilase activity. A new thermostable nitrilase from a novel hyperthermophilic archaea Pyrococcus sp. M24D13 was purified and characterized. The activity of this enzyme increased as the temperatures rise from 70 up to 85°C. Its optimal activity occurred at 85°C and pH 7.5. This new enzyme shows a remarkable resistance to thermal inactivation retaining more than 50% of its activity even after 8 h of incubation at 85°C. In addition, this nitrilase is highly versatile demonstrating activity toward different substrates, such as benzonitrile (60 mM, aromatic nitrile) and butyronitrile (60 mM, aliphatic nitrile), with a specific activity of 3286.7 U mg−1 of protein and 4008.2 U mg−1 of protein, respectively. Moreover the enzyme NitM24D13 also presents cyanidase activity. The apparent Michaelis–Menten constant (Km) and Vmáx of this Nitrilase for benzonitrile were 0.3 mM and 333.3 μM min−1, respectively, and the specificity constant (kcat/Km) for benzonitrile was 2.05 × 105 s−1 M−1. PMID:26973832

  13. Decarboxylation of Pyruvate to Acetaldehyde for Ethanol Production by Hyperthermophiles

    PubMed Central

    Eram, Mohammad S.; Ma, Kesen

    2013-01-01

    Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for the conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes and proteomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles was a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR)/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus. Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE). Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL), and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde in mesophilic organisms. AcDH is present in some mesophilic (such as clostridia) and thermophilic bacteria (e.g., Geobacillus and Thermoanaerobacter). However, no AcDH gene or protein homologs could be found in the released genomes and proteomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles under different assay conditions. In conclusion, no commonly-known PDCs was found in hyperthermophiles. Instead of the commonly-known PDC, it appears that at least one multifunctional enzyme is responsible for catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde in hyperthermophiles. PMID:24970182

  14. Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcription.

    PubMed

    Chen, H T; Legault, P; Glushka, J; Omichinski, J G; Scott, R A

    2000-09-01

    Transcription factor IIB (TFIIB) is an essential component in the formation of the transcription initiation complex in eucaryal and archaeal transcription. TFIIB interacts with a promoter complex containing the TATA-binding protein (TBP) to facilitate interaction with RNA polymerase II (RNA pol II) and the associated transcription factor IIF (TFIIF). TFIIB contains a zinc-binding motif near the N-terminus that is directly involved in the interaction with RNA pol II/TFIIF and plays a crucial role in selecting the transcription initiation site. The solution structure of the N-terminal residues 2-59 of human TFIIB was determined by multidimensional NMR spectroscopy. The structure consists of a nearly tetrahedral Zn(Cys)3(His)1 site confined by type I and "rubredoxin" turns, three antiparallel beta-strands, and disordered loops. The structure is similar to the reported zinc-ribbon motifs in several transcription-related proteins from archaea and eucarya, including Pyrococcus furiosus transcription factor B (PfTFB), human and yeast transcription factor IIS (TFIIS), and Thermococcus celer RNA polymerase II subunit M (TcRPOM). The zinc-ribbon structure of TFIIB, in conjunction with the biochemical analyses, suggests that residues on the beta-sheet are involved in the interaction with RNA pol II/TFIIF, while the zinc-binding site may increase the stability of the beta-sheet. PMID:11045620

  15. Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcription.

    PubMed Central

    Chen, H. T.; Legault, P.; Glushka, J.; Omichinski, J. G.; Scott, R. A.

    2000-01-01

    Transcription factor IIB (TFIIB) is an essential component in the formation of the transcription initiation complex in eucaryal and archaeal transcription. TFIIB interacts with a promoter complex containing the TATA-binding protein (TBP) to facilitate interaction with RNA polymerase II (RNA pol II) and the associated transcription factor IIF (TFIIF). TFIIB contains a zinc-binding motif near the N-terminus that is directly involved in the interaction with RNA pol II/TFIIF and plays a crucial role in selecting the transcription initiation site. The solution structure of the N-terminal residues 2-59 of human TFIIB was determined by multidimensional NMR spectroscopy. The structure consists of a nearly tetrahedral Zn(Cys)3(His)1 site confined by type I and "rubredoxin" turns, three antiparallel beta-strands, and disordered loops. The structure is similar to the reported zinc-ribbon motifs in several transcription-related proteins from archaea and eucarya, including Pyrococcus furiosus transcription factor B (PfTFB), human and yeast transcription factor IIS (TFIIS), and Thermococcus celer RNA polymerase II subunit M (TcRPOM). The zinc-ribbon structure of TFIIB, in conjunction with the biochemical analyses, suggests that residues on the beta-sheet are involved in the interaction with RNA pol II/TFIIF, while the zinc-binding site may increase the stability of the beta-sheet. PMID:11045620

  16. A single amino acid substitution in the DNA-binding domain of Aeropyrum pernix DNA ligase impairs its interaction with proliferating cell nuclear antigen.

    PubMed

    Kiyonari, Shinichi; Kamigochi, Toru; Ishino, Yoshizumi

    2007-09-01

    Proliferating cell nuclear antigen (PCNA) is known as a DNA sliding clamp that acts as a platform for the assembly of enzymes involved in DNA replication and repair. Previously, it was reported that a crenarchaeal PCNA formed a heterotrimeric structure, and that each PCNA subunit has distinct binding specificity to PCNA-binding proteins. Here we describe the PCNA-binding properties of a DNA ligase from the hyperthermophilic crenarchaeon Aeropyrum pernix K1. Based on our findings on the Pyrococcus furiosus DNA ligase-PCNA interaction, we predicted that the aromatic residue, Phe132, in the DNA-binding domain of A. pernix DNA ligase (ApeLig) would play a critical role in binding to A. pernix PCNA (ApePCNA). Surface plasmon resonance analyses revealed that the ApeLig F132A mutant does not interact with an immobilized subunit of ApePCNA. Furthermore, we could not detect any stimulation of the ligation activity of the ApeLig F132A protein by ApePCNA in vitro. These results indicated that the phenylalanine, which is located in our predicted PCNA-binding region in ApeLig, has a critical role for the physical and functional interaction with ApePCNA. PMID:17487442

  17. Analysis of the crystal structure of an active MCM hexamer

    PubMed Central

    Miller, Justin M; Arachea, Buenafe T; Epling, Leslie B; Enemark, Eric J

    2014-01-01

    In a previous Research article (Froelich et al., 2014), we suggested an MCM helicase activation mechanism, but were limited in discussing the ATPase domain because it was absent from the crystal structure. Here we present the crystal structure of a nearly full-length MCM hexamer that is helicase-active and thus has all features essential for unwinding DNA. The structure is a chimera of Sulfolobus solfataricus N-terminal domain and Pyrococcus furiosus ATPase domain. We discuss three major findings: 1) a novel conformation for the A-subdomain that could play a role in MCM regulation; 2) interaction of a universally conserved glutamine in the N-terminal Allosteric Communication Loop with the AAA+ domain helix-2-insert (h2i); and 3) a recessed binding pocket for the MCM ssDNA-binding motif influenced by the h2i. We suggest that during helicase activation, the h2i clamps down on the leading strand to facilitate strand retention and regulate ATP hydrolysis. DOI: http://dx.doi.org/10.7554/eLife.03433.001 PMID:25262915

  18. Accurate placement of substrate RNA by Gar1 in H/ACA RNA-guided pseudouridylation.

    PubMed

    Wang, Peng; Yang, Lijiang; Gao, Yi Qin; Zhao, Xin Sheng

    2015-09-01

    H/ACA RNA-guided ribonucleoprotein particle (RNP), the most complicated RNA pseudouridylase so far known, uses H/ACA guide RNA for substrate capture and four proteins (Cbf5, Nop10, L7Ae and Gar1) for pseudouridylation. Although it was shown that Gar1 not only facilitates the product release, but also enhances the catalytic activity, the chemical role that Gar1 plays in this complicated machinery is largely unknown. Kinetics measurement on Pyrococcus furiosus RNPs at different temperatures making use of fluorescence anisotropy showed that Gar1 reduces the catalytic barrier through affecting the activation entropy instead of enthalpy. Site-directed mutagenesis combined with molecular dynamics simulations demonstrated that V149 in the thumb loop of Cbf5 is critical in placing the target uridine to the right position toward catalytic D85 of Cbf5. The enzyme elegantly aligns the position of uridine in the catalytic site with the help of Gar1. In addition, conversion of uridine to pseudouridine results in a rigid syn configuration of the target nucleotide in the active site and causes Gar1 to pull out the thumb. Both factors guarantee the efficient release of the product. PMID:26206671

  19. Redox-Promoting Protein Motions in Rubredoxin

    SciTech Connect

    Borreguero Calvo, Jose M; He, Junhong; Meilleur, Flora; Weiss, Kevin L; Myles, Dean A A; Herwig, Kenneth W; Agarwal, Pratul K

    2011-01-01

    Proteins are dynamic objects, constantly undergoing conformational fluctuations, yet the linkage between internal protein motion and function is widely debated. This study reports on the characterization of temperature-activated collective and individual atomic motions of oxidized rubredoxin, a small 53 residue protein from thermophilic Pyrococcus furiosus (RdPf). Computational modeling allows detailed investigations of protein motions as a function of temperature, and neutron scattering experiments are used to compare to computational results. Just above the dynamical transition temperature which marks the onset of significant anharmonic motions of the protein, the computational simulations show both a significant reorientation of the average electrostatic force experienced by the coordinated Fe{sup 3+} ion and a dramatic rise in its strength. At higher temperatures, additional anharmonic modes become activated and dominate the electrostatic fluctuations experienced by the ion. At 360 K, close to the optimal growth temperature of P. furiosus, simulations show that three anharmonic modes including motions of two conserved residues located at the protein active site (Ile7 and Ile40) give rise to the majority of the electrostatic fluctuations experienced by the Fe{sup 3+} ion. The motions of these residues undergo displacements which may facilitate solvent access to the ion.

  20. IMAGINE: first neutron protein structure and new capabilities for neutron macromolecular crystallography

    SciTech Connect

    Munshi, Parthapratim; Myles, Dean A A; Robertson, Lee; Stoica, Alexandru Dan; Crow, Lowell; Kovalevskyi, Andrii Y; Koritsanszky, Tibor S; Chakoumakos, Bryan C; Blessing, Robert; Meilleur, Flora

    2013-01-01

    We report the first high resolution neutron protein structure of perdeuterated rubredoxin from Pyrococcus furiosus (PfRd) determined using the new IMAGINE macromolecular neutron crystallography instrument at the Oak Ridge National Laboratory. Neutron diffraction data extending to 1.65 resolution were collected from a relatively small 0.7 mm3 PfRd crystal using 2.5 days (60 h) of beam time. The refined structure contains 371 out of 391, or 95%, of the deuterium atoms of the protein, and 58 solvent molecules. The IMAGINE instrument is designed to provide neutron data at or near atomic resolutions (1.5 ) from crystals with volume < 1.0 mm3 and with unit cell edges < 100 . Beam line features include elliptical focusing mirrors that deliver 3x107 n s-1 cm-2 into a 3.5 x 2.0 mm2 focal spot at the sample position, and variable short and long wavelength cutoff optics that provide automated exchange between multiple wavelength configurations ( min=2.0 , 2.8 , 3.3 - max =3.0 , 4.0 , 4.5 , ~20 ). Notably, the crystal used to collect this PfRd data is 5-10 times smaller than has been previously reported.

  1. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    PubMed Central

    Basen, Mirko; Schut, Gerrit J.; Nguyen, Diep M.; Lipscomb, Gina L.; Benn, Robert A.; Prybol, Cameron J.; Vaccaro, Brian J.; Poole, Farris L.; Kelly, Robert M.; Adams, Michael W. W.

    2014-01-01

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 °C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways. PMID:25368184

  2. The Carboxy-Terminal αN Helix of the Archaeal XerA Tyrosine Recombinase Is a Molecular Switch to Control Site-Specific Recombination

    PubMed Central

    Serre, Marie-Claude; El Arnaout, Toufic; Brooks, Mark A.; Durand, Dominique; Lisboa, Johnny; Lazar, Noureddine; Raynal, Bertrand; van Tilbeurgh, Herman; Quevillon-Cheruel, Sophie

    2013-01-01

    Tyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding. Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers. PMID:23667562

  3. Insights on how the activity of an endoglucanase is affected by physical properties of insoluble celluloses.

    PubMed

    Bragatto, Juliano; Segato, Fernando; Cota, Junio; Mello, Danilo B; Oliveira, Marcelo M; Buckeridge, Marcos S; Squina, Fabio M; Driemeier, Carlos

    2012-05-31

    Cellulose physical properties like crystallinity, porosity, and particle size are known to influence cellulase activity, but knowledge is still insufficient for activity prediction from such measurable substrate characteristics. With the aim of illuminating enzyme-substrate relationships, this work evaluates a purified hyperthermophilic endo-1,4-beta-glucanase (from Pyrococcus furiosus) acting on 13 celluloses characterized for crystallinity and crystal width (by X-ray diffraction), wet porosity (by thermoporometry), and particle size (by light scattering). Activities are analyzed by the Michaelis-Menten kinetic equation, which is justified by low enzyme-substrate affinity. Michaelis-Menten coefficients K(m) and k(cat) are reinterpreted in the context of heterogeneous cellulose hydrolysis. For a set of as-received and milled microcrystalline celluloses, activity is successfully described as a function of accessible substrate concentration, with accessibility proportional to K(m)(-1). Accessibility contribution from external particle areas, pore areas, and crystalline packing are discriminated to have comparable magnitudes, implying that activity prediction demands all these substrate properties to be considered. Results additionally suggest that looser crystalline packing increases the lengths of released cello-oligomers as well as the maximum endoglucanase specific activity (k(cat)). PMID:22577872

  4. Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids.

    PubMed Central

    Hoaki, T; Nishijima, M; Kato, M; Adachi, K; Mizobuchi, S; Hanzawa, N; Maruyama, T

    1994-01-01

    Three hyperthermophilic sulfur-dependent heterotrophs were isolated from a shallow submarine hydrothermal system at an inlet of Kodakara-jima island, Kagoshima, Japan. The isolates grew at 60 to 97 degrees C, with the optimum temperatures at 85 to 90 degrees C. Sensitivity to rifampin and the existence of ether lipids indicated that the isolates are hyperthermophilic archaea. Partial sequencing of the genes coding for 16S rRNA showed that the three isolates are closely related to the genus Thermococcus. They grew on proteinaceous mixtures, such as yeast extract, Casamino Acids, and purified proteins (e.g., casein and gelatin), but not on carbohydrates or organic acids as sole carbon and energy sources. Nine amino acids were essential for growth of isolate KS-1 (Thr, Leu, Ile, Val, Met, Phe, His, Tyr, and Arg). Isolate KS-2 required Lys in addition to the nine amino acids, and KS-8 required Lys instead of Tyr. In comparative studies, it was shown that Thermococcus celer DSM 2476 required 10 amino acids (Thr, Leu, Ile, Val, Met, Phe, Tyr, Trp, Lys, and Arg) while Pyrococcus furiosus DSM 3638 required only Ile and Val. The hyperthermophilic fermentative eubacterium Thermotoga neapolitana DSM 4359 did not require any amino acids for growth. Images PMID:8085828

  5. Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex

    SciTech Connect

    Cocozaki, Alexis I.; Ramia, Nancy F.; Shao, Yaming; Hale, Caryn R.; Terns, Rebecca M.; Terns, Michael P.; Li, Hong

    2012-08-10

    Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3 {angstrom} crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two {alpha}-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.

  6. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes

    SciTech Connect

    Carte, Jason; Wang, Ruiying; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

    2010-11-09

    An RNA-based gene silencing pathway that protects bacteria and archaea from viruses and other genome invaders is hypothesized to arise from guide RNAs encoded by CRISPR loci and proteins encoded by the cas genes. CRISPR loci contain multiple short invader-derived sequences separated by short repeats. The presence of virus-specific sequences within CRISPR loci of prokaryotic genomes confers resistance against corresponding viruses. The CRISPR loci are transcribed as long RNAs that must be processed to smaller guide RNAs. Here we identified Pyrococcus furiosus Cas6 as a novel endoribonuclease that cleaves CRISPR RNAs within the repeat sequences to release individual invader targeting RNAs. Cas6 interacts with a specific sequence motif in the 5{prime} region of the CRISPR repeat element and cleaves at a defined site within the 3{prime} region of the repeat. The 1.8 angstrom crystal structure of the enzyme reveals two ferredoxin-like folds that are also found in other RNA-binding proteins. The predicted active site of the enzyme is similar to that of tRNA splicing endonucleases, and concordantly, Cas6 activity is metal-independent. cas6 is one of the most widely distributed CRISPR-associated genes. Our findings indicate that Cas6 functions in the generation of CRISPR-derived guide RNAs in numerous bacteria and archaea.

  7. Genomic Characterization of Methanomicrobiales Reveals Three Classes of Methanogens

    SciTech Connect

    Anderson, Iain; Ulrich, Luke E.; Lupa, Boguslaw; Susanti, Dwi; Porat, Iris; Hooper, Sean D.; Lykidis, Athanasios; Sieprawska-Lupa, Magdalena; Dharmarajan, Lakshmi; Goltsman, Eugene; Lapidus, Alla; Saunders, Elizabeth; Han, Cliff; Land, Miriam; Lucas, Susan; Mukhopadhyay, Biswarup; Whitman, William B.; Woese, Carl; Bristow, James; Kyrpides, Nikos

    2009-05-01

    Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens. In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales. Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).

  8. A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria

    PubMed Central

    Scott, Israel M.; Rubinstein, Gabe M.; Lipscomb, Gina L.; Basen, Mirko; Schut, Gerrit J.; Rhaesa, Amanda M.; Lancaster, W. Andrew; Poole, Farris L.; Kelly, Robert M.

    2015-01-01

    Caldicellulosiruptor bescii grows optimally at 78°C and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C5 and C6 sugars primarily to hydrogen gas, lactate, acetate, and CO2 and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) toward highly reduced products such as biofuels. During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus. Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, here termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii, XOR represents ca. 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary redox metabolism of this cellulolytic microorganism. PMID:26276113

  9. A euryarchaeal histone modulates strand displacement synthesis by replicative DNA polymerases.

    PubMed

    Sun, Fei; Huang, Li

    2016-07-01

    Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase (PolB), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of PolB and PolD from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPfA1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both PolB and PolD were efficient in strand displacement. HPfA1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPfA1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis. PMID:27333783

  10. Swimming behavior of selected species of Archaea.

    PubMed

    Herzog, Bastian; Wirth, Reinhard

    2012-03-01

    The swimming behavior of Bacteria has been studied extensively, at least for some species like Escherichia coli. In contrast, almost no data have been published for Archaea on this topic. In a systematic study we asked how the archaeal model organisms Halobacterium salinarum, Methanococcus voltae, Methanococcus maripaludis, Methanocaldococcus jannaschii, Methanocaldococcus villosus, Pyrococcus furiosus, and Sulfolobus acidocaldarius swim and which swimming behavior they exhibit. The two Euryarchaeota M. jannaschii and M. villosus were found to be, by far, the fastest organisms reported up to now, if speed is measured in bodies per second (bps). Their swimming speeds, at close to 400 and 500 bps, are much higher than the speed of the bacterium E. coli or of a very fast animal, like the cheetah, each with a speed of ca. 20 bps. In addition, we observed that two different swimming modes are used by some Archaea. They either swim very rapidly, in a more or less straight line, or they exhibit a slower kind of zigzag swimming behavior if cells are in close proximity to the surface of the glass capillary used for observation. We argue that such a "relocate-and-seek" behavior enables the organisms to stay in their natural habitat. PMID:22247169

  11. Crystallization and preliminary X-ray diffraction studies of a hyperthermophilic Rieske protein variant (SDX-triple) with an engineered rubredoxin-like mononuclear iron site

    SciTech Connect

    Iwasaki, Toshio Kounosu, Asako; Ohmori, Daijiro; Kumasaka, Takashi

    2006-10-01

    A hyperthermophilic archaeal Rieske iron–sulfur protein (sulredoxin) variant, SDX-triple (H44I/A45C/H64C), having a rationally designed rubredoxin-like mononuclear iron site in place of a Rieske [2Fe–2S] centre, has been crystallized. The P1 crystals of the SDX-triple variant diffract to 1.63 Å resolution using synchrotron radiation. In place of the Rieske [2Fe–2S] cluster, an archetypal mononuclear iron site has rationally been designed into a hyperthermophilic archaeal Rieske [2Fe–2S] protein (sulredoxin) from Sulfolobus tokodaii by three residue replacements with reference to the Pyrococcus furiosus rubredoxin sequence. The resulting sulredoxin variant, SDX-triple (H44I/A45C/H64C), has been purified and crystallized by the hanging-drop vapour-diffusion method using 65%(v/v) 2-methyl-2,4-pentanediol, 0.025 M citric acid and 0.075 M sodium acetate trihydrate pH 4.3. The crystals diffract to 1.63 Å resolution and belong to the triclinic space group P1, with unit-cell parameters a = 43.56, b = 76.54, c = 80.28 Å, α = 88.12, β = 78.82, γ = 73.46°. The asymmetric unit contains eight protein molecules.

  12. Transcription factor IID in the Archaea: sequences in the Thermococcus celer genome would encode a product closely related to the TATA-binding protein of eukaryotes

    NASA Technical Reports Server (NTRS)

    Marsh, T. L.; Reich, C. I.; Whitelock, R. B.; Olsen, G. J.; Woese, C. R. (Principal Investigator)

    1994-01-01

    The first step in transcription initiation in eukaryotes is mediated by the TATA-binding protein, a subunit of the transcription factor IID complex. We have cloned and sequenced the gene for a presumptive homolog of this eukaryotic protein from Thermococcus celer, a member of the Archaea (formerly archaebacteria). The protein encoded by the archaeal gene is a tandem repeat of a conserved domain, corresponding to the repeated domain in its eukaryotic counterparts. Molecular phylogenetic analyses of the two halves of the repeat are consistent with the duplication occurring before the divergence of the archael and eukaryotic domains. In conjunction with previous observations of similarity in RNA polymerase subunit composition and sequences and the finding of a transcription factor IIB-like sequence in Pyrococcus woesei (a relative of T. celer) it appears that major features of the eukaryotic transcription apparatus were well-established before the origin of eukaryotic cellular organization. The divergence between the two halves of the archael protein is less than that between the halves of the individual eukaryotic sequences, indicating that the average rate of sequence change in the archael protein has been less than in its eukaryotic counterparts. To the extent that this lower rate applies to the genome as a whole, a clearer picture of the early genes (and gene families) that gave rise to present-day genomes is more apt to emerge from the study of sequences from the Archaea than from the corresponding sequences from eukaryotes.

  13. Structure and substrate ion binding in the sodium/proton antiporter PaNhaP

    PubMed Central

    Wöhlert, David; Kühlbrandt, Werner; Yildiz, Özkan

    2014-01-01

    Sodium/proton antiporters maintain intracellular pH and sodium levels. Detailed structures of antiporters with bound substrate ions are essential for understanding how they work. We have resolved the substrate ion in the dimeric, electroneutral sodium/proton antiporter PaNhaP from Pyrococcus abyssi at 3.2 Å, and have determined its structure in two different conformations at pH 8 and pH 4. The ion is coordinated by three acidic sidechains, a water molecule, a serine and a main-chain carbonyl in the unwound stretch of trans-membrane helix 5 at the deepest point of a negatively charged cytoplasmic funnel. A second narrow polar channel may facilitate proton uptake from the cytoplasm. Transport activity of PaNhaP is cooperative at pH 6 but not at pH 5. Cooperativity is due to pH-dependent allosteric coupling of protomers through two histidines at the dimer interface. Combined with comprehensive transport studies, the structures of PaNhaP offer unique new insights into the transport mechanism of sodium/proton antiporters. DOI: http://dx.doi.org/10.7554/eLife.03579.001 PMID:25426802

  14. Development of Novel Sugar Isomerases by Optimization of Active Sites in Phosphosugar Isomerases for Monosaccharides

    PubMed Central

    Yeom, Soo-Jin; Kim, Yeong-Su

    2013-01-01

    Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a d-ribose isomerase, an l-ribose isomerase, and an l-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases. PMID:23204422

  15. Enzymatic production of hydrogen from glucose

    SciTech Connect

    Woodward, J.; Mattingly, S.M.

    1995-06-01

    The objective of this research is to optimize conditions for the enzymatic production of hydrogen gas from biomass-derived glucose. This new project is funded at 0.5 PY level of effort for FY 1995. The rationale for the work is that cellulose is, potentially, a vast source of hydrogen and that enzymes offer a specific and efficient method for its extraction with minimal environmental impact. This work is related to the overall hydrogen program goal of technology development and validation. The approach is based on knowledge that glucose is oxidized by the NADP{sup +} requiring enzyme glucose dehydrogenase (GDH) and that the resulting NADPH can donate its electrons to hydrogenase (H{sub 2}ase) which catalyzes the evolution of H{sub 2}. Thus hydrogen production from glucose was achieved using calf liver GDH and Pyrococcus furiosus H{sub 2}ase yielding 17% of theoretical maximum expected. The cofactor NADP{sup +} for this reaction was regenerated and recycled. Current and future work includes understanding the rate limiting steps of this process and the stabilization/immobilization of the enzymes for long term hydrogen production. Cooperative interactions with the Universities of Georgia and Bath for obtaining thermally stable enzymes are underway.

  16. Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography

    PubMed Central

    Sauguet, Ludovic; Raia, Pierre; Henneke, Ghislaine; Delarue, Marc

    2016-01-01

    Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same ‘double-psi β-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs. PMID:27548043

  17. Enzymatic production of hydrogen from glucose

    NASA Astrophysics Data System (ADS)

    Woodward, J.; Mattingly, S. M.

    The objective of this research is to optimize conditions for the enzymatic production of hydrogen gas from biomass-derived glucose. This new project is funded at 0.5 PY level of effort for FY 1995. The rationale for the work is that cellulose is, potentially, a vast source of hydrogen and that enzymes offer a specific and efficient method for its extraction with minimal environmental impact. This work is related to the overall hydrogen program goal of technology development and validation. The approach is based on knowledge that glucose is oxidized by the NADP(sup +) requiring enzyme glucose dehydrogenase (GDH) and that the resulting NADPH can donate its electrons to hydrogenase (H2ase) which catalyzes the evolution of H2. Thus hydrogen production from glucose was achieved using calf liver GDH and Pyrococcus furiosus H2ase yielding 17% of theoretical maximum expected. The cofactor NADP(sup +) for this reaction was regenerated and recycled. Current and future work includes understanding the rate limiting steps of this process and the stabilization/immobilization of the enzymes for long term hydrogen production. Cooperative interactions with the Universities of Georgia and Bath for obtaining thermally sta

  18. The Cm56 tRNA modification in archaea is catalyzed either by a specific 2′-O-methylase, or a C/D sRNP

    PubMed Central

    RENALIER, MARIE-HÉLÈNE; JOSEPH, NICOLE; GASPIN, CHRISTINE; THEBAULT, PATRICIA; MOUGIN, ANNIE

    2005-01-01

    We identified the first archaeal tRNA ribose 2′-O-methylase, aTrm56, belonging to the Cluster of Orthologous Groups (COG) 1303 that contains archaeal genes only. The corresponding protein exhibits a SPOUT S-adenosylmethionine (AdoMet)-dependent methyltransferase domain found in bacterial and yeast G18 tRNA 2′-O-methylases (SpoU, Trm3). We cloned the Pyrococcus abyssi PAB1040 gene belonging to this COG, expressed and purified the corresponding protein, and showed that in vitro, it specifically catalyzes the AdoMet-dependent 2′-O-ribose methylation of C at position 56 in tRNA transcripts. This tRNA methylation is present only in archaea, and the gene for this enzyme is present in all the archaeal genomes sequenced up to now, except in the crenarchaeon Pyrobaculum aerophilum. In this archaea, the C56 2′-O-methylation is provided by a C/D sRNP. Our work is the first demonstration that, within the same kingdom, two different mechanisms are used to modify the same nucleoside in tRNAs. PMID:15987815

  19. Intra- and interparticle magnetism of cobalt-doped iron-oxide nanoparticles encapsulated in a synthetic ferritin cage

    NASA Astrophysics Data System (ADS)

    Skoropata, E.; Desautels, R. D.; Falvo, E.; Ceci, P.; Kasyutich, O.; Freeland, J. W.; van Lierop, J.

    2014-11-01

    We present an in-depth examination of the composition and magnetism of cobalt (Co2 +)-doped iron-oxide nanoparticles encapsulated in Pyrococcus furiosus ferritin shells. We show that the Co2 + dopant ions were incorporated into the γ -Fe2O3/Fe3O4 core, with small paramagnetic-like clusters likely residing on the surface of the nanoparticle that were observed for all cobalt-doped samples. In addition, element-specific characterization using Mössbauer spectroscopy and polarized x-ray absorption indicated that Co2 + was incorporated exclusively into the octahedral B sites of the spinel-oxide nanoparticle. Comparable superparamagnetic blocking temperatures, coercivities, and effective anisotropies were obtained for 7%, 10%, and 12% cobalt-doped nanoparticles, and were only slightly reduced for 3% cobalt, indicating a strong effect of cobalt incorporation, with a lesser effect of cobalt content. Due to the regular particle size and separation that result from the use of the ferritin cage, a comparison of the effects of interparticle interactions on the disordered assembly of nanoparticles was also obtained that indicated significantly different behaviors between undoped and cobalt-doped nanoparticles.

  20. ATP half-sites in RadA and RAD51 recombinases bind nucleotides.

    PubMed

    Marsh, May E; Scott, Duncan E; Ehebauer, Matthias T; Abell, Chris; Blundell, Tom L; Hyvönen, Marko

    2016-05-01

    Homologous recombination is essential for repair of DNA double-strand breaks. Central to this process is a family of recombinases, including archeal RadA and human RAD51, which form nucleoprotein filaments on damaged single-stranded DNA ends and facilitate their ATP-dependent repair. ATP binding and hydrolysis are dependent on the formation of a nucleoprotein filament comprising RadA/RAD51 and single-stranded DNA, with ATP bound between adjacent protomers. We demonstrate that truncated, monomeric Pyrococcus furiosus RadA and monomerised human RAD51 retain the ability to bind ATP and other nucleotides with high affinity. We present crystal structures of both apo and nucleotide-bound forms of monomeric RadA. These structures reveal that while phosphate groups are tightly bound, RadA presents a shallow, poorly defined binding surface for the nitrogenous bases of nucleotides. We suggest that RadA monomers would be constitutively bound to nucleotides in the cell and that the bound nucleotide might play a structural role in filament assembly. PMID:27419043

  1. Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli.

    PubMed

    Zheng, Wenjun; Wang, Qingsong; Bi, Qun

    2016-04-01

    Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His6-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni(2+) chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future. PMID:26920159

  2. tRNA binding, positioning, and modification by the pseudouridine synthase Pus10.

    PubMed

    Kamalampeta, Rajashekhar; Keffer-Wilkes, Laura C; Kothe, Ute

    2013-10-23

    Pus10 is the most recently identified pseudouridine synthase found in archaea and higher eukaryotes. It modifies uridine 55 in the TΨC arm of tRNAs. Here, we report the first quantitative biochemical analysis of tRNA binding and pseudouridine formation by Pyrococcus furiosus Pus10. The affinity of Pus10 for both substrate and product tRNA is high (Kd of 30nM), and product formation occurs with a Km of 400nM and a kcat of 0.9s(-1). Site-directed mutagenesis was used to demonstrate that the thumb loop in the catalytic domain is important for efficient catalysis; we propose that the thumb loop positions the tRNA within the active site. Furthermore, a new catalytic arginine residue was identified (arginine 208), which is likely responsible for triggering flipping of the target uridine into the active site of Pus10. Lastly, our data support the proposal that the THUMP-containing domain, found in the N-terminus of Pus10, contributes to binding of tRNA. Together, our findings are consistent with the hypothesis that tRNA binding by Pus10 occurs through an induced-fit mechanism, which is a prerequisite for efficient pseudouridine formation. PMID:23743107

  3. MCM ring hexamerization is a prerequisite for DNA-binding

    SciTech Connect

    Froelich, Clifford A.; Nourse, Amanda; Enemark, Eric J.

    2015-09-13

    The hexameric Minichromosome Maintenance (MCM) protein complex forms a ring that unwinds DNA at the replication fork in eukaryotes and archaea. Our recent crystal structure of an archaeal MCM N-terminal domain bound to single-stranded DNA (ssDNA) revealed ssDNA associating across tight subunit interfaces but not at the loose interfaces, indicating that DNA-binding is governed not only by the DNA-binding residues of the subunits (MCM ssDNA-binding motif, MSSB) but also by the relative orientation of the subunits. We now extend these findings to show that DNA-binding by the MCM N-terminal domain of the archaeal organism Pyrococcus furiosus occurs specifically in the hexameric oligomeric form. We show that mutants defective for hexamerization are defective in binding ssDNA despite retaining all the residues observed to interact with ssDNA in the crystal structure. One mutation that exhibits severely defective hexamerization and ssDNA-binding is at a conserved phenylalanine that aligns with the mouse Mcm4(Chaos3) mutation associated with chromosomal instability, cancer, and decreased intersubunit association.

  4. Efficacy of pH elevation as a bactericidal strategy for treating ballast water of freight carriers.

    PubMed

    Starliper, Clifford E; Watten, Barnaby J; Iwanowicz, Deborah D; Green, Phyllis A; Bassett, Noel L; Adams, Cynthia R

    2015-05-01

    Treatment of ship ballast water with sodium hydroxide (NaOH) is one method currently being developed to minimize the risk to introduce aquatic invasive species. The bactericidal capability of sodium hydroxide was determined for 148 bacterial strains from ballast water collected in 2009 and 2010 from the M/V Indiana Harbor, a bulk-freight carrier plying the Laurentian Great Lakes, USA. Primary culture of bacteria was done using brain heart infusion agar and a developmental medium. Strains were characterized based on PCR amplification and sequencing of a portion of the 16S rRNA gene. Sequence similarities (99+ %) were determined by comparison with the National Center for Biotechnology Information (NCBI) GenBank catalog. Flavobacterium spp. were the most prevalent bacteria characterized in 2009, comprising 51.1% (24/47) of the total, and Pseudomonas spp. (62/101; 61.4%) and Brevundimonas spp. (22/101; 21.8%) were the predominate bacteria recovered in 2010; together, comprising 83.2% (84/101) of the total. Testing was done in tryptic soy broth (TSB) medium adjusted with 5 N NaOH. Growth of each strain was evaluated at pH 10.0, pH 11.0 and pH 12.0, and 4 h up to 72 h. The median cell count at 0 h for 148 cultures was 5.20 × 10(6) cfu/mL with a range 1.02 × 10(5)-1.60 × 10(8) cfu/mL. The TSB adjusted to pH 10.0 and incubation for less than 24 h was bactericidal to 52 (35.1%) strains. Growth in pH 11.0 TSB for less than 4 h was bactericidal to 131 (88.5%) strains and pH 11.0 within 12 h was bactericidal to 141 (95.3%). One strain, Bacillus horikoshii, survived the harshest treatment, pH 12.0 for 72 h. PMID:26257948

  5. Efficacy of pH elevation as a bactericidal strategy for treating ballast water of freight carriers

    PubMed Central

    Starliper, Clifford E.; Watten, Barnaby J.; Iwanowicz, Deborah D.; Green, Phyllis A.; Bassett, Noel L.; Adams, Cynthia R.

    2015-01-01

    Treatment of ship ballast water with sodium hydroxide (NaOH) is one method currently being developed to minimize the risk to introduce aquatic invasive species. The bactericidal capability of sodium hydroxide was determined for 148 bacterial strains from ballast water collected in 2009 and 2010 from the M/V Indiana Harbor, a bulk-freight carrier plying the Laurentian Great Lakes, USA. Primary culture of bacteria was done using brain heart infusion agar and a developmental medium. Strains were characterized based on PCR amplification and sequencing of a portion of the 16S rRNA gene. Sequence similarities (99+ %) were determined by comparison with the National Center for Biotechnology Information (NCBI) GenBank catalog. Flavobacterium spp. were the most prevalent bacteria characterized in 2009, comprising 51.1% (24/47) of the total, and Pseudomonas spp. (62/101; 61.4%) and Brevundimonas spp. (22/101; 21.8%) were the predominate bacteria recovered in 2010; together, comprising 83.2% (84/101) of the total. Testing was done in tryptic soy broth (TSB) medium adjusted with 5 N NaOH. Growth of each strain was evaluated at pH 10.0, pH 11.0 and pH 12.0, and 4 h up to 72 h. The median cell count at 0 h for 148 cultures was 5.20 × 106 cfu/mL with a range 1.02 × 105–1.60 × 108 cfu/mL. The TSB adjusted to pH 10.0 and incubation for less than 24 h was bactericidal to 52 (35.1%) strains. Growth in pH 11.0 TSB for less than 4 h was bactericidal to 131 (88.5%) strains and pH 11.0 within 12 h was bactericidal to 141 (95.3%). One strain, Bacillus horikoshii, survived the harshest treatment, pH 12.0 for 72 h. PMID:26257948

  6. On the mineral core of ferritin-like proteins: structural and magnetic characterization

    NASA Astrophysics Data System (ADS)

    García-Prieto, A.; Alonso, J.; Muñoz, D.; Marcano, L.; Abad Díaz de Cerio, A.; Fernández de Luis, R.; Orue, I.; Mathon, O.; Muela, A.; Fdez-Gubieda, M. L.

    2015-12-01

    It is generally accepted that the mineral core synthesized by ferritin-like proteins consists of a ferric oxy-hydroxide mineral similar to ferrihydrite in the case of horse spleen ferritin (HoSF) and an oxy-hydroxide-phosphate phase in plant and prokaryotic ferritins. The structure reflects a dynamic process of deposition and dissolution, influenced by different biological, chemical and physical variables. In this work we shed light on this matter by combining a structural (High Resolution Transmission Electron Microscopy (HRTEM) and Fe K-edge X-ray Absorption Spectroscopy (XAS)) and a magnetic study of the mineral core biomineralized by horse spleen ferritin (HoSF) and three prokaryotic ferritin-like proteins: bacterial ferritin (FtnA) and bacterioferritin (Bfr) from Escherichia coli and archaeal ferritin (PfFtn) from Pyrococcus furiosus. The prokaryotic ferritin-like proteins have been studied under native conditions and inside the cells for the sake of preserving their natural attributes. They share with HoSF a nanocrystalline structure rather than an amorphous one as has been frequently reported. However, the presence of phosphorus changes drastically the short-range order and magnetic response of the prokaryotic cores with respect to HoSF. The superparamagnetism observed in HoSF is absent in the prokaryotic proteins, which show a pure atomic-like paramagnetic behaviour attributed to phosphorus breaking the Fe-Fe exchange interaction.It is generally accepted that the mineral core synthesized by ferritin-like proteins consists of a ferric oxy-hydroxide mineral similar to ferrihydrite in the case of horse spleen ferritin (HoSF) and an oxy-hydroxide-phosphate phase in plant and prokaryotic ferritins. The structure reflects a dynamic process of deposition and dissolution, influenced by different biological, chemical and physical variables. In this work we shed light on this matter by combining a structural (High Resolution Transmission Electron Microscopy (HRTEM

  7. Dual Functions, Clamp Opening and Primer-Template Recognition, Define a Key Clamp Loader Subunit

    PubMed Central

    Coman, Maria Magdalena; Jin, Mi; Ceapa, Razvan; Finkelstein, Jeff; O'Donnell, Michael; Chait, Brian T.; Hingorani, Manju M.

    2010-01-01

    Clamp loader proteins catalyze assembly of circular sliding clamps on DNA to enable processive DNA replication. During the reaction, the clamp loader binds primer-template DNA and positions it in the center of a clamp to form a topological link between the two. Clamp loaders are multi-protein complexes, such as the five protein Escherichia coli, Saccharomyces cerevisiae, and human clamp loaders, and the two protein Pyrococcus furiosus and Methanobacterium thermoautotrophicum clamp loaders, and thus far the site(s) responsible for binding and selecting primer-template DNA as the target for clamp assembly remain unknown. To address this issue, we analyzed the interaction between the E. coli γ complex clamp loader and DNA using UV-induced protein–DNA cross-linking and mass spectrometry. The results show that the δ subunit in the γ complex makes close contact with the primer-template junction. Tryptophan 279 in the δ C-terminal domain lies near the 3′-OH primer end and may play a key role in primer-template recognition. Previous studies have shown that δ also binds and opens the β clamp (hydrophobic residues in the N-terminal domain of δ contact β. The clamp-binding and DNA-binding sites on δ appear positioned for facile entry of primer-template into the center of the clamp and exit of the template strand from the complex. A similar analysis of the S. cerevisiae RFC complex suggests that the dual functionality observed for δ in the γ complex may be true also for clamp loaders from other organisms. PMID:15364574

  8. The enzymology of alanine aminotransferase (AlaAT) isoforms from Hordeum vulgare and other organisms, and the HvAlaAT crystal structure.

    PubMed

    Duff, Stephen M G; Rydel, Timothy J; McClerren, Amanda L; Zhang, Wenlan; Li, Jimmy Y; Sturman, Eric J; Halls, Coralie; Chen, Songyang; Zeng, Jiamin; Peng, Jiexin; Kretzler, Crystal N; Evdokimov, Artem

    2012-12-01

    In this paper we describe the expression, purification, kinetics and biophysical characterization of alanine aminotransferase (AlaAT) from the barley plant (Hordeum vulgare). This dimeric PLP-dependent enzyme is a pivotal element of several key metabolic pathways from nitrogen assimilation to carbon metabolism, and its introduction into transgenic plants results in increased yield. The enzyme exhibits a bi-bi ping-pong reaction mechanism with a K(m) for alanine, 2-oxoglutarate, glutamate and pyruvate of 3.8, 0.3, 0.8 and 0.2 mM, respectively. Barley AlaAT catalyzes the forward (alanine-forming) reaction with a k(cat) of 25.6 s(-1), the reverse (glutamate-forming) reaction with k(cat) of 12.1 s(-1) and an equilibrium constant of ~0.5. The enzyme is also able to utilize aspartate and oxaloacetate with ~10% efficiency as compared to the native substrates, which makes it much more specific than related bacterial/archaeal enzymes (that also have lower K(m) values). We have crystallized barley AlaAT in complex with PLP and l-cycloserine and solved the structure of this complex at 2.7 Å resolution. This is the first example of a plant AlaAT structure, and it reveals a canonical aminotransferase fold similar to structures of the Thermotoga maritima, Pyrococcus furiosus, and human enzymes. This structure bridges our structural understanding of AlaAT mechanism between three kingdoms of life and allows us to shed some light on the specifics of the catalysis performed by these proteins. PMID:22750542

  9. Thermostable β-galactosidases for the synthesis of human milk oligosaccharides.

    PubMed

    Zeuner, Birgitte; Nyffenegger, Christian; Mikkelsen, Jørn Dalgaard; Meyer, Anne S

    2016-05-25

    Human milk oligosaccharides (HMOs) designate a unique family of bioactive lactose-based molecules present in human breast milk. Using lactose as a cheap donor, some β-galactosidases (EC 3.2.1.23) can catalyze transgalactosylation to form the human milk oligosaccharide lacto-N-neotetraose (LNnT; Gal-β(1,4)-GlcNAc-β(1,3)-Gal-β(1,4)-Glc). In order to reduce reaction times and be able to work at temperatures, which are less welcoming to microbial growth, the current study investigates the possibility of using thermostable β-galactosidases for synthesis of LNnT and N-acetyllactosamine (LacNAc; Gal-β(1,4)-GlcNAc), the latter being a core structure in HMOs. Two hyperthermostable GH 1 β-galactosidases, Ttβ-gly from Thermus thermophilus HB27 and CelB from Pyrococcus furiosus, were codon-optimized for expression in Escherichia coli along with BgaD-D, a truncated version of the GH 42 β-galactosidase from Bacillus circulans showing high transgalactosylation activity at low substrate concentrations. The three β-galactosidases were compared in the current study in terms of their transgalactosylation activity in the formation of LacNAc and LNnT. In all cases, BgaD-D was the most potent transgalactosidase, but both thermostable GH 1 β-galactosidases could catalyze formation of LNnT and LacNAc, with Ttβ-gly giving higher yields than CelB. The thermal stability of the three β-galactosidases was elucidated and the results were used to optimize the reaction efficiency in the formation of LacNAc, resulting in 5-6 times higher reaction yields and significantly shorter reaction times. PMID:26802542

  10. Bacterial and archaeal resistance to ionizing radiation

    NASA Astrophysics Data System (ADS)

    Confalonieri, F.; Sommer, S.

    2011-01-01

    Organisms living in extreme environments must cope with large fluctuations of temperature, high levels of radiation and/or desiccation, conditions that can induce DNA damage ranging from base modifications to DNA double-strand breaks. The bacterium Deinococcus radiodurans is known for its resistance to extremely high doses of ionizing radiation and for its ability to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Recently, extreme ionizing radiation resistance was also generated by directed evolution of an apparently radiation-sensitive bacterial species, Escherichia coli. Radioresistant organisms are not only found among the Eubacteria but also among the Archaea that represent the third kingdom of life. They present a set of particular features that differentiate them from the Eubacteria and eukaryotes. Moreover, Archaea are often isolated from extreme environments where they live under severe conditions of temperature, pressure, pH, salts or toxic compounds that are lethal for the large majority of living organisms. Thus, Archaea offer the opportunity to understand how cells are able to cope with such harsh conditions. Among them, the halophilic archaeon Halobacterium sp and several Pyrococcus or Thermococcus species, such as Thermococcus gammatolerans, were also shown to display high level of radiation resistance. The dispersion, in the phylogenetic tree, of radioresistant prokaryotes suggests that they have independently acquired radioresistance. Different strategies were selected during evolution including several mechanisms of radiation byproduct detoxification and subtle cellular metabolism modifications to help cells recover from radiation-induced injuries, protection of proteins against oxidation, an efficient DNA repair tool box, an original pathway of DNA double-strand break repair, a condensed nucleoid that may prevent the dispersion of the DNA fragments and specific radiation-induced proteins involved in

  11. Activation of archaeal transcription mediated by recruitment of transcription factor B.

    PubMed

    Ochs, Simon M; Thumann, Sybille; Richau, Renate; Weirauch, Matt T; Lowe, Todd M; Thomm, Michael; Hausner, Winfried

    2012-05-25

    Archaeal promoters consist of a TATA box and a purine-rich adjacent upstream sequence (transcription factor B (TFB)-responsive element (BRE)), which are bound by the transcription factors TATA box-binding protein (TBP) and TFB. Currently, only a few activators of archaeal transcription have been experimentally characterized. The best studied activator, Ptr2, mediates activation by recruitment of TBP. Here, we present a detailed biochemical analysis of an archaeal transcriptional activator, PF1088, which was identified in Pyrococcus furiosus by a bioinformatic approach. Operon predictions suggested that an upstream gene, pf1089, is polycistronically transcribed with pf1088. We demonstrate that PF1088 stimulates in vitro transcription by up to 7-fold when the pf1089 promoter is used as a template. By DNase I and hydroxyl radical footprinting experiments, we show that the binding site of PF1088 is located directly upstream of the BRE of pf1089. Mutational analysis indicated that activation requires the presence of the binding site for PF1088. Furthermore, we show that activation of transcription by PF1088 is dependent upon the presence of an imperfect BRE and is abolished when the pf1089 BRE is replaced with a BRE from a strong archaeal promoter. Gel shift experiments showed that TFB recruitment to the pf1089 operon is stimulated by PF1088, and TFB seems to stabilize PF1088 operator binding even in the absence of TBP. Taken together, these results represent the first biochemical evidence for a transcriptional activator working as a TFB recruitment factor in Archaea, for which the designation TFB-RF1 is suggested. PMID:22496454

  12. Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals

    PubMed Central

    Zeldes, Benjamin M.; Keller, Matthew W.; Loder, Andrew J.; Straub, Christopher T.; Adams, Michael W. W.; Kelly, Robert M.

    2015-01-01

    Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high

  13. The box C/D sRNP dimeric architecture is conserved across domain Archaea

    PubMed Central

    Bower-Phipps, Kathleen R.; Taylor, David W.; Wang, Hong-Wei; Baserga, Susan J.

    2012-01-01

    Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze RNA-guided 2′-O-ribose methylation in two of the three domains of life. Recent structural studies have led to a controversy over whether box C/D sRNPs functionally assemble as monomeric or dimeric macromolecules. The archaeal box C/D sRNP from Methanococcus jannaschii (Mj) has been shown by glycerol gradient sedimentation, gel filtration chromatography, native gel analysis, and single-particle electron microscopy (EM) to adopt a di-sRNP architecture, containing four copies of each box C/D core protein and two copies of the Mj sR8 sRNA. Subsequently, investigators used a two-stranded artificial guide sRNA, CD45, to assemble a box C/D sRNP from Sulfolobus solfataricus with a short RNA methylation substrate, yielding a crystal structure of a mono-sRNP. To more closely examine box C/D sRNP architecture, we investigate the role of the omnipresent sRNA loop as a structural determinant of sRNP assembly. We show through sRNA mutagenesis, native gel electrophoresis, and single-particle EM that a di-sRNP is the near exclusive architecture obtained when reconstituting box C/D sRNPs with natural or artificial sRNAs containing an internal loop. Our results span three distantly related archaeal species—Sulfolobus solfataricus, Pyrococcus abyssi, and Archaeoglobus fulgidus—indicating that the di-sRNP architecture is broadly conserved across the entire archaeal domain. PMID:22753779

  14. Crystal structure of the C-terminal globular domain of oligosaccharyltransferase from Archaeoglobus fulgidus at 1.75 Å resolution.

    PubMed

    Matsumoto, Shunsuke; Igura, Mayumi; Nyirenda, James; Matsumoto, Masaki; Yuzawa, Satoru; Noda, Nobuo; Inagaki, Fuyuhiko; Kohda, Daisuke

    2012-05-22

    Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers glycan to asparagine in the N-glycosylation sequon. The catalytic subunit of OST is called STT3 in eukaryotes, AglB in archaea, and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three AglB paralogs. Two of them are the shortest AglBs across all domains of life. We determined the crystal structure of the C-terminal globular domain of the smallest AglB to identify the minimal structural unit. The Archaeoglobus AglB lacked a β-barrel-like structure, which had been found in other AglB and PglB structures. In agreement, the deletion in a larger Pyrococcus AglB confirmed its dispensability for the activity. By contrast, the Archaeoglobus AglB contains a kinked helix bearing a conserved motif, called DK/MI motif. The lysine and isoleucine residues in the motif participate in the Ser/Thr recognition in the sequon. The Archaeoglobus AglB structure revealed that the kinked helix contained an unexpected insertion. A revised sequence alignment based on this finding identified a variant type of the DK motif with the insertion. A mutagenesis study of the Archaeoglobus AglB confirmed the contribution of this particular type of the DK motif to the activity. When taken together with our previous results, this study defined the classification of OST: one group consisting of eukaryotes and most archaea possesses the DK-type Ser/Thr pocket, and the other group consisting of eubacteria and the remaining archaea possesses the MI-type Ser/Thr pocket. This classification provides a useful framework for OST studies. PMID:22559858

  15. The metabolism of hydrogen by extremely thermophilic bacteria

    SciTech Connect

    Adams, M.W.W.

    1991-01-01

    The novel archaebacterium, Pyrococcus furiosus, grows optimally at 100{degree}C by a fermentative metabolism and produces hydrogen (H{sub 2}). We have shown that this organism appears to ferment glucose and evolve H{sub 2} by a novel pathway. The following metalloenzymes and proteins involved in H{sub 2} metabolism have been purified and characterized: hydrogenase (NiFeS), ferredoxin (FeS), pyruvate ferredoxin oxidoreductase (FeS), and an new enzyme which contains tungsten, glyceraldehyde ferredoxin oxidoreductase (WFeS). A rubredoxin was also purified, and it and the ferredoxin have been sequenced. In addition, a second new enzyme has been identified, glucose ferredoxin oxidoreductase. These represent the first enzymes and proteins to be purified from any organism able to grow optimally above 90{degree}C. All are remarkably thermostable and show maximal catalytic activity >95{degree}C. The ferredoxin has several unique properties and is potentially an extremely thermostable model for the catalytic sites of a variety of mesophilic metalloenzymes. In addition, the FeS-containing enzymes, hydrogenase and pyruvate ferredoxin oxidoreductase, and a ferredoxin, have been purified from the most thermophilic eubacterium currently known, Thermotoga maritima. This organism grows up to 90{degree}C, also by fermentation. The hydrogenases of T. maritima and P. furiosus each have many unique properties in comparison with mesophilic hydrogenases, and both appear to contain new types of metal centers that are specifically adapted to catalyze H{sub 2} production at the extreme temperatures. Hydrogenase activity has also been measured in four other extremely thermophilic organisms, one of which is capable of growth at 120{degree}C. 1 fig.

  16. A novel microbial habitat in the mid-ocean ridge subseafloor

    PubMed Central

    Summit, Melanie; Baross, John A.

    2001-01-01

    The subseafloor at the mid-ocean ridge is predicted to be an excellent microbial habitat, because there is abundant space, fluid flow, and geochemical energy in the porous, hydrothermally influenced oceanic crust. These characteristics also make it a good analog for potential subsurface extraterrestrial habitats. Subseafloor environments created by the mixing of hot hydrothermal fluids and seawater are predicted to be particularly energy-rich, and hyperthermophilic microorganisms that broadly reflect such predictions are ejected from these systems in low-temperature (≈15°C), basalt-hosted diffuse effluents. Seven hyperthermophilic heterotrophs isolated from low-temperature diffuse fluids exiting the basaltic crust in and near two hydrothermal vent fields on the Endeavour Segment, Juan de Fuca Ridge, were compared phylogenetically and physiologically to six similarly enriched hyperthermophiles from samples associated with seafloor metal sulfide structures. The 13 organisms fell into four distinct groups: one group of two organisms corresponding to the genus Pyrococcus and three groups corresponding to the genus Thermococcus. Of these three groups, one was composed solely of sulfide-derived organisms, and the other two related groups were composed of subseafloor organisms. There was no evidence of restricted exchange of organisms between sulfide and subseafloor habitats, and therefore this phylogenetic distinction indicates a selective force operating between the two habitats. Hypotheses regarding the habitat differences were generated through comparison of the physiology of the two groups of hyperthermophiles; some potential differences between these habitats include fluid flow stability, metal ion concentrations, and sources of complex organic matter. PMID:11226209

  17. Anomalous behavior of water inside the SecY translocon.

    PubMed

    Capponi, Sara; Heyden, Matthias; Bondar, Ana-Nicoleta; Tobias, Douglas J; White, Stephen H

    2015-07-21

    The heterotrimeric SecY translocon complex is required for the cotranslational assembly of membrane proteins in bacteria and archaea. The insertion of transmembrane (TM) segments during nascent-chain passage through the translocon is generally viewed as a simple partitioning process between the water-filled translocon and membrane lipid bilayer, suggesting that partitioning is driven by the hydrophobic effect. Indeed, the apparent free energy of partitioning of unnatural aliphatic amino acids on TM segments is proportional to accessible surface area, which is a hallmark of the hydrophobic effect [Öjemalm K, et al. (2011) Proc Natl Acad Sci USA 108(31):E359-E364]. However, the apparent partitioning solvation parameter is less than one-half the value expected for simple bulk partitioning, suggesting that the water in the translocon departs from bulk behavior. To examine the state of water in a SecY translocon complex embedded in a lipid bilayer, we carried out all-atom molecular-dynamics simulations of the Pyrococcus furiosus SecYE, which was determined to be in a "primed" open state [Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107(40):17182-17187]. Remarkably, SecYE remained in this state throughout our 450-ns simulation. Water molecules within SecY exhibited anomalous diffusion, had highly retarded rotational dynamics, and aligned their dipoles along the SecY transmembrane axis. The translocon is therefore not a simple water-filled pore, which raises the question of how anomalous water behavior affects the mechanism of translocon function and, more generally, the partitioning of hydrophobic molecules. Because large water-filled cavities are found in many membrane proteins, our findings may have broader implications. PMID:26139523

  18. Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.

    PubMed

    Graziadei, Andrea; Masiewicz, Pawel; Lapinaite, Audrone; Carlomagno, Teresa

    2016-05-01

    RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product. PMID:26925607

  19. Mechanisms for Two-Step Proton Transfer Reactions in the Outward-Facing Form of MATE Transporter.

    PubMed

    Nishima, Wataru; Mizukami, Wataru; Tanaka, Yoshiki; Ishitani, Ryuichiro; Nureki, Osamu; Sugita, Yuji

    2016-03-29

    Bacterial pathogens or cancer cells can acquire multidrug resistance, which causes serious clinical problems. In cells with multidrug resistance, various drugs or antibiotics are extruded across the cell membrane by multidrug transporters. The multidrug and toxic compound extrusion (MATE) transporter is one of the five families of multidrug transporters. MATE from Pyrococcus furiosus uses H(+) to transport a substrate from the cytoplasm to the outside of a cell. Crystal structures of MATE from P. furiosus provide essential information on the relevant H(+)-binding sites (D41 and D184). Hybrid quantum mechanical/molecular mechanical simulations and continuum electrostatic calculations on the crystal structures predict that D41 is protonated in one structure (Straight) and, both D41 and D184 protonated in another (Bent). All-atom molecular dynamics simulations suggest a dynamic equilibrium between the protonation states of the two aspartic acids and that the protonation state affects hydration in the substrate binding cavity and lipid intrusion in the cleft between the N- and C-lobes. This hypothesis is examined in more detail by quantum mechanical/molecular mechanical calculations on snapshots taken from the molecular dynamics trajectories. We find the possibility of two proton transfer (PT) reactions in Straight: the 1st PT takes place between side-chains D41 and D184 through a transient formation of low-barrier hydrogen bonds and the 2nd through another H(+) from the headgroup of a lipid that intrudes into the cleft resulting in a doubly protonated (both D41 and D184) state. The 1st PT affects the local hydrogen bond network and hydration in the N-lobe cavity, which would impinge on the substrate-binding affinity. The 2nd PT would drive the conformational change from Straight to Bent. This model may be applicable to several prokaryotic H(+)-coupled MATE multidrug transporters with the relevant aspartic acids. PMID:27028644

  20. Identification of 2D-gel proteins : a comparison of MALDI/TOF peptide mass mapping to {mu} LC-ESI tandem mass spectrometry.

    SciTech Connect

    Lim, H.; Hays, L. G.; Eng, J.; Tollaksen, S. L.; Giometti, C. S.; Holden, J. F.; Adams, M. W. W.; Reich, C. I.; Olsen, G. J.; Yates, J. R.; Biosciences Division; The Scripps Research Inst.; Univ. of Georgia; Univ. of Illinois

    2003-09-01

    A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

  1. Self-assembly Is Prerequisite for Catalysis of Fe(II) Oxidation by Catalytically Active Subunits of Ferritin*

    PubMed Central

    Ebrahimi, Kourosh Honarmand; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

    2015-01-01

    Fe(III) storage by ferritin is an essential process of the iron homeostasis machinery. It begins by translocation of Fe(II) from outside the hollow spherical shape structure of the protein, which is formed as the result of self-assembly of 24 subunits, to a di-iron binding site, the ferroxidase center, buried in the middle of each active subunit. The pathway of Fe(II) to the ferroxidase center has remained elusive, and the importance of self-assembly for the functioning of the ferroxidase center has not been investigated. Here we report spectroscopic and metal ion binding studies with a mutant of ferritin from Pyrococcus furiosus (PfFtn) in which self-assembly was abolished by a single amino acid substitution. We show that in this mutant metal ion binding to the ferroxidase center and Fe(II) oxidation at this site was obliterated. However, metal ion binding to a conserved third site (site C), which is located in the inner surface of each subunit in the vicinity of the ferroxidase center and is believed to be the path for Fe(II) to the ferroxidase center, was not disrupted. These results are the basis of a new model for Fe(II) translocation to the ferroxidase center: self-assembly creates channels that guide the Fe(II) ions toward the ferroxidase center directly through the protein shell and not via the internal cavity and site C. The results may be of significance for understanding the molecular basis of ferritin-related disorders such as neuroferritinopathy in which the 24-meric structure with 432 symmetry is distorted. PMID:26370076

  2. The key to the extraordinary thermal stability of P. furiosus holo-rubredoxin: iron binding-guided packing of a core aromatic cluster responsible for high kinetic stability of the native structure.

    PubMed

    Prakash, Satya; Sundd, Monica; Guptasarma, Purnananda

    2014-01-01

    Pyrococcus furiosus rubredoxin (PfRd), a small, monomeric, 53 residues-long, iron-containing, electron-transfer protein of known structure is sometimes referred to as being the most structurally-stable protein known to man. Here, using a combination of mutational and spectroscopic (CD, fluorescence, and NMR) studies of differently made holo- and apo-forms of PfRd, we demonstrate that it is not the presence of iron, or even the folding of the PfRd chain into a compact well-folded structure that causes holo-PfRd to display its extraordinary thermal stability, but rather the correct iron binding-guided packing of certain residues (specifically, Trp3, Phe29, Trp36, and also Tyr10) within a tight aromatic cluster of six residues in PfRd's hydrophobic core. Binding of the iron atom appears to play a remarkable role in determining subtle details of residue packing, forcing the chain to form a hyper-thermally stable native structure which is kinetically stable enough to survive (subsequent) removal of iron. On the other hand, failure to bind iron causes the same chain to adopt an equally well-folded native-like structure which, however, has a differently-packed aromatic cluster in its core, causing it to be only as stable as any other ordinary mesophile-derived rubredoxin. Our studies demonstrate, perhaps for the very first time ever that hyperthermal stability in proteins can owe to subtle differences in residue packing vis a vis mesostable proteins, without there being any underlying differences in either amino acid sequence, or bound ligand status. PMID:24603898

  3. Microbial metalloproteomes are largely uncharacterized.

    PubMed

    Cvetkovic, Aleksandar; Menon, Angeli Lal; Thorgersen, Michael P; Scott, Joseph W; Poole, Farris L; Jenney, Francis E; Lancaster, W Andrew; Praissman, Jeremy L; Shanmukh, Saratchandra; Vaccaro, Brian J; Trauger, Sunia A; Kalisiak, Ewa; Apon, Junefredo V; Siuzdak, Gary; Yannone, Steven M; Tainer, John A; Adams, Michael W W

    2010-08-01

    Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms. PMID:20639861

  4. 1H nuclear-magnetic-resonance investigation of oxidized Fe4S4 ferredoxin from Thermotoga maritima. Hyperfine-shifted resonances, sequence-specific assignments and secondary structure.

    PubMed

    Wildegger, G; Bentrop, D; Ejchart, A; Alber, M; Hage, A; Sterner, R; Rösch, P

    1995-05-01

    The oxidized Fe4S4 ferredoxin from the hyperthermophilic bacterium Thermotoga maritima has been investigated by one- and two-dimensional NMR in order to characterize its hyperfine-shifted resonances originating from the cysteinyl cluster ligands and to assign its resonances in the diamagnetic shift range. The chemical shift and relaxation time pattern of the hyperfine-shifted signals is very similar to other oxidized Fe4S4 ferredoxins. A tentative sequence-specific assignment of these resonances according to a general pattern of chemical shift of cysteine protons versus sequence position of cluster ligand is presented. Furthermore, sequence-specific assignments for 85% of the amino acid residues that were obtained without any guidance by known X-ray structures of ferredoxins are given. They reveal the formation of at least two elements of secondary structure by the polypeptide chain of T. maritima ferredoxin: an alpha-helix comprising residues C43-D49 and a double-stranded antiparallel beta-sheet consisting of the N- and C-terminal parts of the protein. This folding pattern is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibrio gigas [Kissinger, C.R., Sieker, L.C., Adman E.T. & Jensen, L.H. (1991) J. Mol. Biol. 219, 693-715] and therefore suggesting different mechanisms of stabilization for T. maritima ferredoxin and the ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus that was recently investigated by NMR [Teng, Q., Zhou, Z.H., Smith, E.T., Busse, S. C., Howard, J.B., Adams M.W.W. & La Mar, G.N. (1994) Biochemistry 33, 6316-6326]. PMID:7758460

  5. Evidence of Molecular Adaptation to Extreme Environments and Applicability to Space Environments

    NASA Astrophysics Data System (ADS)

    Filipovic, M. D.; Ognjanovic, S.; Ognjanovic, M.

    2008-06-01

    This is initial investigation of gene signatures responsible for adapting microscopic life to the extreme Earth environments. We present preliminary results on identification of the clusters of orthologous groups (COGs) common to several hyperthermophiles and exclusion of those common to a mesophile (non-hyperthermophile): Escherichia coli (E. coli K12), will yield a group of proteins possibly involved in adaptation to life under extreme temperatures. Comparative genome analyses represent a powerful tool in discovery of novel genes responsible for adaptation to specific extreme environments. Methanogens stand out as the only group of organisms that have species capable of growth at 0° C (Metarhizium frigidum (M.~frigidum) and Methanococcoides burtonii (M.~burtonii)) and 110° C (Methanopyrus kandleri (M.~kandleri)). Although not all the components of heat adaptation can be attributed to novel genes, the chaperones known as heat shock proteins stabilize the enzymes under elevated temperature. However, highly conserved chaperons found in bacteria and eukaryots are not present in hyperthermophilic Archea, rather, they have a unique chaperone TF55. Our aim was to use software which we specifically developed for extremophile genome comparative analyses in order to search for additional novel genes involved in hyperthermophile adaptation. The following hyperthermophile genomes incorporated in this software were used for these studies: Methanocaldococcus jannaschii (M.~jannaschii), M.~kandleri, Archaeoglobus fulgidus (A.~fulgidus) and three species of Pyrococcus. Common genes were annotated and grouped according to their roles in cellular processes where such information was available and proteins not previously implicated in the heat-adaptation of hyperthermophiles were identified. Additional experimental data are needed in order to learn more about these proteins. To address non-gene based components of thermal adaptation, all sequenced extremophiles were analysed for

  6. 2D 1H and 3D 1H-15N NMR of zinc-rubredoxins: contributions of the beta-sheet to thermostability.

    PubMed Central

    Richie, K. A.; Teng, Q.; Elkin, C. J.; Kurtz, D. M.

    1996-01-01

    Based on 2D 1H-1H and 2D and 3D 1H-15N NMR spectroscopies, complete 1H NMR assignments are reported for zinc-containing Clostridium pasteurianum rubredoxin (Cp ZnRd). Complete 1H NMR assignments are also reported for a mutated Cp ZnRd, in which residues near the N-terminus, namely, Met 1, Lys 2, and Pro 15, have been changed to their counterparts, (-), Ala and Glu, respectively, in rubredoxin from the hyperthermophilic archaeon, Pyrococcus furiosus (Pf Rd). The secondary structure of both wild-type and mutated Cp ZnRds, as determined by NMR methods, is essentially the same. However, the NMR data indicate an extension of the three-stranded beta-sheet in the mutated Cp ZnRd to include the N-terminal Ala residue and Glu 15, as occurs in Pf Rd. The mutated Cp Rd also shows more intense NOE cross peaks, indicating stronger interactions between the strands of the beta-sheet and, in fact, throughout the mutated Rd. However, these stronger interactions do not lead to any significant increase in thermostability, and both the mutated and wild-type Cp Rds are much less thermostable than Pf Rd. These correlations strongly suggest that, contrary to a previous proposal [Blake PR et al., 1992, Protein Sci 1:1508-1521], the thermostabilization mechanism of Pf Rd is not dominated by a unique set of hydrogen bonds or electrostatic interactions involving the N-terminal strand of the beta-sheet. The NMR results also suggest that an overall tighter protein structure does not necessarily lead to increased thermostability. PMID:8732760

  7. Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: construction of a 16-residue ion-pair network at the subunit interface.

    PubMed

    Lebbink, J H; Knapp, S; van der Oost, J; Rice, D; Ladenstein, R; de Vos, W M

    1999-06-01

    The role of an 18-residue ion-pair network, that is present in the glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus, in conferring stability to other, less stable homologous enzymes, has been studied by introducing four new charged amino acid residues into the subunit interface of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. These two GDHs are 55 % identical in amino acid sequence, differ greatly in thermo-activity and stability and derive from microbes with different phylogenetic positions. Amino acid substitutions were introduced as single mutations as well as in several combinations. Elucidation of the crystal structure of the quadruple mutant S128R/T158E/N117R/S160E T. maritima glutamate dehydrogenase showed that all anticipated ion-pairs are formed and that a 16-residue ion-pair network is present. Enlargement of existing networks by single amino acid substitutions unexpectedly resulted in a decrease in resistance towards thermal inactivation and thermal denaturation. However, combination of destabilizing single mutations in most cases restored stability, indicating the need for balanced charges at subunit interfaces and high cooperativity between the different members of the network. Combination of the three destabilizing mutations in triple mutant S128R/T158E/N117R resulted in an enzyme with a 30 minutes longer half-life of inactivation at 85 degrees C, a 3 degrees C higher temperature optimum for catalysis, and a 0.5 degrees C higher apparent melting temperature than that of wild-type glutamate dehydrogenase. These findings confirm the hypothesis that large ion-pair networks do indeed stabilize enzymes from hyperthermophilic organisms. PMID:10366510

  8. A Novel Family of Soluble Minimal Scaffolds Provides Structural Insight into the Catalytic Domains of Integral Membrane Metallopeptidases*

    PubMed Central

    López-Pelegrín, Mar; Cerdà-Costa, Núria; Martínez-Jiménez, Francisco; Cintas-Pedrola, Anna; Canals, Albert; Peinado, Juan R.; Marti-Renom, Marc A.; López-Otín, Carlos; Arolas, Joan L.; Gomis-Rüth, F. Xavier

    2013-01-01

    In the search for structural models of integral-membrane metallopeptidases (MPs), we discovered three related proteins from thermophilic prokaryotes, which we grouped into a novel family called “minigluzincins.” We determined the crystal structures of the zymogens of two of these (Pyrococcus abyssi proabylysin and Methanocaldococcus jannaschii projannalysin), which are soluble and, with ∼100 residues, constitute the shortest structurally characterized MPs to date. Despite relevant sequence and structural similarity, the structures revealed two unique mechanisms of latency maintenance through the C-terminal segments previously unseen in MPs as follows: intramolecular, through an extended tail, in proabylysin, and crosswise intermolecular, through a helix swap, in projannalysin. In addition, structural and sequence comparisons revealed large similarity with MPs of the gluzincin tribe such as thermolysin, leukotriene A4 hydrolase relatives, and cowrins. Noteworthy, gluzincins mostly contain a glutamate as third characteristic zinc ligand, whereas minigluzincins have a histidine. Sequence and structural similarity further allowed us to ascertain that minigluzincins are very similar to the catalytic domains of integral membrane MPs of the MEROPS database families M48 and M56, such as FACE1, HtpX, Oma1, and BlaR1/MecR1, which are provided with trans-membrane helices flanking or inserted into a minigluzincin-like catalytic domain. In a time where structural biochemistry of integral-membrane proteins in general still faces formidable challenges, the minigluzincin soluble minimal scaffold may contribute to our understanding of the working mechanisms of these membrane MPs and to the design of novel inhibitors through structure-aided rational drug design approaches. PMID:23733187

  9. Unambiguous determination of H-atom positions: comparing results from neutron and high-resolution X-ray crystallography

    SciTech Connect

    Gardberg, Anna S.; Del Castillo, Alexis R.; Weiss, Kevin L.; Meilleur, Flora; Blakeley, Matthew P.; Myles, Dean A.A.

    2010-11-19

    The locations of H atoms in biological structures can be difficult to determine using X-ray diffraction methods. Neutron diffraction offers a relatively greater scattering magnitude from H and D atoms. Here, 1.65 {angstrom} resolution neutron diffraction studies of fully perdeuterated and selectively CH{sub 3}-protonated perdeuterated crystals of Pyrococcus furiosus rubredoxin (D-rubredoxin and HD-rubredoxin, respectively) at room temperature (RT) are described, as well as 1.1 {angstrom} resolution X-ray diffraction studies of the same protein at both RT and 100 K. The two techniques are quantitatively compared in terms of their power to directly provide atomic positions for D atoms and analyze the role played by atomic thermal motion by computing the {sigma} level at the D-atom coordinate in simulated-annealing composite D-OMIT maps. It is shown that 1.65 {angstrom} resolution RT neutron data for perdeuterated rubredoxin are {approx}8 times more likely overall to provide high-confidence positions for D atoms than 1.1 {angstrom} resolution X-ray data at 100 K or RT. At or above the 1.0{sigma} level, the joint X-ray/neutron (XN) structures define 342/378 (90%) and 291/365 (80%) of the D-atom positions for D-rubredoxin and HD-rubredoxin, respectively. The X-ray-only 1.1 {angstrom} resolution 100 K structures determine only 19/388 (5%) and 8/388 (2%) of the D-atom positions above the 1.0{sigma} level for D-rubredoxin and HD-rubredoxin, respectively. Furthermore, the improved model obtained from joint XN refinement yielded improved electron-density maps, permitting the location of more D atoms than electron-density maps from models refined against X-ray data only.

  10. Mre11 ATLD17/18 mutation retains Tel1/ATM activity but blocks DNA double-strand break repair.

    PubMed

    Limbo, Oliver; Moiani, Davide; Kertokalio, Aryandi; Wyman, Claire; Tainer, John A; Russell, Paul

    2012-12-01

    The Mre11 complex (Mre11-Rad50-Nbs1 or MRN) binds double-strand breaks where it interacts with CtIP/Ctp1/Sae2 and ATM/Tel1 to preserve genome stability through its functions in homology-directed repair, checkpoint signaling and telomere maintenance. Here, we combine biochemical, structural and in vivo functional studies to uncover key properties of Mre11-W243R, a mutation identified in two pediatric cancer patients with enhanced ataxia telangiectasia-like disorder. Purified human Mre11-W243R retains nuclease and DNA binding activities in vitro. X-ray crystallography of Pyrococcus furiosus Mre11 indicates that an analogous mutation leaves the overall Mre11 three-dimensional structure and nuclease sites intact but disorders surface loops expected to regulate DNA and Rad50 interactions. The equivalent W248R allele in fission yeast allows Mre11 to form an MRN complex that efficiently binds double-strand breaks, activates Tel1/ATM and maintains telomeres; yet, it causes hypersensitivity to ionizing radiation and collapsed replication forks, increased Rad52 foci, defective Chk1 signaling and meiotic failure. W248R differs from other ataxia telangiectasia-like disorder analog alleles by the reduced stability of its interaction with Rad50 in cell lysates. Collective results suggest a separation-of-function mutation that disturbs interactions amongst the MRN subunits and Ctp1 required for DNA end processing in vivo but maintains interactions sufficient for Tel1/ATM checkpoint and telomere maintenance functions. PMID:23080121

  11. Comparative analysis of three hyperthermophilic GH1 and GH3 family members with industrial potential.

    PubMed

    Cota, Junio; Corrêa, Thamy L R; Damásio, André R L; Diogo, José A; Hoffmam, Zaira B; Garcia, Wanius; Oliveira, Leandro C; Prade, Rolf A; Squina, Fabio M

    2015-01-25

    Beta-glucosidases (BGLs) are enzymes of great potential for several industrial processes, since they catalyze the cleavage of glucosidic bonds in cellobiose and other short cellooligosaccharides. However, features such as good stability to temperature, pH, ions and chemicals are required characteristics for industrial applications. This work aimed to provide a comparative biochemical analysis of three thermostable BGLs from Pyrococcus furiosus and Thermotoga petrophila. The genes PfBgl1 (GH1 from P. furiosus), TpBgl1 (GH1 from T. petrophila) and TpBgl3 (GH3 from T. petrophila) were cloned and proteins were expressed in Escherichia coli. The purified enzymes are hyperthermophilic, showing highest activity at temperatures above 80°C at acidic (TpBgl3 and PfBgl1) and neutral (TpBgl1) pHs. The BGLs showed greatest stability to temperature mainly at pH 6.0. Activities using a set of different substrates suggested that TpBgl3 (GH3) is more specific than GH1 family members. In addition, the influence of six monosaccharides on BGL catalysis was assayed. While PfBgl1 and TpBgl3 seemed to be weakly inhibited by monosaccharides, TpBgl1 was activated, with xylose showing the strongest activation. Under the conditions tested, TpBgl1 showed the highest inhibition constant (Ki=1100.00mM) when compared with several BGLs previously characterized. The BGLs studied have potential for industrial use, specifically the enzymes belonging to the GH1 family, due to its broad substrate specificity and weak inhibition by glucose and other saccharides. PMID:25102284

  12. Anomalous behavior of water inside the SecY translocon

    PubMed Central

    Capponi, Sara; Heyden, Matthias; Bondar, Ana-Nicoleta; Tobias, Douglas J.; White, Stephen H.

    2015-01-01

    The heterotrimeric SecY translocon complex is required for the cotranslational assembly of membrane proteins in bacteria and archaea. The insertion of transmembrane (TM) segments during nascent-chain passage through the translocon is generally viewed as a simple partitioning process between the water-filled translocon and membrane lipid bilayer, suggesting that partitioning is driven by the hydrophobic effect. Indeed, the apparent free energy of partitioning of unnatural aliphatic amino acids on TM segments is proportional to accessible surface area, which is a hallmark of the hydrophobic effect [Öjemalm K, et al. (2011) Proc Natl Acad Sci USA 108(31):E359–E364]. However, the apparent partitioning solvation parameter is less than one-half the value expected for simple bulk partitioning, suggesting that the water in the translocon departs from bulk behavior. To examine the state of water in a SecY translocon complex embedded in a lipid bilayer, we carried out all-atom molecular-dynamics simulations of the Pyrococcus furiosus SecYE, which was determined to be in a “primed” open state [Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107(40):17182–17187]. Remarkably, SecYE remained in this state throughout our 450-ns simulation. Water molecules within SecY exhibited anomalous diffusion, had highly retarded rotational dynamics, and aligned their dipoles along the SecY transmembrane axis. The translocon is therefore not a simple water-filled pore, which raises the question of how anomalous water behavior affects the mechanism of translocon function and, more generally, the partitioning of hydrophobic molecules. Because large water-filled cavities are found in many membrane proteins, our findings may have broader implications. PMID:26139523

  13. The complete genome sequence of Staphylothermus marinus reveals differences in sulfur metabolism among heterotrophic Crenarchaeota

    SciTech Connect

    Anderson, iain J.; Dharmarajan, Lakshmi; Rodriguez, Jason; Hooper, Sean; Porat, Iris; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Sun, Hui; Land, Miriam; Lapidus, Alla; Lucas, Susan; Barry, Kerrie; Huber, Harald; Zhulin, Igor B.; Whitman, William B.; Mukhopadhyay, Biswarup; Woese, Carl; Bristow, James; Kyrpides, Nikos

    2008-09-05

    Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced - Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.

  14. Non-contact measurement technique for enzymatic reaction of glucokinase

    NASA Astrophysics Data System (ADS)

    Staforelli, Juan P.; Gallardo, María. J.; Meza, Pablo; Torres, Sergio; Mella, Héctor; Reyes, Claudio

    2014-03-01

    A non-contact infrared imaging-based measurement technique is applied to quantify the enzymatic reaction of glucokinase. The method is implemented by a long-wave (8-12 [μm]) infrared microbolometer imaging array and a germanium-based infrared optical vision system adjusted to the size of a small biological sample. The enzymatic reaction is carried out by the glucokinase enzyme, which is representative of the internal dynamics of the cell. Such reactions produce a spontaneous exothermal release of energy detected by the infrared imaging system as a non-contact measurement technique. It is shown by stoichiometry computations and infrared thermal resolution metrics that the infrared imaging system can detect the energy release at the [mK] range. This allows to quantify the spontaneity of the enzymatic reaction in a three dimensional (surface and time) single and noncontact real- time measurement. The camera is characterized for disclosing its sensibility, and the fixed pattern noise is compensated by a two point calibration method. On the other hand, the glucokinase enzyme is isolated from Pyrococcus furiosus. Therefore, the experiment is carried out by manual injection with graduated micropipettes using 40 [μl] of glucokinase at the surface of the substrate contained in an eppendorf tube. For recording, the infrared camera is adjusted in-focus at 25.4 [mm] from the superficial level of the substrate. The obtained values of energy release are 139 +/- 22 [mK] at room temperature and 274 +/- 22 [mK] for a bath temperature of 334 [K].

  15. Solution nuclear magnetic resonance structure of the GATase subunit and structural basis of the interaction between GATase and ATPPase subunits in a two-subunit-type GMPS from Methanocaldococcus jannaschii.

    PubMed

    Ali, Rustam; Kumar, Sanjeev; Balaram, Hemalatha; Sarma, Siddhartha P

    2013-06-25

    The solution structure of the monomeric glutamine amidotransferase (GATase) subunit of the Methanocaldococcus janaschii (Mj) guanosine monophosphate synthetase (GMPS) has been determined using high-resolution nuclear magnetic resonance methods. Gel filtration chromatography and ¹⁵N backbone relaxation studies have shown that the Mj GATase subunit is present in solution as a 21 kDa (188-residue) monomer. The ensemble of 20 lowest-energy structures showed root-mean-square deviations of 0.35 ± 0.06 Å for backbone atoms and 0.8 ± 0.06 Å for all heavy atoms. Furthermore, 99.4% of the backbone dihedral angles are present in the allowed region of the Ramachandran map, indicating the stereochemical quality of the structure. The core of the tertiary structure of the GATase is composed of a seven-stranded mixed β-sheet that is fenced by five α-helices. The Mj GATase is similar in structure to the Pyrococcus horikoshi (Ph) GATase subunit. Nuclear magnetic resonance (NMR) chemical shift perturbations and changes in line width were monitored to identify residues on GATase that were responsible for interaction with magnesium and the ATPPase subunit, respectively. These interaction studies showed that a common surface exists for the metal ion binding as well as for the protein-protein interaction. The dissociation constant for the GATase-Mg(2+) interaction has been found to be ∼1 mM, which implies that interaction is very weak and falls in the fast chemical exchange regime. The GATase-ATPPase interaction, on the other hand, falls in the intermediate chemical exchange regime on the NMR time scale. The implication of this interaction in terms of the regulation of the GATase activity of holo GMPS is discussed. PMID:23724776

  16. Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance.

    PubMed

    Matsumi, Rie; Manabe, Kenji; Fukui, Toshiaki; Atomi, Haruyuki; Imanaka, Tadayuki

    2007-04-01

    We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg(Tk)) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu(Tk)) or a gene cluster which includes apu(Tk) and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 microM simvastatin were isolated. The transformants exhibited growth in the presence of 20 microM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg(Tk) locus when the endogenous hmg(Tk) gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg(Pf)) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Deltaapu(Tk) strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu(Tk) is a major polysaccharide-degrading enzyme in T. kodakaraensis. PMID:17259314

  17. Comparative Analysis of Archaeal Lipid-linked Oligosaccharides That Serve as Oligosaccharide Donors for Asn Glycosylation.

    PubMed

    Taguchi, Yuya; Fujinami, Daisuke; Kohda, Daisuke

    2016-05-20

    The glycosylation of asparagine residues is the predominant protein modification in all three domains of life. An oligosaccharide chain is preassembled on a lipid-phospho carrier and transferred onto asparagine residues by the action of a membrane-bound enzyme, oligosaccharyltransferase. The oligosaccharide donor for the oligosaccharyl transfer reaction is dolichol-diphosphate-oligosaccharide in Eukaryota and polyprenol-diphosphate-oligosaccharide in Eubacteria. The donor in some archaeal species was reportedly dolichol-monophosphate-oligosaccharide. Thus, the difference in the number of phosphate groups aroused interest in whether the use of the dolichol-monophosphate type donors is widespread in the domain Archaea. Currently, all of the archaeal species with identified oligosaccharide donors have belonged to the phylum Euryarchaeota. Here, we analyzed the donor structures of two species belonging to the phylum Crenarchaeota, Pyrobaculum calidifontis and Sulfolobus solfataricus, in addition to two species from the Euryarchaeota, Pyrococcus furiosus and Archaeoglobus fulgidus The electrospray ionization tandem mass spectrometry analyses confirmed that the two euryarchaeal oligosaccharide donors were the dolichol-monophosphate type and newly revealed that the two crenarchaeal oligosaccharide donors were the dolichol-diphosphate type. This novel finding is consistent with the hypothesis that the ancestor of Eukaryota is rooted within the TACK (Thaum-, Aig-, Cren-, and Korarchaeota) superphylum, which includes Crenarchaea. Our comprehensive study also revealed that one archaeal species could contain two distinct oligosaccharide donors for the oligosaccharyl transfer reaction. The A. fulgidus cells contained two oligosaccharide donors bearing oligosaccharide moieties with different backbone structures, and the S. solfataricus cells contained two oligosaccharide donors bearing stereochemically different dolichol chains. PMID:27015803

  18. Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

    SciTech Connect

    Keller, MW; Schut, GJ; Lipscomb, GL; Menon, AL; Iwuchukwu, IJ; Leuko, TT; Thorgersen, MP; Nixon, WJ; Hawkins, AS; Kelly, RM; Adams, MWW

    2013-04-09

    Microorganisms can be engineered to produce useful. products, including chemicals and fuels from sugars derived from renewable feedstocks, such as plant biomass. An alternative method is to use low potential reducing power from nonbiomass sources, such as hydrogen gas or electricity, to reduce carbon dioxide directly into products. This approach circumvents the overall low efficiency of photosynthesis and the production of sugar intermediates. Although significant advances have been made in manipulating microorganisms to produce useful products from organic substrates, engineering them to use carbon dioxide and hydrogen gas has not been reported. Herein, we describe a unique temperature-dependent approach that confers on a microorganism (the archaeon Pyrococcus furiosus, which grows optimally on carbohydrates at 100 degrees C) the capacity to use carbon dioxide, a reaction that it does not accomplish naturally. This was achieved by the heterologous expression of five genes of the carbon fixation cycle of the archaeon Metallosphaera sedula, which grows autotrophically at 73 degrees C. The engineered P. furiosus strain is able to use hydrogen gas and incorporate carbon dioxide into 3-hydroxypropionic acid, one of the top 12 industrial chemical building blocks. The reaction can be accomplished by cell-free extracts and by whole cells of the recombinant P. furiosus strain. Moreover, it is carried out some 30 degrees C below the optimal growth temperature of the organism in conditions that support only minimal growth but maintain sufficient metabolic activity to sustain the production of 3-hydroxypropionate. The approach described here can be expanded to produce important organic chemicals, all through biological activation of carbon dioxide.

  19. A c Subunit with Four Transmembrane Helices and One Ion (Na+)-binding Site in an Archaeal ATP Synthase

    PubMed Central

    Mayer, Florian; Leone, Vanessa; Langer, Julian D.; Faraldo-Gómez, José D.; Müller, Volker

    2012-01-01

    The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling with DCCD as well as Na+-DCCD competition experiments revealed only one binding site for DCCD and Na+, indicating that the mature c subunit of this A1AO ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na+-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na+-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na+-specific under in vivo conditions, comparable with the Na+-dependent V1VO ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na+-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A1AO ATP synthases. PMID:23007388

  20. Use of a Tn5-based transposon system to create a cost-effective Zymomonas mobilis for ethanol production from lignocelluloses

    PubMed Central

    2013-01-01

    Background Current methods of ethanol production from lignocelluloses generate a mixture of sugars, primarily glucose and xylose; the fermentation cells are always exposed to stresses like high temperature and low nutritional conditions that affect their growth and productivity. Stress-tolerant strains capable of using both glucose and xylose to produce ethanol with high yield are highly desirable. Results A recombinant Zymomonas mobilis (Z. mobilis) designated as HYMX was constructed by integrating seven genes (Pfu-sHSP, yfdZ, metB, xylA, xylB, tktA and talB) into the genome of Z. mobilis CP4 (CP4) via Tn5 transposon in the present study. The small heat shock protein gene (Pfu-sHSP) from Pyrococcus furious (P. furious) was used to increase the heat-tolerance, the yfdZ and metB genes from E. coli were used to decrease the nutritional requirement. To overcome the bottleneck of CP4 being unable to use pentose, xylose catabolic genes (xylA, xylB, tktA and talB) from E. coli were integrated into CP4 also for construction of the xylose utilizing metabolic pathway. Conclusions The genomic integration confers on Z. mobilis the ability to grow in medium containing xylose as the only carbon source, and to grow in simple chemical defined medium without addition of amino acid. The HYMX demonstrated not only the high tolerance to unfavorable stresses like high temperature and low nutrient, but also the capability of converting both glucose and xylose to ethanol with high yield at high temperature. What’s more, these genetic characteristics were stable up to 100 generations on nonselective medium. Although significant improvements were achieved, yeast extract is needed for ethanol production. PMID:23635356

  1. Differential Interaction Kinetics of a Bipolar Structure-Specific Endonuclease with DNA Flaps Revealed by Single-Molecule Imaging

    PubMed Central

    Rezgui, Rachid; Lestini, Roxane; Kühn, Joëlle; Fave, Xenia; McLeod, Lauren; Myllykallio, Hannu; Alexandrou, Antigoni; Bouzigues, Cedric

    2014-01-01

    As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 5′ and 3′-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 5′ or 3′ extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases. PMID:25412080

  2. Autonomous Microbial Sampler (AMS), a device for the uncontaminated collection of multiple microbial samples from submarine hydrothermal vents and other aquatic environments

    NASA Astrophysics Data System (ADS)

    Taylor, Craig D.; Doherty, Kenneth W.; Molyneaux, Stephen J.; Morrison, Archie T.; Billings, John D.; Engstrom, Ivory B.; Pfitsch, Don W.; Honjo, Susumu

    2006-05-01

    An Autonomous Microbial Sampler (AMS) is described that will obtain uncontaminated and exogenous DNA-free microbial samples from most marine, freshwater and hydrothermal ecosystems. Sampling with the AMS may be conducted using manned submersibles, remotely operated vehicles (ROVs), autonomous underwater vehicles (AUVs), or when tethered to a hydrowire during hydrocast operations on research vessels. The modular device consists of a titanium nozzle for sampling in potentially hot environments (>350 °C) and fluid-handling components for the collection of six independent filtered or unfiltered samples. An onboard microcomputer permits sampling to be controlled by the investigator, by external devices (e.g., AUV computer), or by internal programming. Temperature, volume pumped and other parameters are recorded during sampling. Complete protection of samples from microbial contamination was observed in tests simulating deployment of the AMS in coastal seawater, where the sampling nozzle was exposed to seawater containing 1×10 6 cells ml -1 of a red pigmented tracer organism, Serratia marinorubra. Field testing of the AMS at a hydrothermal vent field was successfully undertaken in 2000. Results of DNA destruction studies have revealed that exposure of samples of the Eukaryote Euglena and the bacterium S. marinorubra to 0.5 N sulfuric acid at 23 °C for 1 h was sufficient to remove polymerase chain reaction (PCR) amplifiable DNA. Studies assessing the suitability of hydrogen peroxide as a sterilizing and DNA-destroying agent showed that 20% or 30% hydrogen peroxide sterilized samples of Serratia in 1 h and destroyed the DNA of Serratia in 3 h, but not 1 or 2 h. DNA AWAY™ killed Serratia and destroyed the DNA of both Serratia and the vent microbe (GB-D) of the genus Pyrococcus in 1 h.

  3. An extremely thermostable amylopullulanase from Staphylothermus marinus displays both pullulan- and cyclodextrin-degrading activities.

    PubMed

    Li, Xiaolei; Li, Dan; Park, Kwan-Hwa

    2013-06-01

    A gene encoding an amylopullulanase of the glycosyl hydrolase (GH) family 57 from Staphylothermus marinus (SMApu) was heterologously expressed in Escherichia coli. SMApu consisted of 639 amino acids with a molecular mass of 75.3 kDa. It only showed maximal amino acid identity of 17.1 % with that of Pyrococcus furiosus amylopullulanase in all identified amylases. Not like previously reported amylopullulanases, SMApu has no signal peptide but contains a continuous GH57N_Apu domain. It had the highest catalytic efficiency toward pullulan (k cat/K m , 342.34 s(-1) mL mg(-1)) and was extremely thermostable with maximal pullulan-degrading activity (42.1 U/mg) at 105 °C and pH 5.0 and a half-life of 50 min at 100 °C. Its activity increased to 116 % in the presence of 5 mM CaCl2. SMApu could also degrade cyclodextrins, which are resistant to the other amylopullulanases. The initial hydrolytic products from pullulan, γ-CD, and 6-O-maltooligosyl-β-CD were [6)-α-D-Glcp-(1 → 4)-α-D-Glcp-(1 → 4)-α-D-Glcp-(1→]n, maltooctaose, and single maltooligosaccharide plus β-CD, respectively. The final hydrolytic products from above-mentioned substrates were maltose and glucose. These results confirm that SMApu is a novel amylopullulanase of the family GH57 possessing the cyclodextrin-degrading activity of cyclomaltodextrinase. PMID:23001056

  4. Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications.

    PubMed

    Brown, J R; Doolittle, W F

    1995-03-28

    Universal trees based on sequences of single gene homologs cannot be rooted. Iwabe et al. [Iwabe, N., Kuma, K.-I., Hasegawa, M., Osawa, S. & Miyata, T. (1989) Proc. Natl. Acad. Sci. USA 86, 9355-9359] circumvented this problem by using ancient gene duplications that predated the last common ancestor of all living things. Their separate, reciprocally rooted gene trees for elongation factors and ATPase subunits showed Bacteria (eubacteria) as branching first from the universal tree with Archaea (archaebacteria) and Eucarya (eukaryotes) as sister groups. Given its topical importance to evolutionary biology and concerns about the appropriateness of the ATPase data set, an evaluation of the universal tree root using other ancient gene duplications is essential. In this study, we derive a rooting for the universal tree using aminoacyl-tRNA synthetase genes, an extensive multigene family whose divergence likely preceded that of prokaryotes and eukaryotes. An approximately 1600-bp conserved region was sequenced from the isoleucyl-tRNA synthetases of several species representing deep evolutionary branches of eukaryotes (Nosema locustae), Bacteria (Aquifex pyrophilus and Thermotoga maritima) and Archaea (Pyrococcus furiosus and Sulfolobus acidocaldarius). In addition, a new valyl-tRNA synthetase was characterized from the protist Trichomonas vaginalis. Different phylogenetic methods were used to generate trees of isoleucyl-tRNA synthetases rooted by valyl- and leucyl-tRNA synthetases. All isoleucyl-tRNA synthetase trees showed Archaea and Eucarya as sister groups, providing strong confirmation for the universal tree rooting reported by Iwabe et al. As well, there was strong support for the monophyly (sensu Hennig) of Archaea. The valyl-tRNA synthetase gene from Tr. vaginalis clustered with other eukaryotic ValRS genes, which may have been transferred from the mitochondrial genome to the nuclear genome, suggesting that this amitochondrial trichomonad once harbored an

  5. Fundamental Studies of Recombinant Hydrogenases

    SciTech Connect

    Adams, Michael W

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  6. Modeling of flap endonuclease interactions with DNA substrate.

    PubMed

    Allawi, Hatim T; Kaiser, Michael W; Onufriev, Alexey V; Ma, Wu-Po; Brogaard, Andrew E; Case, David A; Neri, Bruce P; Lyamichev, Victor I

    2003-05-01

    Structure-specific 5' nucleases play an important role in DNA replication and repair uniquely recognizing an overlap flap DNA substrate and processing it into a DNA nick. However, in the absence of a high-resolution structure of the enzyme/DNA complex, the mechanism underlying this recognition and substrate specificity, which is key to the enzyme's function, remains unclear. Here, we propose a three-dimensional model of the structure-specific 5' flap endonuclease from Pyrococcus furiosus in its complex with DNA. The model is based on the known X-ray structure of the enzyme and a variety of biochemical and molecular dynamics (MD) data utilized in the form of distance restraints between the enzyme and the DNA. Contacts between the 5' flap endonuclease and the sugar-phosphate backbone of the overlap flap substrate were identified using enzyme activity assays on substrates with methylphosphonate or 2'-O-methyl substitutions. The enzyme footprint extends two to four base-pairs upstream and eight to nine base-pairs downstream of the cleavage site, thus covering 10-13 base-pairs of duplex DNA. The footprint data are consistent with a model in which the substrate is bound in the DNA-binding groove such that the downstream duplex interacts with the helix-hairpin-helix motif of the enzyme. MD simulations to identify the substrate orientation in this model are consistent with the results of the enzyme activity assays on the methylphosphonate and 2'-O-methyl-modified substrates. To further refine the model, 5' flap endonuclease variants with alanine point substitutions at amino acid residues expected to contact phosphates in the substrate and one deletion mutant were tested in enzyme activity assays on the methylphosphonate-modified substrates. Changes in the enzyme footprint observed for two point mutants, R64A and R94A, and for the deletion mutant in the enzyme's beta(A)/beta(B) region, were interpreted as being the result of specific interactions in the enzyme/DNA complex

  7. A neutron crystallographic analysis of a rubredoxin mutant at 1.6 A resolution.

    PubMed

    Chatake, Toshiyuki; Kurihara, Kazuo; Tanaka, Ichiro; Tsyba, Irina; Bau, Robert; Jenney, Francis E; Adams, Michael W W; Niimura, Nobuo

    2004-08-01

    A neutron diffraction study has been carried out at 1.6 A resolution on a mutant rubredoxin from Pyrococcus furiosus using the BIX-3 single-crystal diffractometer at the JRR-3 reactor of the Japan Atomic Energy Research Institute. In order to study the unusual thermostability of rubredoxin from P. furiosus (an organism that grows optimally at 373 K), the hydrogen-bonding patterns were compared between the wild-type protein and a 'triple-mutant' variant. In this mutant protein, three residues were changed (Trp3-->Tyr3, Ile23-->Val23, Leu32-->Ile32) so that they are identical to those in a mesophilic rubredoxin from Clostridium pasteurianum. In the present study, some minor changes were found between the wild-type and mutant proteins in the hydrogen-bonding patterns of the Trp3/Tyr3 region. In this investigation, the H/D-exchange ratios in the protein were also studied. Because the target protein was soaked in D2O during the crystallization procedure, most of the N-H and O-H bonds have become deuterated, while essentially all of the C-H bonds have not. In particular, the H/D-exchange pattern of the N-H amide bonds of the protein backbone is of interest because it may contain some indirect information about the mechanism of unfolding of this small protein. The results are in broad agreement with those from solution NMR studies, which suggest that the backbone amide bonds near the four Cys residues of the FeS4 redox center are most resistant to H/D exchange. Finally, the detailed geometries of the water molecules of hydration around the rubredoxin molecule are also reported. The 1.6 A resolution of the present neutron structure determination has revealed a more detailed picture than previously available of some portions of the water structure, including ordered and disordered O-D bonds. Crystallographic details: space group P2(1)2(1)2(1) (orthorhombic), unit-cell parameters a = 34.48, b = 35.70, c = 43.16 A; final agreement factors R = 0.196 and Rfree = 0.230 for 19

  8. Marine Subsurface Microbial Communities Across a Hydrothermal Gradient in Okinawa Trough Sediments

    NASA Astrophysics Data System (ADS)

    Brandt, L. D.; Hser Wah Saw, J.; Ettema, T.; House, C. H.

    2015-12-01

    IODP Expedition 331 to the Okinawa backarc basin provided an opportunity to study the microbial stratigraphy within the sediments surrounding a hydrothermal vent. The Okinawa backarc basin is a sedimented region of the seafloor located on a continental margin, and also hosts a hydrothermal network within the subsurface. Site C0014 within the Iheya North hydrothermal field is located 450 m east of the active vent and has a surface temperature of 5°C with no evidence of hydrothermal alteration within the top 10 meters below sea floor (mbsf). Temperature increases with depth at an estimated rate of 3°C/m and transitions from non-hydrothermal margin sediments to a hydrothermally altered regime below 10 mbsf. In this study, we utilized deep 16S rRNA sequencing of DNA from IODP Expedition 331 Site C0014 sediment horizons in order to assess diversity throughout the sediment column as well as determine the potential limits of the biosphere. Analysis of the amplicon data shows a shift over 15 mbsf from a heterogeneous community of cosmopolitan marine subsurface taxa toward an archaeal-dominated community in the deepest horizons of the predicted biosphere. Notably, the phylum Chloroflexi represents a substantial taxon through most horizons, where it appears to be replaced below 10 mbsf by punctuations of thermophilic and methanotrophic Archaea and Miscellaneous Crenarchaeotic Group abundances. DNA from the aforementioned transition horizons was further analyzed using metagenomic sequencing. Preliminary taxonomic analysis of the metagenomic data agrees well with amplicon data in capturing the shift in relative abundance of Archaea increasing with depth. Additionally, reverse gyrase, a gene found exclusively in hyperthermophilic microorganisms, was recovered only in the metagenome of the deepest horizon. A BLAST search of this protein sequence against the GenBank non-redudnant protein database produced top hits with reverse gyrase from Thermococcus and Pyrococcus, which are

  9. Modeling the Growth of Hyperthermophiles in Deep-sea Hydrothermal Diffuse Fluids and Sulfide Deposits

    NASA Astrophysics Data System (ADS)

    Ver Eecke, H. C.; Oslowski, D. M.; Butterfield, D. A.; Olson, E. J.; Lilley, M. D.; Holden, J. F.

    2009-12-01

    In 2008 and 2009, 534 hydrothermal fluid samples and 5 actively-venting black smoker chimneys were collected using Alvin for correlative microbiological and chemical analyses as part of the Endeavour Segment and Axial Volcano Geochemistry and Ecology Research (EAGER) program. Hyperthermophilic, autotrophic Fe(III) oxide reducers, methanogens, and sulfur-reducing heterotrophs were enriched for at 85 and 95°C using most-probable-number estimates from 28 diffuse fluid and 8 chimney samples. Heterotrophs were the most abundant of the three groups in both diffuse fluids and black-smoker chimneys. Iron reducers were more abundant than methanogens, and more abundant in sulfide-hosted vents than in basalt-hosted vents. Fluid chemistry suggests that there is net biogenic methanogenesis at the Marker 113/62 diffuse vent at Axial Volcano but nowhere else sampled. The growth of hyperthermophilic methanogens and heterotrophs was modeled in the lab using pure cultures. Methanocaldococcus jannaschii grew at 82°C in a 2-liter reactor with continuous gas flow at H2 concentrations between 20 and 225 µM with a H2 km of 100 µM. Correlating H2 end-member mixing curves from vent fluids and seawater with our laboratory modeling study suggests that H2 concentrations are limiting for Methanocaldococcus growth at most Mothra, Main Field, and High Rise vent sites at Endeavour but sufficient to support growth at some Axial Volcano vents. Therefore, hyperthermophilic methanogens may depend on H2 syntrophy at low H2 sites. Twenty-one pure hyperthermophilic heterotroph strains each grew on α-1,4 and β-1,4 linked sugars and polypeptides with concomitant H2 production. The H2 production rate (cell-1 doubling-1) for Pyrococcus furiosus at 95°C without sulfur was 29 fmol, 36 fmol, and 53 fmol for growth on α-1,4 sugars, β-1,4 sugars, and peptides, respectively. The CH4 production rate for M. jannaschii was 390 fmol cell-1 doubling-1; therefore, we estimate that it would take approximately

  10. Microbial Geochemistry in Shallow-Sea Hydrothermal Systems

    NASA Astrophysics Data System (ADS)

    Amend, J. P.; Pichler, T.

    2006-12-01

    Shallow-sea hydrothermal systems are far more ubiquitous than generally recognized. Approximately 50-60 systems are currently known, occurring world-wide in areas of high heat flow, such as, volcanic island arcs, near-surface mid-ocean ridges, and intraplate oceanic volcanoes. In contrast to deep-sea systems, shallow- sea vent fluids generally include a meteoric component, they experience phase separation near the sediment- water interface, and they discharge into the photic zone (<200 m). They also are characterized by wide ranges in chemical composition, hundreds of redox disequilibria that translate to potential metabolisms, and broad phylogenetic diversity among the thermophilic bacteria and archaea. Perhaps because deep-sea smokers and continental hot springs are visually more stunning, shallow-sea systems are often overlooked study sites. We will discuss their particular features that afford unique opportunities in microbial geochemistry. Two of the better studied examples are at Vulcano Island (Italy) and Ambitle Island (Papua New Guinea). The vents and sediment seeps at Vulcano are the "type locality" for numerous cultured hyperthermophiles, including the bacteria Aquifex and Thermotoga, the crenarchaeon Pyrodictium, and the Euryarchaeota Archaeoglobus and Pyrococcus. Isotope-labeled incubation experiments of heated sediments and an array of culturing studies have shown that simple organic compounds are predominantly fermented or anaerobically respired with sulfate. 16S rRNA gene surveys, together with fluorescent in situ hybridization studies, demonstrated the dominance of key thermophilic bacteria and archaea (e.g., Aquificales, Thermotogales, Thermococcales, Archaeoglobales) in the sediments and the presence of a broad spectrum of mostly uncultured crenarchaeota in several vent waters, sediment samples, and geothermal wells. Thermodynamic modeling quantified potential energy yields from aerobic and anaerobic respiration reactions and fermentation

  11. Linking genome content to biofuel production yields: a meta-analysis of major catabolic pathways among select H2 and ethanol-producing bacteria

    PubMed Central

    2012-01-01

    Background Fermentative bacteria offer the potential to convert lignocellulosic waste-streams into biofuels such as hydrogen (H2) and ethanol. Current fermentative H2 and ethanol yields, however, are below theoretical maxima, vary greatly among organisms, and depend on the extent of metabolic pathways utilized. For fermentative H2 and/or ethanol production to become practical, biofuel yields must be increased. We performed a comparative meta-analysis of (i) reported end-product yields, and (ii) genes encoding pyruvate metabolism and end-product synthesis pathways to identify suitable biomarkers for screening a microorganism’s potential of H2 and/or ethanol production, and to identify targets for metabolic engineering to improve biofuel yields. Our interest in H2 and/or ethanol optimization restricted our meta-analysis to organisms with sequenced genomes and limited branched end-product pathways. These included members of the Firmicutes, Euryarchaeota, and Thermotogae. Results Bioinformatic analysis revealed that the absence of genes encoding acetaldehyde dehydrogenase and bifunctional acetaldehyde/alcohol dehydrogenase (AdhE) in Caldicellulosiruptor, Thermococcus, Pyrococcus, and Thermotoga species coincide with high H2 yields and low ethanol production. Organisms containing genes (or activities) for both ethanol and H2 synthesis pathways (i.e. Caldanaerobacter subterraneus subsp. tengcongensis, Ethanoligenens harbinense, and Clostridium species) had relatively uniform mixed product patterns. The absence of hydrogenases in Geobacillus and Bacillus species did not confer high ethanol production, but rather high lactate production. Only Thermoanaerobacter pseudethanolicus produced relatively high ethanol and low H2 yields. This may be attributed to the presence of genes encoding proteins that promote NADH production. Lactate dehydrogenase and pyruvate:formate lyase are not conducive for ethanol and/or H2 production. While the type(s) of encoded hydrogenases appear

  12. Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N-acyl-D-amino acid amidohydrolase responsible for D-amino acid production.

    PubMed

    Lin, Pei-Hsun; Su, Shiun-Cheng; Tsai, Ying-Chieh; Lee, Chia-Yin

    2002-10-01

    An N-acyl-d-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-d-methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN-D-AAase showed significant amino acid similarity to the N-acyl-d-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44-56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N-D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U.mg-1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 degrees C, respectively. After 30 min heat treatment at 45 degrees C, between pH 6 and pH 8, 80% activity remained. The N-D-AAase had higher hydrolysing activity against N-acetyl-d-amino acid derivates containing d-methionine, d-leucine and d-alanine and against N-chloroacetyl-d-phenylalanine. Importantly, the enzyme does not act on the N-acetyl-l-amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N-D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme. PMID:12354118

  13. Hydrogen Production and Enzyme Activities in the Hyperthermophile Thermococcus paralvinellae Grown on Maltose, Tryptone, and Agricultural Waste.

    PubMed

    Hensley, Sarah A; Moreira, Emily; Holden, James F

    2016-01-01

    Thermococcus may be an important alternative source of H2 in the hot subseafloor in otherwise low H2 environments such as some hydrothermal vents and oil reservoirs. It may also be useful in industry for rapid agricultural waste treatment and concomitant H2 production. Thermococcus paralvinellae grown at 82°C without sulfur produced up to 5 mmol of H2 L(-1) at rates of 5-36 fmol H2 cell(-1) h(-1) on 0.5% (wt vol(-1)) maltose, 0.5% (wt vol(-1)) tryptone, and 0.5% maltose + 0.05% tryptone media. Two potentially inhibiting conditions, the presence of 10 mM acetate and low pH (pH 5) in maltose-only medium, did not significantly affect growth or H2 production. Growth rates, H2 production rates, and cell yields based on H2 production were the same as those for Pyrococcus furiosus grown at 95°C on the same media for comparison. Acetate, butyrate, succinate, isovalerate, and formate were also detected as end products. After 100 h, T. paralvinellae produced up to 5 mmol of H2 L(-1) of medium when grown on up to 70% (vol vol(-1)) waste milk from cows undergoing treatment for mastitis with the bacterial antibiotic Ceftiofur and from untreated cows. The amount of H2 produced by T. paralvinellae increased with increasing waste concentrations, but decreased in P. furiosus cultures supplemented with waste milk above 1% concentration. All mesophilic bacteria from the waste milk that grew on Luria Bertani, Sheep's Blood (selective for Staphylococcus, the typical cause of mastitis), and MacConkey (selective for Gram-negative enteric bacteria) agar plates were killed by heat during incubation at 82°C. Ceftiofur, which is heat labile, was below the detection limit following incubation at 82°C. T. paralvinellae also produced up to 6 mmol of H2 L(-1) of medium when grown on 0.1-10% (wt vol(-1)) spent brewery grain while P. furiosus produced < 1 mmol of H2 L(-1). Twelve of 13 enzyme activities in T. paralvinellae showed significant (p < 0.05) differences across six different growth

  14. Sulfur K-Edge X-Ray Absorption Spectroscopy And Density Functional Theory Calculations on Superoxide Reductase: Role of the Axial Thiolate in Reactivity

    SciTech Connect

    Dey, A.; Jenney, F.E.; Jr.; Adams, M.W.W.; Johnson, M.K.; Hodgson, K.O.; Hedman, B.; Solomon, E.I.

    2009-06-02

    Superoxide reductase (SOR) is a non-heme iron enzyme that reduces superoxide to peroxide at a diffusion-controlled rate. Sulfur K-edge X-ray absorption spectroscopy (XAS) is used to investigate the ground-state electronic structure of the resting high-spin and CN- bound low-spin FeIII forms of the 1Fe SOR from Pyrococcus furiosus. A computational model with constrained imidazole rings (necessary for reproducing spin states), H-bonding interaction to the thiolate (necessary for reproducing Fe-S bond covalency of the high-spin and low-spin forms), and H-bonding to the exchangeable axial ligand (necessary to reproduce the ground state of the low-spin form) was developed and then used to investigate the enzymatic reaction mechanism. Reaction of the resting ferrous site with superoxide and protonation leading to a high-spin FeIII-OOH species and its subsequent protonation resulting in H2O2 release is calculated to be the most energetically favorable reaction pathway. Our results suggest that the thiolate acts as a covalent anionic ligand. Replacing the thiolate with a neutral noncovalent ligand makes protonation very endothermic and greatly raises the reduction potential. The covalent nature of the thiolate weakens the FeIII bond to the proximal oxygen of this hydroperoxo species, which raises its pKa by an additional 5 log units relative to the pKa of a primarily anionic ligand, facilitating its protonation. A comparison with cytochrome P450 indicates that the stronger equatorial ligand field from the porphyrin results in a low-spin FeIII-OOH species that would not be capable of efficient H2O2 release due to a spin-crossing barrier associated with formation of a high-spin 5C FeIII product. Additionally, the presence of the dianionic porphyrin pi ring in cytochrome P450 allows O-O heterolysis, forming an FeIV-oxo porphyrin radical species, which is calculated to be extremely unfavorable for the non-heme SOR ligand environment. Finally, the 5C FeIII site that results

  15. Crystal structure of a subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis: requirement of a C-terminal beta-jelly roll domain for hyperstability.

    PubMed

    Foophow, Tita; Tanaka, Shun-ichi; Angkawidjaja, Clement; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2010-07-23

    Tk-SP is a hyperthermostable subtilisin-like serine protease from Thermococcus kodakaraensis and is autoprocessed from its precursor (Pro-Tk-SP) with N- and C-propeptides. The crystal structure of the active-site mutant of Pro-Tk-SP lacking C-propeptide, ProN-Tk-S359A, was determined at 2.0 A resolution. ProN-Tk-S359A consists of the N-propeptide, subtilisin, and beta-jelly roll domains. Two Ca(2+) ions bind to the beta-jelly roll domain. The overall structure of ProN-Tk-S359A without the beta-jelly roll domain is similar to that of the bacterial propeptide:subtilisin complex, except that it does not contain Ca(2+) ions. To analyze the role of the beta-jelly roll domain of Tk-SP, we constructed a series of the active-site mutants of Tk-SP with (Tk-S359A/C) and without (Tk-S359A/CDeltaJ) beta-jelly roll domain. Both Tk-S359C and Tk-S359CDeltaJ exhibited protease activities in gel assay, indicating that the beta-jelly roll domain is not required for folding or activity. However, the T(m) value of Tk-S359ADeltaJ determined by far-UV CD spectroscopy in the presence of 10-mM CaCl(2) was lower than that of Tk-S359A by 29.4 degrees C. The T(m) value of Tk-S359A was decreased by 29.5 degrees C by the treatment with 10 mM ethylenediaminetetraacetic acid, indicating that the beta-jelly roll domain contributes to the stabilization of Tk-S359A only in a Ca(2+)-bound form. Tk-SP highly resembles subtilisin-like serine proteases from Pyrococcus furiosus, Thermococcus gammatolerans, and Thermococcus onnurineus in size and amino acid sequence. We propose that attachment of a beta-jelly roll domain to the C-terminus is one of the strategies of the proteins from hyperthermophiles to adapt to high-temperature environment. PMID:20595040

  16. Comparative Study of Two Box H/ACA Ribonucleoprotein Pseudouridine-Synthases: Relation between Conformational Dynamics of the Guide RNA, Enzyme Assembly and Activity

    PubMed Central

    Leclerc, Fabrice; Branlant, Christiane; Charpentier, Bruno

    2013-01-01

    Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10–L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21. PMID:23922977

  17. Hydrogen Production and Enzyme Activities in the Hyperthermophile Thermococcus paralvinellae Grown on Maltose, Tryptone, and Agricultural Waste

    PubMed Central

    Hensley, Sarah A.; Moreira, Emily; Holden, James F.

    2016-01-01

    Thermococcus may be an important alternative source of H2 in the hot subseafloor in otherwise low H2 environments such as some hydrothermal vents and oil reservoirs. It may also be useful in industry for rapid agricultural waste treatment and concomitant H2 production. Thermococcus paralvinellae grown at 82°C without sulfur produced up to 5 mmol of H2 L−1 at rates of 5–36 fmol H2 cell−1 h−1 on 0.5% (wt vol−1) maltose, 0.5% (wt vol−1) tryptone, and 0.5% maltose + 0.05% tryptone media. Two potentially inhibiting conditions, the presence of 10 mM acetate and low pH (pH 5) in maltose-only medium, did not significantly affect growth or H2 production. Growth rates, H2 production rates, and cell yields based on H2 production were the same as those for Pyrococcus furiosus grown at 95°C on the same media for comparison. Acetate, butyrate, succinate, isovalerate, and formate were also detected as end products. After 100 h, T. paralvinellae produced up to 5 mmol of H2 L−1 of medium when grown on up to 70% (vol vol−1) waste milk from cows undergoing treatment for mastitis with the bacterial antibiotic Ceftiofur and from untreated cows. The amount of H2 produced by T. paralvinellae increased with increasing waste concentrations, but decreased in P. furiosus cultures supplemented with waste milk above 1% concentration. All mesophilic bacteria from the waste milk that grew on Luria Bertani, Sheep's Blood (selective for Staphylococcus, the typical cause of mastitis), and MacConkey (selective for Gram-negative enteric bacteria) agar plates were killed by heat during incubation at 82°C. Ceftiofur, which is heat labile, was below the detection limit following incubation at 82°C. T. paralvinellae also produced up to 6 mmol of H2 L−1 of medium when grown on 0.1–10% (wt vol−1) spent brewery grain while P. furiosus produced < 1 mmol of H2 L−1. Twelve of 13 enzyme activities in T. paralvinellae showed significant (p < 0.05) differences across six different

  18. Community Response to a Heavy Precipitation Event in High Temperature, Chemosynthetic Biofilms and Sediments

    NASA Astrophysics Data System (ADS)

    Meyer-Dombard, D. R.; Loiacono, S. T.; Shock, E.

    2012-12-01

    Coordinated analysis of the "Bison Pool" (BP) Environmental Genome and a complementary contextual geochemical dataset of ~75 parameters revealed biogeochemical cycling and metabolic and microbial community shifts in a Yellowstone National Park hot spring ecosystem (1). The >22m outflow of BP is a gradient of decreasing temperature, increasing dissolved oxygen, and changing availability of nutrients. Microbial life at BP transitions from a 92°C chemosynthetic community in the BP source pool to a 56°C photosynthetic mat community. Metagenomic data at BP showed the potential for both heterotrophic and autotrophic carbon metabolism (rTCA and acetyl-CoA cycles) in the highest temperature, chemosynthetic regions (1). This region of the outflow is dominated by Aquificales and Pyrococcus relatives, with smaller contributions of heterotrophic Bacteria. Following a 2h heavy precipitation event we observed an influx of exogenous organic material into the source pool supplied from the meadow surrounding the BP area. We sampled biomass and fluid at several locations within the outflow immediately following the event, and on several occasions for the next eight days. Elemental analysis and carbon and nitrogen isotopic analyses were conducted on biomass and sediment, and dissolved organic and inorganic carbon content and δ13C of fluids were analyzed. DNA and RNA were extracted, and following RT-PCR, nitrogen cycle functional gene expression was evaluated. Previous work at BP has shown that chemosynthetic biomass may carry isotopic signatures of fractionation during carbon fixation, via the acetyl-CoA and rTCA cycles (2). However, the addition of exogenous organic carbon during the rain event had an immediate and dramatic effect on the sediments and biofilms in the chemosynthetic zone of the outflow. Dissolved organic carbon was the highest measured in six years. Chemosynthetic biomass responded by incorporating the organic carbon. Carbon isotopic signatures in chemosynthetic

  19. Sulfur K-Edge X-Ray Absorption Spectroscopy And Density Functional Theory Calculations on Superoxide Reduc Tase: Role of the Axial Thiolate in Reactivity

    SciTech Connect

    Dey, A.; Jenney, F.E., Jr.; Adams, M.W.; Johnson, M.K.; Hodgson, K.O.; Hedman, B.; Solomon, E.I.; /Stanford U., Chem. Dept. /Athens U. /SLAC, SSRL

    2007-10-26

    Superoxide reductase (SOR) is a non-heme iron enzyme that reduces superoxide to peroxide at a diffusion-controlled rate. Sulfur K-edge X-ray absorption spectroscopy (XAS) is used to investigate the ground-state electronic structure of the resting high-spin and CN{sup -} bound low-spin Fe{sup III} forms of the 1Fe SOR from Pyrococcus furiosus. A computational model with constrained imidazole rings (necessary for reproducing spin states), H-bonding interaction to the thiolate (necessary for reproducing Fe-S bond covalency of the high-spin and low-spin forms), and H-bonding to the exchangeable axial ligand (necessary to reproduce the ground state of the low-spin form) was developed and then used to investigate the enzymatic reaction mechanism. Reaction of the resting ferrous site with superoxide and protonation leading to a high-spin Fe{sup III}-OOH species and its subsequent protonation resulting in H2O2 release is calculated to be the most energetically favorable reaction pathway. Our results suggest that the thiolate acts as a covalent anionic ligand. Replacing the thiolate with a neutral noncovalent ligand makes protonation very endothermic and greatly raises the reduction potential. The covalent nature of the thiolate weakens the Fe{sup III} bond to the proximal oxygen of this hydroperoxo species, which raises its pKa by an additional 5 log units relative to the pK{sub a} of a primarily anionic ligand, facilitating its protonation. A comparison with cytochrome P450 indicates that the stronger equatorial ligand field from the porphyrin results in a low-spin Fe{sup III}-OOH species that would not be capable of efficient H2O2 release due to a spin-crossing barrier associated with formation of a high-spin 5C Fe{sup III} product. Additionally, the presence of the dianionic porphyrin {pi} ring in cytochrome P450 allows O-O heterolysis, forming an Fe{sup IV}-oxo porphyrin radical species, which is calculated to be extremely unfavorable for the non-heme SOR ligand

  20. Extreme Tolerance to Elevated Pressure in a Thermococcus isolate from the Mid-Cayman Rise

    NASA Astrophysics Data System (ADS)

    Narasingarao, P.; Huber, J. A.; Schrenk, M. O.; Bartlett, D.

    2013-12-01

    Hydrothermal systems are windows into the deep biosphere. Venting fluids with temperatures up to 400°C containing gases such as H2, CO2, H2S and CH4 provide an oasis of life in the deep ocean primarily based on chemosynthesis. The Mid-Cayman Rise (MCR) includes the deepest hydrothermal vent system known thus far, and is characterized by two venting sites Piccard (4950m) and Von Damm (2350m). Here we demonstrate the remarkable high pressure tolerance limits of a Thermococcus sp. designated strain 175, isolated from samples collected from Piccard during an expedition in 2012. Diffuse venting fluids collected at the site resulted in the isolation of several Thermococcus strains capable of growth in basal salts medium supplemented with H2/CO2 and yeast extract, along with sulfur as an electron acceptor. Given the importance of pressure as an environmental parameter influencing evolution and adaptation of deep-sea life, the pressure tolerance of Thermococcus strain 175 was tested. High pressure incubations were originally conducted in serum vials filled completely with growth medium and therefore lacking all headspace gas. To test for growth with H2/CO2 , modified hungate tubes with a piston mechanism were used (Bowles et al. 2011) . The results indicate that strain 175 can grow at 90°C up to 120 megapascal (MPa). Growth rates are comparable when the strain is grown at atmospheric pressure or at 120 MPa pressure. Morphologically, the strain is irregular cocci and does not show any changes in its cellular structure when switched between atmospheric pressure and elevated pressure. This wide range of pressure tolerance has not been previously observed in other microorganisms, including Pyrococcus yayanosii CH1 (Zeng et al., 2009) which is also capable of growth at 120MPa but does not grow below 15 MPa. Thermococcus strain 175 represents an excellent model system to study high pressure adaptation due to its high growth rate and broad range of growth pressures. The

  1. Molecular Assemblies, Genes and Genomics Integrated Efficiently (MAGGIE)

    SciTech Connect

    Baliga, Nitin S

    2011-05-26

    Final report on MAGGIE. We set ambitious goals to model the functions of individual organisms and their community from molecular to systems scale. These scientific goals are driving the development of sophisticated algorithms to analyze large amounts of experimental measurements made using high throughput technologies to explain and predict how the environment influences biological function at multiple scales and how the microbial systems in turn modify the environment. By experimentally evaluating predictions made using these models we will test the degree to which our quantitative multiscale understanding wilt help to rationally steer individual microbes and their communities towards specific tasks. Towards this end we have made substantial progress towards understanding evolution of gene families, transcriptional structures, detailed structures of keystone molecular assemblies (proteins and complexes), protein interactions, biological networks, microbial interactions, and community structure. Using comparative analysis we have tracked the evolutionary history of gene functions to understand how novel functions evolve. One level up, we have used proteomics data, high-resolution genome tiling microarrays, and 5' RNA sequencing to revise genome annotations, discover new genes including ncRNAs, and map dynamically changing operon structures of five model organisms: For Desulfovibrio vulgaris Hildenborough, Pyrococcus furiosis, Sulfolobus solfataricus, Methanococcus maripaludis and Haiobacterium salinarum NROL We have developed machine learning algorithms to accurately identify protein interactions at a near-zero false positive rate from noisy data generated using tagfess complex purification, TAP purification, and analysis of membrane complexes. Combining other genome-scale datasets produced by ENIGMA (in particular, microarray data) and available from literature we have been able to achieve a true positive rate as high as 65% at almost zero false positives when