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Sample records for pyrococcus horikoshii tryptophanyl-trna

  1. Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-Å resolution

    PubMed Central

    Du, Xinlin; Choi, In-Geol; Kim, Rosalind; Wang, Weiru; Jancarik, Jaru; Yokota, Hisao; Kim, Sung-Hou

    2000-01-01

    The intracellular protease from Pyrococcus horikoshii (PH1704) and PfpI from Pyrococcus furiosus are members of a class of intracellular proteases that have no sequence homology to any other known protease family. We report the crystal structure of PH1704 at 2.0-Å resolution. The protease is tentatively identified as a cysteine protease based on the presence of cysteine (residue 100) in a nucleophile elbow motif. In the crystal, PH1704 forms a hexameric ring structure, and the active sites are formed at the interfaces between three pairs of monomers. PMID:11114201

  2. Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough.

    PubMed

    González, J M; Masuchi, Y; Robb, F T; Ammerman, J W; Maeder, D L; Yanagibayashi, M; Tamaoka, J; Kato, C

    1998-05-01

    A hyperthermophilic, anaerobic archaeon was isolated from hydrothermal fluid samples obtained at the Okinawa Trough vents in the NE Pacific Ocean, at a depth of 1395m. The strain is obligately heterotrophic, and utilizes complex proteinaceous media (peptone, tryptone, or yeast extract), or a 21-amino-acid mixture supplemented with vitamins, as growth substrates. Sulfur greatly enhances growth. The cells are irregular cocci with a tuft of flagella, growing optimally at 98 degrees C (maximum growth temperature 102 degrees C), but capable of prolonged survival at 105 degrees C. Optimum growth was at pH 7 (range 5-8) and NaCl concentration 2.4% (range 1%-5%). Tryptophan was required for growth, in contrast to the closely related strains Pyrococcus furiosus and P. abyssi. Thin sections of the cell, viewed by transmission electron microscopy, revealed a periplasmic space similar in appearance to the envelope of P. furiosus. The predominant cell membrane component was tetraether lipid, with minor amounts of diether lipids. Treatment of the cells by mild osmotic shock released an extract that contained a Zn(2+)-dependent alkaline phosphatase. Phylogenetic analysis of the sequences encoding 16S rRNA and glutamate dehydrogenase places the isolate with certainty within the genus Pyrococcus although there is relatively low DNA-DNA hybridization (< 63%) with described species of this genus. Based on the reported results, we propose a new species, to be named Pyrococcus horikoshii sp.nov.

  3. 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.

  4. Cloning, purification, crystallization and preliminary crystallographic analysis of acylphosphatase from Pyrococcus horikoshii OT3.

    PubMed

    Miyazono, Ken Ichi; Kudo, Norio; Tanokura, Masaru

    2004-06-01

    Acylphosphatase is one of the smallest enzymes and catalyzes the hydrolysis of the carboxy-phosphate bond. An extremely thermostable acylphosphatase from a hyperthermophilic archaea, Pyrococcus horikoshii OT3, has been cloned, expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with potassium/sodium tartrate as the precipitant at pH 5.5. X-ray diffraction data have been collected to a highest resolution of 1.72 angstroms on a synchrotron-radiation source. The crystals belong to space group P3(2)21, with approximate unit-cell parameters a = b = 86.6, c = 75.4 angstroms and two monomers in the asymmetric unit.

  5. Crystal Structural and Functional Analysis of the Putative Dipeptidase from Pyrococcus horikoshii OT3.

    PubMed

    Jeyakanthan, Jeyaraman; Takada, Katsumi; Sawano, Masahide; Ogasahara, Kyoko; Mizutani, Hisashi; Kunishima, Naoki; Yokoyama, Shigeyuki; Yutani, Katsuhide

    2009-01-01

    The crystal structure of a putative dipeptidase (Phdpd) from Pyrococcus horikoshii OT3 was solved using X-ray data at 2.4 A resolution. The protein is folded into two distinct entities. The N-terminal domain consists of the general topology of the alpha/beta fold, and the C-terminal domain consists of five long mixed strands, four helices, and two 3(10) helices. The structure of Phdpd is quite similar to reported structures of prolidases from P. furiosus (Zn-Pfprol) and P. horikoshii (Zn-Phdpd), where Zn ions are observed in the active site resulting in an inactive form. However, Phdpd did not contain metals in the crystal structure and showed prolidase activity in the absence of additional Co ions, whereas the specific activities increased by 5 times in the presence of a sufficient concentration (1.2 mM) of Co ions. The substrate specificities (X-Pro) of Phdpd were broad compared with those of Zn-Phdpd in the presence of Co ions, whose relative activities are 10% or less for substrates other than Met-Pro, which is the most favorable substrate. The binding constants of Zn-Phdpd with three metals (Zn, Co, and Mn) were higher than those of Phdpd and that with Zn was higher by greater than 2 orders, which were determined by DSC experiments. From the structural comparison of both forms and the above experimental results, it could be elucidated why the protein with Zn(2+) ions is inactive.

  6. Characterization of the archaeal ribonuclease P proteins from Pyrococcus horikoshii OT3.

    PubMed

    Terada, Atsushi; Honda, Takashi; Fukuhara, Hideo; Hada, Kazumasa; Kimura, Makoto

    2006-08-01

    Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5'-leader sequence of precursor tRNA (pre-tRNA). Our earlier study revealed that RNase P RNA (pRNA) and five proteins (PhoPop5, PhoRpp38, PhoRpp21, PhoRpp29, and PhoRpp30) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 reconstituted RNase P activity that exhibits enzymatic properties like those of the authentic enzyme. In present study, we investigated involvement of the individual proteins in RNase P activity. Two particles (R-3Ps), in which pRNA was mixed with three proteins, PhoPop5, PhoRpp30, and PhoRpp38 or PhoPop5, PhoRpp30, and PhoRpp21 showed a detectable RNase P activity, and five reconstituted particles (R-4Ps) composed of pRNA and four proteins exhibited RNase P activity, albeit at reduced level compared to that of the reconstituted particle (R-5P) composed of pRNA and five proteins. Time-course analysis of the RNase P activities of R-4Ps indicated that the R-4Ps lacking PhoPop5, PhoRpp21, or PhoRpp30 had virtually reduced activity, while omission of PhoRpp29 or PhoRpp38 had a slight effect on the activity. The results indicate that the proteins contribute to RNase P activity in order of PhoPop5 > PhoRpp30 > PhoRpp21 > PhoRpp29 > PhoRpp38. It was further found that R-4Ps showed a characteristic Mg2+ ion dependency approximately identical to that of R-5P. However, R-4Ps had optimum temperature of around at 55 degrees C which is lower than 70 degrees C for R-5P. Together, it is suggested that the P. horikoshii RNase P proteins are predominantly involved in optimization of the pRNA conformation, though they are individually dispensable for RNase P activity in vitro.

  7. 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.

  8. 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

  9. Crystal structure of an archaeal Ski2p-like protein from Pyrococcus horikoshii OT3.

    PubMed

    Zhang, Xiaodong; Nakashima, Takashi; Kakuta, Yoshimitsu; Yao, Min; Tanaka, Isao; Kimura, Makoto

    2008-01-01

    The Ski complex composed of Ski2p, Ski3p, and Ski8p plays an essential role in the 3' to 5' cytoplasmic mRNA degradation pathway in yeast. Ski2p is a putative RNA helicase, belonging in the DExD/H-box protein families and conserved in eukarya as well as in archaea. The gene product (Ph1280p) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 shows sequence homology with Ski2p, sharing 22.6% identical amino acids with a central region of Ski2p. In order to gain structural information about the Ski2p-like RNA helicase, we overproduced Ph1280p in Escherichia coli cells, and purified it to apparent homogeneity. Ph1280p exhibits DNA/RNA-dependent ATPase activity with an optimal temperature at approximately 90 degrees C. The crystal structure of Ph1280p has been solved at a resolution of 3.5 A using single-wavelength anomalous dispersion (SAD) and selenomethionyl (Se-Met)-substituted protein. Ph1280p comprises four subdomains; the two N-terminal subdomains (N1 and N2) fold into an RecA-like architecture with the conserved helicase motifs, while the two C-terminal subdomains (C1 and C2) fold into alpha-helical structures containing a winged helix (WH)-fold and helix-hairpin-helix (HhH)-fold, respectively. Although the structure of each of the Ph1280p subdomains can be individually superimposed on the corresponding domains in other helicases, such as the Escherichia coli DNA helicase RecQ, the relative orientation of the helicase and C-terminal subdomains in Ph1280p is significantly different from that of other helicases. This structural feature is implicated in substrate specificity for the Ski2-like helicase and would play a critical role in the 3' to 5' cytoplasmic mRNA degradation in the Ski complex. PMID:18042682

  10. Thermostable flap endonuclease from the archaeon, Pyrococcus horikoshii, cleaves the replication fork-like structure endo/exonucleolytically.

    PubMed

    Matsui, E; Kawasaki, S; Ishida, H; Ishikawa, K; Kosugi, Y; Kikuchi, H; Kawarabayashi, Y; Matsui, I

    1999-06-25

    The flap endonuclease gene homologue from the hyperthermophilic archaeon, Pyrococcus horikoshii, was overexpressed in Escherichia coli and purified. The results of gel filtration indicated that this protein was a 41-kDa monomer. P. horikoshii flap endonuclease (phFEN) cleaves replication fork-like substrates (RF) and 5' double-strand flap structures (DF) using both flap endonuclease and 5'-3'-exonuclease activities. The mammalian flap endonuclease (mFEN) is a single-strand flap-specific endonuclease (Harrington, J. J., and Lieber, M. R. (1994) EMBO J. 13, 1235-1246), but the action patterns of phFEN appear to be quite different from those of mFEN at this point. The DF-specific flap endonuclease and 5'-exonuclease activities have not yet been reported. Therefore, this is the first report of the specific endo/exonuclease activities of phFEN. The DF-specific 5'-exonuclease activity degraded the downstream primer of 3' single-flap structure and was 15 times higher than the activities against nicked substrates without 3' flap strand. DF-specific flap endonuclease cleaved the 5' double-flap strand in DF and the lagging strand in RF at the junction portion. Because the RF appears to be the intermediate structure, due to the arrest of the replication fork, the double strand breaks after the arrests of the replication forks are probably caused by phFEN.

  11. 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.

  12. Characterization of the PH1704 Protease from Pyrococcus horikoshii OT3 and the Critical Functions of Tyr120

    PubMed Central

    Yu, Lei; Han, Weiwei; Feng, Yan

    2014-01-01

    The PH1704 protease from hyperthermophilic archaean Pyrococcus horikoshii OT3 is a member of DJ-1/ThiJ/PfpI superfamily with diverse functional subclasses. The recombinant PH1704 was efficiently purified and was systematically characterized by a combination of substrate specificity analysis, steady-state kinetics study and molecular docking research. The homogeneous protease was obtained as a presumed dodecamer with molecular weight of ∼240 kDa. Iodoacetamide strongly inhibited the peptidase activity, confirming that Cys100 is a nucleophilic residue. The recombinant protein was identified as both an aminopeptidase and an endopeptidase. Experimental data showed that L-R-amc was the best substrate of PH1704. Structural interaction fingerprint analysis (SIFt) indicated the binding pose of PH1704 and showed that Tyr120 is important in substrate binding. Kinetic parameters Kcat and Kcat/Km of the Y120P mutant with L-R-amc was about 7 and 7.8 times higher than that of the wild type (WT). For the endopeptidase Y120P with AAFR-amc, Kcat and Kcat/Km is 10- and 21- fold higher than that of WT. Experimental data indicate the important functions of Tyr120: involvement in enzyme activity to form a hydrogen bond with Cys100 and as an entrance gate of the substrate with Lys43. The results of this study can be used to investigate the DJ-1/ThiJ/PfpI superfamily. PMID:25192005

  13. Crystal structure of product-bound complex of UDP-N-acetyl-D-mannosamine dehydrogenase from Pyrococcus horikoshii OT3

    SciTech Connect

    Pampa, K.J.; Lokanath, N.K.; Girish, T.U.; Kunishima, N.; Rai, V.R.

    2014-10-24

    Highlights: • Determined the structure of UDP-D-ManNAcADH to a resolution of 1.55 Å. • First complex structure of PhUDP-D-ManNAcADH with UDP-D-ManMAcA. • The monomeric structure consists of three distinct domains. • Cys258 acting as catalytic nucleophilic and Lys204 acts as acid/base catalyst. • Oligomeric state plays an important role for the catalytic function. - Abstract: UDP-N-acetyl-D-mannosamine dehydrogenase (UDP-D-ManNAcDH) belongs to UDP-glucose/GDP-mannose dehydrogenase family and catalyzes Uridine-diphospho-N-acetyl-D-mannosamine (UDP-D-ManNAc) to Uridine-diphospho-N-acetyl-D-mannosaminuronic acid (UDP-D-ManNAcA) through twofold oxidation of NAD{sup +}. In order to reveal the structural features of the Pyrococcus horikoshii UDP-D-ManNAcADH, we have determined the crystal structure of the product-bound enzyme by X-ray diffraction to resolution of 1.55 Å. The protomer folds into three distinct domains; nucleotide binding domain (NBD), substrate binding domain (SBD) and oligomerization domain (OD, involved in the dimerization). The clear electron density of the UDP-D-ManNAcA is observed and the residues binding are identified for the first time. Crystal structures reveal a tight dimeric polymer chains with product-bound in all the structures. The catalytic residues Cys258 and Lys204 are conserved. The Cys258 acts as catalytic nucleophile and Lys204 as acid/base catalyst. The product is directly interacts with residues Arg211, Thr249, Arg244, Gly255, Arg289, Lys319 and Arg398. In addition, the structural parameters responsible for thermostability and oligomerization of the three dimensional structure are analyzed.

  14. 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.

  15. Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by D-amino acids.

    PubMed

    Kawakami, Ryushi; Ohmori, Taketo; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2015-08-01

    To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of D-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using (14)C-labeled D-Trp showed that D-Trp in the medium was used as a protein component in the cells, suggesting the presence of D-amino acid metabolic enzymes. Pyridoxal 5'-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with D-amino acids, especially D-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of D-amino acids for growth.

  16. The TET2 and TET3 aminopeptidases from Pyrococcus horikoshii form a hetero-subunit peptidasome with enhanced peptide destruction properties.

    PubMed

    Appolaire, Alexandre; Durá, M Asunción; Ferruit, Mylène; Andrieu, Jean-Pierre; Godfroy, Anne; Gribaldo, Simonetta; Franzetti, Bruno

    2014-11-01

    TET aminopeptidases assemble as large homo-dodecameric complexes. The reason why prokaryotic genomes often encode a diverse set of TET peptidases homologues remains unclear. In the archaeon Pyrococcus horikoshii, PhTET1, PhTET2 and PhTET3 homo-oligomeric particles have been proposed to work in concert to breakdown intracellular polypeptides. When coexpressed in Escherichia coli, the PhTET2 and PhTET3 proteins were found to assemble efficiently as heteromeric complexes. Biophysical analysis demonstrated that these particles possess the same quaternary structure as the homomeric TET dodecamers. The same hetero-oligomeric complexes were immunodetected in P. horikoshii cell extracts analysed by sucrose gradient fractionation and ion exchange chromatography. The biochemical activity of a purified hetero-oligomeric TET particle, assessed on chromogenic substrates and on a complex mixture of peptides, reveals that it displays higher efficiency than an equivalent combination of homo-oligomeric TET particles. Interestingly, phylogenetic analysis shows that PhTET2 and PhTET3 are paralogous proteins that arose from gene duplication in the ancestor of Thermococcales. Together, these results establish that the PhTET2 and PhTET3 proteins are two subunits of the same enzymatic complex aimed at the destruction of polypeptidic chains of very different composition. This is the first report for such a mechanism intended to improve multi-enzymatic complex efficiency among exopeptidases.

  17. 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

  18. 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.

  19. Identification of a glycolytic regulon in the archaea Pyrococcus and Thermococcus.

    PubMed

    van de Werken, Harmen J G; Verhees, Corné H; Akerboom, Jasper; de Vos, Willem M; van der Oost, John

    2006-07-01

    The glycolytic pathway of the hyperthermophilic archaea that belong to the order Thermococcales (Pyrococcus, Thermococcus and Palaeococcus) differs significantly from the canonical Embden-Meyerhof pathway in bacteria and eukarya. This archaeal glycolysis variant consists of several novel enzymes, some of which catalyze unique conversions. Moreover, the enzymes appear not to be regulated allosterically, but rather at transcriptional level. To elucidate details of the gene expression control, the transcription initiation sites of the glycolytic genes in Pyrococcus furiosus have been mapped by primer extension analysis and the obtained promoter sequences have been compared with upstream regions of non-glycolytic genes. Apart from consensus sequences for the general transcription factors (TATA-box and BRE) this analysis revealed the presence of a potential transcription factor binding site (TATCAC-N(5)-GTGATA) in glycolytic and starch utilizing promoters of P. furiosus and several thermococcal species. The absence of this inverted repeat in Pyrococcus abyssi and Pyrococcus horikoshii probably reflects that their reduced catabolic capacity does not require this regulatory system. Moreover, this phyletic pattern revealed a TrmB-like regulator (PF0124 and TK1769) which may be involved in recognizing the repeat. This Thermococcales glycolytic regulon, with more than 20 genes, is the largest regulon that has yet been described for Archaea.

  20. 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

  1. Identification of two [4Fe-4S]-cluster-containing hydro-lyases from Pyrococcus furiosus.

    PubMed

    van Vugt-Lussenburg, Barbara M A; van der Weel, Laura; Hagen, Wilfred R; Hagedoorn, Peter-Leon

    2009-09-01

    The hyperthermophilic archaeon Pyrococcus furiosus is a strict anaerobe. It is therefore not expected to use the oxidative tricarboxylic acid (TCA) cycle for energy transduction. Nonetheless, its genome encodes more putative TCA cycle enzymes than the closely related Pyrococcus horikoshii and Pyrococcus abyssi, including an aconitase (PF0201). Furthermore, a two-subunit fumarase (PF1755 and PF1754) is encoded on the Pyr. furiosus genome. In the present study, these three genes were heterologously overexpressed in Escherichia coli to enable characterization of the enzymes. PF1755 and PF1754 were shown to form a [4Fe-4S]-cluster-containing heterodimeric enzyme, able to catalyse the reversible hydratation of fumarate. The aconitase PF0201 also contained an Fe-S cluster, and catalysed the conversion from citrate to isocitrate. The fumarase belongs to the class of two-subunit, [4Fe-4S]-cluster-containing fumarate hydratases exemplified by MmcBC from Pelotomaculum thermopropionicum; the aconitase belongs to the aconitase A family. Aconitase probably plays a role in amino acid synthesis when the organism grows on carbohydrates. However, the function of the seemingly metabolically isolated fumarase in Pyr. furiosus has yet to be established.

  2. 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

  3. 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

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

    PubMed

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

    2016-07-14

    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.

  5. Pyrococcus furiosus strains and methods of using same

    SciTech Connect

    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.

  6. 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

  7. 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

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

    DOE PAGES

    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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

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

    PubMed Central

    Biarrotte-Sorin, Sabrina; Mayer, Claudine

    2005-01-01

    The GCN5-related N-acetyltransferase (GNAT) superfamily has a primordial role in cellular processes such as transcription initiation and regulation by histone acetylation, aminoglycoside resistance and melatonin metabolism. To date, no acetyltransferase from the archaeal domain of life has been studied. This paper describes the cloning, expression, purification and crystallization of a Pyrococcus furiosus hypothetical acetyltransferase PfGNAT (MW = 22 007 Da). The crystals belong to space group P622, with one molecule in the asymmetric unit and unit-cell parameters a = b = 82.6, c = 105.92 Å, α = β = 90, γ = 120°. Crystals diffract X-rays to 3.0 Å resolution on a synchrotron-radiation source. Determination of this structure will provide new insights into the substrate-specificity of this acetyltransferase and the thermal stability of the N-acetyltransferase domain. PMID:16511014

  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 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

  2. 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

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

    DOE PAGES

    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. Molybdenum Incorporation in Tungsten Aldehyde Oxidoreductase Enzymes from Pyrococcus furiosus▿ †

    PubMed Central

    Sevcenco, Ana-Maria; Bevers, Loes E.; Pinkse, Martijn W. H.; Krijger, Gerard C.; Wolterbeek, Hubert T.; Verhaert, Peter D. E. M.; Hagen, Wilfred R.; Hagedoorn, Peter-Leon

    2010-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is not incorporated in the active site of these enzymes. Application of the radioisotope 99Mo in metal isotope native radioautography in gel electrophoresis (MIRAGE) technology to P. furiosus shows that molybdenum can in fact be incorporated in all five AOR enzymes. Mo(V) signals characteristic for molybdopterin were observed in formaldehyde oxidoreductase (FOR) in electron paramagnetic resonance (EPR)-monitored redox titrations. Our finding that the aldehyde oxidation activity of FOR and WOR5 (W-containing oxidoreductase 5) correlates only with the residual tungsten content suggests that the Mo-containing AORs are most likely inactive. An observed W/Mo antagonism is indicative of tungstate-dependent negative feedback of the expression of the tungstate/molybdate ABC transporter. An intracellular selection mechanism for tungstate and molybdate processing has to be present, since tungsten was found to be preferentially incorporated into the AORs even under conditions with comparable intracellular concentrations of tungstate and molybdate. Under the employed growth conditions of starch as the main carbon source in a rich medium, no tungsten- and/or molybdenum-associated proteins are detected in P. furiosus other than the high-affinity transporter, the proteins of the metallopterin insertion machinery, and the five W-AORs. PMID:20562313

  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. Expression and in vitro assembly of recombinant glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed Central

    Diruggiero, J; Robb, F T

    1995-01-01

    The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus furiosus, was expressed in Escherichia coli by using the pET11-d system. The recombinant GDH was soluble and constituted 15% of the E. coli cell extract. The N-terminal amino acid sequence of the recombinant protein was identical to the sequence of the P. furiosus enzyme, except for the presence of an initial methionine which was absent from the enzyme purified from P. furiosus. By molecular exclusion chromatography we showed that the recombinant GDH was composed of equal amounts of monomeric and hexameric forms. Heat treatment of the recombinant protein triggered in vitro assembly of inactive monomers into hexamers, resulting in increased GDH activity. The specific activity of the recombinant enzyme, purified by heat treatment and affinity chromatography, was equivalent to that of the native enzyme from P. furiosus. The recombinant GDH displayed a slightly lower level of thermostability, with a half-life of 8 h at 100 degrees C, compared with 10.5 h for the enzyme purified from P. furiosus. PMID:7887598

  7. SurR regulates hydrogen production in Pyrococcus furiosus by a sulfur-dependent redox switch

    PubMed Central

    Yang, Hua; Lipscomb, Gina L.; Keese, Annette M.; Schut, Gerrit J.; Thomm, Michael; Adams, Michael W. W.; Wang, Bi Cheng; Scott, Robert A.

    2010-01-01

    SUMMARY We present structural and biochemical evidence for a redox switch in the archaeal transcriptional regulator SurR of Pyrococcus furiosus, a hyperthermophilic anaerobe. P. furiosus produces H2 during fermentation, but undergoes a metabolic shift to produce H2S when elemental sulfur (S0) becomes available. Changes in gene expression occur within minutes of S0 addition, and the majority of these S0-responsive genes are regulatory targets of SurR, a key regulator involved in primary S0 response. SurR was shown in vitro to have dual functionality, activating transcription of some of these genes, notably the hydrogenase operons, and repressing others, including a gene encoding sulfur reductase. This work demonstrates via biochemical and structural evidence that the activity of SurR is modulated by cysteine residues in a CxxC motif that constitute a redox switch. Oxidation of the switch with S0 inhibits sequence-specific DNA binding by SurR, leading to deactivation of genes related to H2 production and derepression of genes involved in S0 metabolism. PMID:20598080

  8. Differential signal transduction via TrmB, a sugar sensing transcriptional repressor of Pyrococcus furiosus.

    PubMed

    Lee, Sung-Jae; Surma, Melanie; Seitz, Sabine; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2007-06-01

    TrmB is a transcriptional repressor of the hyperthermophilic archaeon Pyrococcus furiosus serving at least two operons. TrmB represses genes encoding an ABC transporter for trehalose and maltose (the TM system) with trehalose and maltose as inducers. TrmB also represses genes encoding another ABC transporter for maltodextrins (the MD system) with maltotriose and sucrose as inducers. Here we report that glucose which was also bound by TrmB acted as a corepressor (causing stronger repression) for both the TM and the MD system. Binding of glucose by TrmB was increased in the presence of TM promoter DNA. Maltose which acted as inducer for the TM system acted as a corepressor for the MD system intensifying repression. We propose that the differential conformational changes of TrmB in response to binding the different sugars governs the ability of TrmB to interact with the promoter region and represents a simple mechanism for selecting the usage of one carbon source over the other, reminiscent of catabolite repression in bacteria.

  9. 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

  10. Expression of Pyrococcus furiosus Superoxide Reductase in Arabidopsis Enhances Heat Tolerance1[C][W][OA

    PubMed Central

    Im, Yang Ju; Ji, Mikyoung; Lee, Alice; Killens, Rushyannah; Grunden, Amy M.; Boss, Wendy F.

    2009-01-01

    Plants produce reactive oxygen species (ROS) in response to environmental stresses sending signaling cues, which, if uncontrolled, result in cell death. Like other aerobic organisms, plants have ROS-scavenging enzymes, such as superoxide dismutase (SOD), which removes superoxide anion radical (O2−) and prevents the production and buildup of toxic free radicals. However, increasing the expression of cytosolic SODs is complex, and increasing their production in vivo has proven to be challenging. To avoid problems with endogenous regulation of gene expression, we expressed a gene from the archaeal hyperthermophile Pyrococcus furiosus that reduces O2−. P. furiosus uses superoxide reductase (SOR) rather than SOD to remove superoxide. SOR is a thermostable enzyme that reduces O2− in a one-electron reduction without producing oxygen. We show that P. furiosus SOR can be produced as a functional enzyme in planta and that plants producing SOR have enhanced tolerance to heat, light, and chemically induced ROS. Stress tolerance in the SOR-producing plants correlates positively with a delayed increase in ROS-sensitive transcripts and a decrease in ascorbate peroxidase activity. The SOR plants provide a good model system to study the impact of cytosolic ROS on downstream signaling in plant growth and development. Furthermore, this work demonstrates that this synthetic approach for reducing cytosolic ROS holds promise as a means for improving stress tolerance in crop plants. PMID:19684226

  11. 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.

  12. 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

  13. Novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus

    SciTech Connect

    Aono, S.; Bryant, F.O.; Adams, M.W.W. )

    1989-06-01

    The archaebacterium Pyrococcus furiosus is a strict anaerobe that grows optimally at 100{degree}C by a fermentative-type metabolism in which H{sub 2} and CO{sub 2} are the only detectable products. A ferredoxin, which functions as the electron donor to the hydrogenase of this organism was purified under anaerobic reducing conditions. The study shows that P. furiosus ferredoxin is therefore distinct from other ferredoxins in that the bulk of its iron is not present as iron-sulfur clusters with an S = 1/2 ground state. The apoferredoxin was reconstituted with iron and sulfide to give a protein that was indistinguishable from the native ferredoxin by its iron content and electron paramagnetic resonance properties, which showed that the novel iron-sulfur clusters were not artifacts of purification. The reduced ferredoxin also functioned as an electron donor for H{sub 2} evolution catalyzed by the hydrogenase of the mesophilic eubacterium Clostridium pasteurianum. P. furiosus ferredoxin was resistant to denaturation by sodium dodecyl sulfate (20%, wt/vol) and was remarkably thermostable. Its UV-visible absorption spectrum and electron carrier activity to P. furiosus hydrogenase were unaffected by a 12-h incubation at 95{degree}C.

  14. 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.

  15. 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

  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.

  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

    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

  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. 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

  20. 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.

  1. Preliminary crystallographic analysis of an extremely thermostable glutamate dehydrogenase from the archaeon Pyrococcus woesei.

    PubMed

    Knapp, S; Karshikoff, A; Waldkötter, K; Rüdiger, A; Antranikian, G; Ladenstein, R

    1995-05-01

    The extremely heat-stable glutamate dehydrogenase (GluDH) from Pyrococcus woesei was crystallized by the hanging-drop vapour-diffusion method. Crystals suitable for X-ray crystallographic investigations were obtained using polyethylene glycol (PEG) 4000 and ammonium acetate as precipitating agents. The crystals obtained diffract to a resolution of 2.8 A, have a prismatic shape and grow up to 0.5 mm in their maximal dimension. They belong to the triclinic system (space group P1; a = 90.9, b = 92.8, c = 107.1 A, alpha = 69.1, beta = 80.7, Vgamma = 65.0 degrees ) with a unit-cell volume of 765052 A(3) which accommodates one GluDH hexamer of 276 kDa. The averaged density of the crystal determined by Ficoll-gradient centrifugation is 1.15 g cm(-3), which corresponds to a molecular mass of 255 kDa in the unit cell. A native data set has been collected on an MAR image-plate system using Cu Kalpha radiation. The completeness of the data set in the range 316-3 A is 74%, and contains 64% of the data in the outer shell (3.4-2.8 A), with an average R(merge) value of 9%. Calculation of self-rotation functions revealed the 32 symmetry of the hexamer; 3 non-crystallographic twofold axes were found at a distance of 120 degrees in a plane perpendicular to the non-crystallographic threefold axis. PMID:15299309

  2. 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

  3. The RNA- and DNA-targeting CRISPR–Cas immune systems of Pyrococcus furiosus

    PubMed Central

    Terns, Rebecca M.; Terns, Michael P.

    2014-01-01

    Using the hyperthermophile Pyrococcus furiosus, we have delineated several key steps in CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) invader defence pathways. P. furiosus has seven transcriptionally active CRISPR loci that together encode a total of 200 crRNAs (CRISPR RNAs). The 27 Cas proteins in this organism represent three distinct pathways and are primarily encoded in two large gene clusters. The Cas6 protein dices CRISPR locus transcripts to generate individual invader-targeting crRNAs. The mature crRNAs include a signature sequence element (the 5′ tag) derived from the CRISPR locus repeat sequence that is important for function. crRNAs are tailored into distinct species and integrated into three distinct crRNA–Cas protein complexes that are all candidate effector complexes. The complex formed by the Cmr [Cas module RAMP (repeat-associated mysterious proteins)] (subtype III-B) proteins cleaves complementary target RNAs and can be programmed to cleave novel target RNAs in a prokaryotic RNAi-like manner. Evidence suggests that the other two CRISPR–Cas systems in P. furiosus, Csa (Cas subtype Apern) (subtype I-A) and Cst (Cas subtype Tneap) (subtype I-B), target invaders at the DNA level. Studies of the CRISPR–Cas systems from P. furiosus are yielding fundamental knowledge of mechanisms of crRNA biogenesis and silencing for three of the diverse CRISPR–Cas pathways, and reveal that organisms such as P. furiosus possess an arsenal of multiple RNA-guided mechanisms to resist diverse invaders. Our knowledge of the fascinating CRISPR–Cas pathways is leading in turn to our ability to co-opt these systems for exciting new biomedical and biotechnological applications. PMID:24256230

  4. Identification of Membrane Proteins in the Hyperthermophilic Archaeon Pyrococcus Furiosus Using Proteomics and Prediction Programs

    PubMed Central

    Holden, James F.; Poole II, Farris L.; Tollaksen, Sandra L.; Giometti, Carol S.; Lim, Hanjo; Yates III, John R.

    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 α-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 β-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. PMID:18629240

  5. Mechanism of oxygen detoxification by the surprisingly oxygen-tolerant hyperthermophilic archaeon, Pyrococcus furiosus.

    PubMed

    Thorgersen, Michael P; Stirrett, Karen; Scott, Robert A; Adams, Michael W W

    2012-11-01

    The anaerobic archaeon Pyrococcus furiosus grows by fermenting carbohydrates producing H(2), CO(2), and acetate. We show here that it is surprisingly tolerant to oxygen, growing well in the presence of 8% (vol/vol) O(2). Although cell growth and acetate production were not significantly affected by O(2), H(2) production was reduced by 50% (using 8% O(2)). The amount of H(2) produced decreased in a linear manner with increasing concentrations of O(2) over the range 2-12% (vol/vol), and for each mole of O(2) consumed, the amount of H(2) produced decreased by approximately 2 mol. The recycling of H(2) by the two cytoplasmic hydrogenases appeared not to play a role in O(2) resistance because a mutant strain lacking both enzymes was not more sensitive to O(2) than the parent strain. Decreased H(2) production was also not due to inactivation of the H(2)-producing, ferredoxin-dependent membrane-bound hydrogenase because its activity was unaffected by O(2) exposure. Electrons from carbohydrate oxidation must therefore be diverted to relieve O(2) stress at the level of reduced ferredoxin before H(2) production. Deletion strains lacking superoxide reductase (SOR) and putative flavodiiron protein A showed increased sensitivity to O(2), indicating that these enzymes play primary roles in resisting O(2). However, a mutant strain lacking the proposed electron donor to SOR, rubredoxin, was unaffected in response to O(2). Hence, electrons from sugar oxidation normally used to produce H(2) are diverted to O(2) detoxification by SOR and putative flavodiiron protein A, but the electron flow pathway from ferredoxin does not necessarily involve rubredoxin.| PMID:23093671

  6. Recombinant production of hyperthermostable CelB from Pyrococcus furiosus in Lactobacillus sp.

    PubMed

    Böhmer, N; Lutz-Wahl, S; Fischer, L

    2012-11-01

    Lactic acid bacteria (LAB) are used widespread in the food industry as traditional starters for various fermented foods. For recombinant protein production, LAB would be superior with view from the food safety demands since most of them are Generally Recognized As Safe organisms. We investigated the two pSIP expression systems, pSIP403 and pSIP409 (Sørvig et al. 2005), to produce a hyper-thermophilic β-glycosidase (CelB) from Pyrococcus furiosus in Lactobacillus plantarum NC8 and Lactobacillus casei as hosts, respectively. Both lactobacilli harboring the pSIP409-celB vector produced active CelB in batch bioreactor cultivations (MRS medium) while the specific CelB activity of the cell free extract was about 44 % higher with L. plantarum (1,590 ± 90 nkat/mg(protein)) than with L. casei (1,070 ± 66 nkat/mg(protein)) using p-nitrophenyl-β-galactoside (pNPGal) as the substrate. A fed-batch bioreactor cultivation of L. plantarum NC8 pSIP409-celB resulted in a specific CelB activity of 2,500 ± 120 nkat ( pNPGal)/mg(protein) after 28 h. A repeated dosage of the inducer spp-IP did not increase the enzyme expression further. As alternative for the cost intensive MRS medium, a basal whey medium with supplements (yeast extract, Tween 80, NH(4)-citrate) was developed. In bioreactor cultivations using this medium, about 556 ± 29 nkat ( pNPGal)/mg(protein) of CelB activity was achieved. It was shown that both LAB were potential expression hosts for recombinant enzyme production. The pSIP expression system can be applied in L. casei.

  7. Accumulation of Mannosylglycerate and Di-myo-Inositol-Phosphate by Pyrococcus furiosus in Response to Salinity and Temperature

    PubMed Central

    Martins, L. O.; Santos, H.

    1995-01-01

    (sup13)C and (sup1)H nuclear magnetic resonance spectroscopy was used to identify and quantify organic solutes accumulated by the hyperthermophilic archaeon Pyrococcus furiosus in response to temperature and salinity. Di-myo-inositol-phosphate and 2-O-(beta)-mannosylglycerate were the major organic solutes accumulated in these cells. The total intracellular organic solutes increased significantly in response either to an increase in temperature or to an increase in salinity, but (beta)-mannosylglycerate accumulated mainly at high salinities, whereas the concentration of di-myo-inositol-phosphate increased dramatically at supraoptimal growth temperatures. Glutamate was present at concentrations detectable by nuclear magnetic resonance only in cells grown in low-salinity media. The intracellular levels of K(sup+) are clearly dependent on the salinity of the medium, and the concentrations of this cation are high enough to counterbalance the negative charges of (beta)-mannosylglycerate and di-myo-inositol-phosphate in the cell. The results presented here together with those previously reported for Pyrococcus woesei (S. Scholz, J. Sonnenbichler, W. Schafer, and R. Hensel, FEBS Lett. 306:239-242, 1992) strongly support a role for di-myo-inositol-phosphate in thermoprotection. PMID:16535119

  8. 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.

  9. 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.

  10. Regulation and Mechanism of Action of the Small Heat Shock Protein from the Hyperthermophilic Archaeon Pyrococcus furiosus†

    PubMed Central

    Laksanalamai, Pongpan; Maeder, Dennis L.; Robb, Frank T.

    2001-01-01

    The small heat shock protein (sHSP) from the hyperthermophile Pyrococcus furiosus was specifically induced at the level of transcription by heat shock at 105°C. The gene encoding this protein was cloned and overexpressed in Escherichia coli. The recombinant sHSP prevented the majority of E. coli proteins from aggregating in vitro for up to 40 min at 105°C. The sHSP also prevented bovine glutamate dehydrogenase from aggregating at 56°C. Survivability of E. coli overexpressing the sHSP was enhanced approximately sixfold during exposure to 50°C for 2 h compared with the control culture, which did not express the sHSP. Apparently, the sHSP confers a survival advantage on mesophilic bacteria by preventing protein aggregation at supraoptimal temperatures. PMID:11489874

  11. 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

  12. 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

  13. 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

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

    DOE PAGES

    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

  15. Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

    PubMed

    Dong, G; Vieille, C; Zeikus, J G

    1997-09-01

    The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.

  16. 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.

  17. 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.

  18. 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

  19. Structures of the Signal Recognition Particle Receptor from the Archaeon Pyrococcus furiosus: Implications for the Targeting Step at the Membrane

    PubMed Central

    Egea, Pascal F.; Tsuruta, Hiro; de Leon, Gladys P.; Napetschnig, Johanna; Walter, Peter; Stroud, Robert M.

    2008-01-01

    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•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•SR targeting complexes. PMID:18978942

  20. 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

  1. 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

  2. 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

  3. Physiological aggregation of maltodextrin phosphorylase from Pyrococcus furiosus and its application in a process of batch starch degradation to alpha-D-glucose-1-phosphate.

    PubMed

    Nahálka, Jozef

    2008-04-01

    Maltodextrin phosphorylase from Pyrococcus furiosus (PF1535) was fused with the cellulose-binding domain of Clostridium cellulovorans serving as an aggregation module. After molecular cloning of the corresponding gene fusion construct and controlled expression in Escherichia coli BL21, 83% of total maltodextrin phosphorylase activity (0.24 U/mg of dry cell weight) was displayed in active inclusion bodies. These active inclusion bodies were easily isolated by nonionic detergent treatment and directly used for maltodextrin conversion to alpha-D-glucose-1-phosphate in a repetitive batch mode. Only 10% of enzyme activity was lost after ten conversion cycles at optimum conditions.

  4. 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

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

    PubMed

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

    2014-12-01

    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 Mg(2+)-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.

  6. 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

  7. Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli.

    PubMed

    Purcarea, C; Hervé, G; Ladjimi, M M; Cunin, R

    1997-07-01

    The genes coding for aspartate transcarbamylase (ATCase) in the deep-sea hyperthermophilic archaeon Pyrococcus abyssi were cloned by complementation of a pyrB Escherichia coli mutant. The sequence revealed the existence of a pyrBI operon, coding for a catalytic chain and a regulatory chain, as in Enterobacteriaceae. Comparison of primary sequences of the polypeptides encoded by the pyrB and pyrI genes with those of homologous eubacterial and eukaryotic chains showed a high degree of conservation of the residues which in E. coli ATCase are involved in catalysis and allosteric regulation. The regulatory chain shows more-extensive divergence with respect to that of E. coli and other Enterobacteriaceae than the catalytic chain. Several substitutions suggest the existence in P. abyssi ATCase of additional hydrophobic interactions and ionic bonds which are probably involved in protein stabilization at high temperatures. The catalytic chain presents a secondary structure similar to that of the E. coli enzyme. Modeling of the tridimensional structure of this chain provides a folding close to that of the E. coli protein in spite of several significant differences. Conservation of numerous pairs of residues involved in the interfaces between different chains or subunits in E. coli ATCase suggests that the P. abyssi enzyme has a quaternary structure similar to that of the E. coli enzyme. P. abyssi ATCase expressed in transgenic E. coli cells exhibited reduced cooperativity for aspartate binding and sensitivity to allosteric effectors, as well as a decreased thermostability and barostability, suggesting that in P. abyssi cells this enzyme is further stabilized through its association with other cellular components.

  8. Electronic, Magnetic, and Redox Properties of [MFe(3)S(4)] Clusters (M = Cd, Cu, Cr) in Pyrococcus furiosus Ferredoxin.

    PubMed

    Staples, Christopher R.; Dhawan, Ish K.; Finnegan, Michael G.; Dwinell, Derek A.; Zhou, Zhi Hao; Huang, Heshu; Verhagen, Marc F. J. M.; Adams, Michael W. W.; Johnson, Michael K.

    1997-12-01

    The ground- and excited-state properties of heterometallic [CuFe(3)S(4)](2+,+), [CdFe(3)S(4)](2+,+), and [CrFe(3)S(4)](2+,+) cubane clusters assembled in Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR and variable-temperature/variable-field magnetic circular dichroism (MCD) studies. The results indicate Cd(2+) incorporation into [Fe(3)S(4)](0,-) cluster fragments to yield S = 2 [CdFe(3)S(4)](2+) and S = (5)/(2) [CdFe(3)S(4)](+) clusters and Cu(+) incorporation into [Fe(3)S(4)](+,0) cluster fragments to yield S = (1)/(2) [CuFe(3)S(4)](2+) and S = 2 [CuFe(3)S(4)](+) clusters. This is the first report of the preparation of cubane type [CrFe(3)S(4)](2+,+) clusters, and the combination of EPR and MCD results indicates S = 0 and S = (3)/(2) ground states for the oxidized and reduced forms, respectively. Midpoint potentials for the [CdFe(3)S(4)](2+,+), [CrFe(3)S(4)](2+,+), and [CuFe(3)S(4)](2+,+) couples, E(m) = -470 +/- 15, -440 +/- 10, and +190 +/- 10 mV (vs NHE), respectively, were determined by EPR-monitored redox titrations or direct electrochemistry at a glassy carbon electrode. The trends in redox potential, ground-state spin, and electron delocalization of [MFe(3)S(4)](2+,+) clusters in P. furiosus ferredoxin are discussed as a function of heterometal (M = Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd, and Tl).

  9. Inhibition and stimulation of formation of the ferroxidase center and the iron core in Pyrococcus furiosus ferritin.

    PubMed

    Honarmand Ebrahimi, Kourosh; Hagedoorn, Peter-Leon; Hagen, Wilfred R

    2010-11-01

    Ferritin is a ubiquitous iron-storage protein that has 24 subunits. Each subunit of ferritins that exhibit high Fe(II) oxidation rates has a diiron binding site, the so-called ferroxidase center (FC). The role of the FC appears to be essential for the iron-oxidation catalysis of ferritins. Studies of the iron oxidation by mammalian, bacterial, and archaeal ferritin have indicated different mechanisms are operative for Fe(II) oxidation, and for inhibition of the Fe(II) oxidation by Zn(II). These differences are presumably related to the variations in the amino acid residues of the FC and/or transport channels. We have used a combination of UV-vis spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry to study the inhibiting action of Zn(II) ions on the iron-oxidation process by apoferritin and by ferritin aerobically preloaded with 48 Fe(II) per 24-meric protein, and to study a possible role of phosphate in initial iron mineralization by Pyrococcus furiosus ferritin (PfFtn). Although the empty FC can accommodate two zinc ions, binding of one zinc ion to the FC suffices to essentially abolish iron-oxidation activity. Zn(II) no longer binds to the FC nor does it inhibit iron core formation once the FC is filled with two Fe(III). Phosphate and vanadate facilitate iron oxidation only after formation of a stable FC, whereupon they become an integral part of the core. These results corroborate our previous proposal that the FC in PfFtn is a stable prosthetic group, and they suggest that its formation is essential for iron-oxidation catalysis by the protein.

  10. 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

  11. 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

  12. 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

  13. Intact Functional Fourteen-subunit Respiratory Membrane-bound [NiFe]-Hydrogenase Complex of the Hyperthermophilic Archaeon Pyrococcus furiosus*

    PubMed Central

    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.

    2014-01-01

    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

  14. 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

  15. Influence of ionic liquid cosolvent on transgalactosylation reactions catalyzed by thermostable beta-glycosylhydrolase CelB from Pyrococcus Furiosus.

    PubMed

    Lang, Markus; Kamrat, Thomas; Nidetzky, Bernd

    2006-12-20

    The synthesis of glycosides by enzymatic transglycosylation is a kinetically controlled reaction performed in the context of a non-favorable thermodynamic equilibrium. An unreactive organic cosolvent which increases the selectivity of the enzyme for glycosyl transfer to the acceptor nucleophile compared with water (Ksel) could improve maximum product yield. Here we report on the effect of the ionic liquid 1,3-dimethylimidazoliummethylsulfate on hydrolase and transferase activities of the hyperthermostable beta-glycosidase CelB from the archaeon Pyrococcus furiosus. CelB retained full catalytic efficiency for lactose hydrolysis at 80 degrees C in a 50% (by vol.) solution of ionic liquid in sodium citrate buffer, pH 5.5. It was inactive but not irreversibly denatured at 70% ionic liquid. Using lactose (0.15 M) as galactosyl donor, values of Ksel for a representative series of eight acceptor alcohols were determined in kinetic assays at 80 degrees C and found to increase between 1.3-fold (D-xylose) and 3.1-fold (glycerol) in 45% ionic liquid. Enhancement of Ksel was dependent on ionic liquid concentration and higher than expected from the decrease in water activity caused by the cosolvent. Experimental molar ratios of D-glucose and D-galactose produced during enzymatic conversion of lactose (75-150 mM) in the presence of D-xylose (0.5 M) or glycerol (0.5 M) showed excellent agreement with predictions based on Ksel values and confirm a significant, yet moderate effect of 45% ionic liquid on increasing the yield of D-galactoside product, by < or = 10%.

  16. 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.

  17. Mannosylglycerate and Di-myo-Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress

    PubMed Central

    Esteves, Ana M.; Chandrayan, Sanjeev K.; McTernan, Patrick M.; Adams, Michael W. W.; Santos, Helena

    2014-01-01

    Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far. PMID:24795373

  18. 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.

  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. 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

  1. Characterization of a tungsten-iron-sulfur protein exhibiting novel spectroscopic and redox properties from the hyperthermophilic archaebacterium Pyrococcus furiosus.

    PubMed

    Mukund, S; Adams, M W

    1990-07-15

    The archaebacterium, Pyrococcus furiosus, is a strict anaerobe that grows optimally at 100 degrees C by a fermentative-type metabolism in which H2 and CO2 are the only detectable products. Tungsten is known to stimulate the growth of this organism. A red-colored tungsten-containing protein (abbreviated RTP) that is redox-active and extremely thermostable has been purified. RTP is a monomer of Mr = 85,000 and contains approximately 6 iron, 1 tungsten, and 4 acid-labile sulfide atoms/molecule. Titrations using visible spectroscopy were consistent with the oxidation and reduction of the protein each requiring two electrons/molecule, suggesting that these metals and the sulfide are arranged in two redox active centers. P. furiosus ferredoxin served as an electron acceptor for the protein. Dithionite-reduced RTP exhibited a remarkable and complex EPR spectrum at 6 K with g values ranging from 1.3 to 10.0. This was shown to arise from the spin-coupling interaction of two paramagnetic centers. One (center A) has a S = 3/2 spin system (effective g values: gx = 3.33, gy = 4.75, and gz = 1.92, where D = 4.3 cm-1 and lambda = 0.135), whereas the EPR properties of the other (center B) could not be deduced. Nevertheless, theoretical analyses show how the redox properties of both centers may be determined using EPR spectroscopy. Their midpoint potentials (Em) at 20 degrees C and pH 8.0 are -410 mV (center A) and -500 mV (center B) with an effective potential for the spin coupled system (Em, A + B) of -505 mV. The Em values are dependent on temperature (delta Em/delta T = -2 mV/degrees C between 20 and 70 degrees C) and pH with pK alpha values of 8.0 (A) and approximately 8.5 (B). The Em values at 100 degrees C, the growth temperature, were estimated at -590, -650, and -660 mV for centers A, B, and A + B, respectively. These data indicate that RTP catalyzes a dehydrogenase-type reaction of extremely low potential, which involves the transfer of two protons and of two electrons

  2. Evidence for the operation of a novel Embden-Meyerhof pathway that involves ADP-dependent kinases during sugar fermentation by Pyrococcus furiosus.

    PubMed

    Kengen, S W; de Bok, F A; van Loo, N D; Dijkema, C; Stams, A J; de Vos, W M

    1994-07-01

    The main pathway for the fermentation of maltose or cellobiose by the hyperthermophile Pyrococcus furiosus was investigated by in vivo NMR and by enzyme measurements. Addition of [1-13C]glucose to cell suspensions resulted in the formation of C2-labeled acetate and C3-labeled alanine. No label was recovered in CO2 or HCO3-. In the presence of [3-13C]glucose, the label ended up in the C1 atom of alanine and in HCO3- and CO2. These labeling patterns indicate that glucose is converted along an Embden-Meyerhof pathway, and they disagree with the previously proposed nonphosphorylated Entner-Doudoroff pathway (pyroglycolysis). The NMR data were supported by enzyme measurements. Hexokinase (8.7 units/mg), phosphoglucose isomerase (6.8 units/mg), phosphofructokinase (0.81 unit/mg), and aldolase (0.26 unit/mg) were present in cell-free extracts (specific activities at 90 degrees C). Remarkably, the two kinases required ADP as the phosphoryl group donor instead of ATP. No activity was found with pyrophosphate. These are the first descriptions of ADP-dependent (AMP-forming) kinases to date. Since P. furiosus is a phylogenetically ancient organism, these enzymes may represent an ancestral kind of metabolism.

  3. 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

  4. '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

  5. Isolation and characterization of a heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli.

    PubMed Central

    Rüdiger, A; Jorgensen, P L; Antranikian, G

    1995-01-01

    The gene encoding an extremely heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei was cloned and expressed in Escherichia coli. Purification of the enzyme to homogeneity was achieved after heat treatment of the recombinant E. coli cells, affinity chromatography on a maltotriose-coupled Sepharose 6B column, and anion-exchange chromatography on Mono Q. The pullulanase, which was purified 90-fold with a final yield of 15%, is composed of a single polypeptide chain with a molecular mass of 90 kDa. The enzyme is optimally active at 100 degrees C and pH 6.0 and shows 40% activity at 120 degrees C. Enzyme activation up to 370% is achieved in the presence of calcium ions and reducing agents such as beta-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and alpha-cyclodextrin are inhibitory. The high rigidity of the heat-stable enzyme is demonstrated by fluorescence spectroscopic studies in the presence of denaturing agents such as sodium dodecyl sulfate. At temperatures above 80 degrees C, the enzyme seems to switch from the compact to the unfolded form, which is accompanied by an apparent shift in the molecular mass from 45 to 90 kDa. PMID:7574598

  6. The role of TrmB and TrmB-like transcriptional regulators for sugar transport and metabolism in the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed

    Lee, Sung-Jae; Surma, Melanie; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2008-09-01

    TrmB of Pyrococcus furiosus was discovered as the trehalose/maltose-specific repressor for the genes encoding the trehalose/maltose high-affinity ABC transporter (the TM system). TrmB also represses the genes encoding the high affinity maltodextrin-specific ABC transporter (the MD system) with maltodextrin and sucrose as inducers. In addition, TrmB binds glucose leading to an increased repression of both, the TM and the MD system. Thus, TrmB recognizes different promoters and depending on the promoter it will be activated or inactivated for promoter binding by different sugar effectors. The TrmB-like protein TrmBL1 of P. furiosus is a global regulator and recognizes preferentially, but not exclusively, the TGM (for Thermococcales-glycolytic motif) sequence that is found upstream of the MD system as well as of genes encoding enzymes involved in the glycolytic and the gluconeogenic pathway. It responds to maltose and maltotriose as inducers and functions as repressor for the genes encoding the MD system and glycolytic enzymes, but as activator for genes encoding gluconeogenic enzymes. The TrmB-like protein TrmBL2 of P. furiosus lacks the sugar-binding domain that has been determined in TrmB. It recognizes the MD promoter, but not all TGM harboring promoters. It is evolutionary the most conserved among the Thermococcales. The regulatory range of TrmBL2 remains unclear.

  7. Uncovering the stoichiometry of Pyrococcus furiosus RNase P, a multi-subunit catalytic ribonucleoprotein complex, by surface-induced dissociation and ion mobility mass spectrometry.

    PubMed

    Ma, Xin; Lai, Lien B; Lai, Stella M; Tanimoto, Akiko; Foster, Mark P; Wysocki, Vicki H; Gopalan, Venkat

    2014-10-20

    We demonstrate that surface-induced dissociation (SID) coupled with ion mobility mass spectrometry (IM-MS) is a powerful tool for determining the stoichiometry of a multi-subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg(2+). We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5' maturation. Previous step-wise, Mg(2+)-dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM-MS in resolving conformational heterogeneity and yielding insights on RNP assembly.

  8. The ferredoxin-dependent conversion of glyceraldehyde-3-phosphate in the hyperthermophilic archaeon Pyrococcus furiosus represents a novel site of glycolytic regulation.

    PubMed

    van der Oost, J; Schut, G; Kengen, S W; Hagen, W R; Thomm, M; de Vos, W M

    1998-10-23

    The fermentative conversion of glucose in anaerobic hyperthermophilic Archaea is a variant of the classical Embden-Meyerhof pathway found in Bacteria and Eukarya. A major difference of the archaeal glycolytic pathway concerns the conversion of glyceraldehyde-3-phosphate. In the hyperthermophilic archaeon Pyrococcus furiosus, this reaction is catalyzed by an unique enzyme, glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). Here, we report the isolation, characterization, and transcriptional analysis of the GAPOR-encoding gene. GAPOR is related to a family of ferredoxin-dependent tungsten enzymes in (hyper)thermophilic Archaea and, in addition, to a hypothetical protein in Escherichia coli. Electron paramagnetic resonance analysis of the purified P. furiosus GAPOR protein confirms the anticipated involvement of tungsten in catalysis. During glycolysis in P. furiosus, GAPOR gene expression is induced, whereas the activity of glyceraldehyde-3-phosphate dehydrogenase is repressed. It is discussed that this unprecedented unidirectional reaction couple in the pyrococcal glycolysis and gluconeogenesis gives rise to a novel site of glycolytic regulation that might be widespread among Archaea.

  9. Structural features underlying the selective cleavage of a novel exo-type maltose-forming amylase from Pyrococcus sp. ST04.

    PubMed

    Park, Kwang-Hyun; Jung, Jong-Hyun; Park, Sung-Goo; Lee, Myeong-Eun; Holden, James F; Park, Cheon-Seok; Woo, Eui-Jeon

    2014-06-01

    A novel maltose-forming α-amylase (PSMA) was recently found in the hyperthermophilic archaeon Pyrococcus sp. ST04. This enzyme shows <13% amino-acid sequence identity to other known α-amylases and displays a unique enzymatic property in that it hydrolyzes both α-1,4-glucosidic and α-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner. Here, the crystal structure of PSMA at a resolution of 1.8 Å is reported, showing a tight ring-shaped tetramer with monomers composed of two domains: an N-domain (amino acids 1-341) with a typical GH57 family (β/α)7-barrel fold and a C-domain (amino acids 342-597) composed of α-helical bundles. A small closed cavity observed in proximity to the catalytic residues Glu153 and Asp253 at the domain interface has the appropriate volume and geometry to bind a maltose unit, accounting for the selective exo-type maltose hydrolysis of the enzyme. A narrow gate at the putative subsite +1 formed by residue Phe218 and Phe452 is essential for specific cleavage of glucosidic bonds. The closed cavity at the active site is connected to a short substrate-binding channel that extends to the central hole of the tetramer, exhibiting a geometry that is significantly different from classical maltogenic amylases or β-amylases. The structural features of this novel exo-type maltose-forming α-amylase provide a molecular basis for its unique enzymatic characteristics and for its potential use in industrial applications and protein engineering.

  10. 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

  11. Pyrococcus furiosus flagella: biochemical and transcriptional analyses identify the newly detected flaB0 gene to encode the major flagellin

    PubMed Central

    Näther-Schindler, Daniela J.; Schopf, Simone; Bellack, Annett; Rachel, Reinhard; Wirth, Reinhard

    2014-01-01

    We have described previously that the flagella of the Euryarchaeon Pyrococcus furiosus are multifunctional cell appendages used for swimming, adhesion to surfaces and formation of cell-cell connections. Here, we characterize these organelles with respect to their biochemistry and transcription. Flagella were purified by shearing from cells followed by CsCl-gradient centrifugation and were found to consist mainly of a ca. 30 kDa glycoprotein. Polymerization studies of denatured flagella resulted in an ATP-independent formation of flagella-like filaments. The N-terminal sequence of the main flagellin was determined by Edman degradation, but none of the genes in the complete genome code for a protein with that N-terminus. Therefore, we resequenced the respective region of the genome, thereby discovering that the published genome sequence is not correct. A total of 771 bp are missing in the data base, resulting in the correction of the previously unusual N-terminal sequence of flagellin FlaB1 and in the identification of a third flagellin. To keep in line with the earlier nomenclature we call this flaB0. Very interestingly, the previously not identified flaB0 codes for the major flagellin. Transcriptional analyses of the revised flagellar operon identified various different cotranscripts encoding only a single protein in case of FlaB0 and FlaJ or up to five proteins (FlaB0-FlaD). Analysing the RNA of cells from different growth phases, we found that the length and number of detected cotranscript increased over time suggesting that the flagellar operon is transcribed mostly in late exponential and stationary growth phase. PMID:25566211

  12. TrmB, a sugar sensing regulator of ABC transporter genes in Pyrococcus furiosus exhibits dual promoter specificity and is controlled by different inducers.

    PubMed

    Lee, Sung-Jae; Moulakakis, Christina; Koning, Sonja M; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2005-09-01

    TrmB is the transcriptional repressor for the gene cluster of the trehalose/maltose ABC transporter of the hyperthermophilic archaea Thermococcus litoralis and Pyrococcus furiosus (malE or TM operon), with maltose and trehalose acting as inducers. We found that TrmB (the protein is identical in both organisms) also regulated the transcription of genes encoding a separate maltodextrin ABC transporter in P. furiosus (mdxE or MD operon) with maltotriose, longer maltodextrins and sucrose acting as inducers, but not with maltose or trehalose. In vitro transcription of the malE and the mdxE operons was inhibited by TrmB binding to the different operator sequences. Inhibition of the TM operon was released by maltose and trehalose whereas inhibition of the MD operon was released by maltotriose and larger maltodextrins as well as by sucrose. Scanning mutagenesis of the TM operator revealed the role of the palindromic TACTNNNAGTA sequence for TrmB recognition. TrmB exhibits a broad spectrum of sugar-binding specificity, binding maltose, sucrose, maltotriose and trehalose in decreasing order of affinity, half-maximal binding occurring at 20, 60, 250 and 500 microM substrate concentration respectively. Of all substrates, only maltose shows sigmoidal binding characteristics with a Hill coefficient of 2. As measured by molecular sieve chromatography and cross-linking TrmB behaved as dimer in dilute buffer solution at room temperature. We conclude that TrmB acts as a bifunctional transcriptional regulator acting on two different promoters and being differentially controlled by binding to different sugars. We believe this to represent a novel strategy of prokaryotic transcription regulation.

  13. Impact of Substrate Glycoside Linkage and Elemental Sulfur on Bioenergetics of and Hydrogen Production by the Hyperthermophilic Archaeon Pyrococcus furiosus▿ †

    PubMed Central

    Chou, Chung-Jung; Shockley, Keith R.; Conners, Shannon B.; Lewis, Derrick L.; Comfort, Donald A.; Adams, Michael W. W.; Kelly, Robert M.

    2007-01-01

    Glycoside linkage (cellobiose versus maltose) dramatically influenced bioenergetics to different extents and by different mechanisms in the hyperthermophilic archaeon Pyrococcus furiosus when it was grown in continuous culture at a dilution rate of 0.45 h−1 at 90°C. In the absence of S0, cellobiose-grown cells generated twice as much protein and had 50%-higher specific H2 generation rates than maltose-grown cultures. Addition of S0 to maltose-grown cultures boosted cell protein production fourfold and shifted gas production completely from H2 to H2S. In contrast, the presence of S0 in cellobiose-grown cells caused only a 1.3-fold increase in protein production and an incomplete shift from H2 to H2S production, with 2.5 times more H2 than H2S formed. Transcriptional response analysis revealed that many genes and operons known to be involved in α- or β-glucan uptake and processing were up-regulated in an S0-independent manner. Most differentially transcribed open reading frames (ORFs) responding to S0 in cellobiose-grown cells also responded to S0 in maltose-grown cells; these ORFs included ORFs encoding a membrane-bound oxidoreductase complex (MBX) and two hypothetical proteins (PF2025 and PF2026). However, additional genes (242 genes; 108 genes were up-regulated and 134 genes were down-regulated) were differentially transcribed when S0 was present in the medium of maltose-grown cells, indicating that there were different cellular responses to the two sugars. These results indicate that carbohydrate characteristics (e.g., glycoside linkage) have a major impact on S0 metabolism and hydrogen production in P. furiosus. Furthermore, such issues need to be considered in designing and implementing metabolic strategies for production of biofuel by fermentative anaerobes. PMID:17827328

  14. Cloning and sequencing of the gene encoding glutamine synthetase I from the archaeum Pyrococcus woesei: anomalous phylogenies inferred from analysis of archaeal and bacterial glutamine synthetase I sequences.

    PubMed Central

    Tiboni, O; Cammarano, P; Sanangelantoni, A M

    1993-01-01

    The gene glnA encoding glutamine synthetase I (GSI) from the archaeum Pyrococcus woesei was cloned and sequenced with the Sulfolobus solfataricus glnA gene as the probe. An operon reading frame of 448 amino acids was identified within a DNA segment of 1,528 bp. The encoded protein was 49% identical with the GSI of Methanococcus voltae and exhibited conserved regions characteristic of the GSI family. The P. woesei GSI was aligned with available homologs from other archaea (S. solfataricus, M. voltae) and with representative sequences from cyanobacteria, proteobacteria, and gram-positive bacteria. Phylogenetic trees were constructed from both the amino acid and the nucleotide sequence alignments. In accordance with the sequence similarities, archaeal and bacterial sequences did not segregate on a phylogeny. On the basis of sequence signatures, the GSI trees could be subdivided into two ensembles. One encompassed the GSI of cyanobacteria and proteobacteria, but also that of the high-G + C gram-positive bacterium Streptomyces coelicolor (all of which are regulated by the reversible adenylylation of the enzyme subunits); the other embraced the GSI of the three archaea as well as that of the low-G + C gram-positive bacteria (Clostridium acetobutilycum, Bacillus subtilis) and Thermotoga maritima (none of which are regulated by subunit adenylylation). The GSIs of the Thermotoga and the Bacillus-Clostridium lineages shared a direct common ancestor with that of P. woesei and the methanogens and were unrelated to their homologs from cyanobacteria, proteobacteria, and S. coelicolor. The possibility is presented that the GSI gene arose among the archaea and was then laterally transferred from some early methanogen to a Thermotoga-like organism. However, the relationship of the cyanobacterial-proteobacterial GSIs to the Thermotoga GSI and the GSI of low-G+C gram-positive bacteria remains unexplained. PMID:8098326

  15. 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.

  16. 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

    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.

  17. 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.

  18. 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

  19. Survival and growth of two heterotrophic hydrothermal vent archaea, Pyrococcus strain GB-D and Thermococcus fumicolans, under low pH and high sulfide concentrations in combination with high temperature and pressure regimes.

    PubMed

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

    2007-03-01

    Growth and survival of hyperthermophilic archaea in their extreme hydrothermal vent and subsurface environments are controlled by chemical and physical key parameters. This study examined the effects of elevated sulfide concentrations, temperature, and acidic pH on growth and survival of two hydrothermal vent archaea (Pyrococcus strain GB-D and Thermococcus fumicolans) under high temperature and pressure regimes. These two strains are members of the Thermococcales, a family of hyperthermophilic, heterotrophic, sulfur-reducing archaea that occur in high densities at vent sites. As actively growing cells, these two strains tolerated regimes of pH, pressure, and temperature that were in most cases not tolerated under severe substrate limitation. A moderate pH of 5.5-7 extends their survival and growth range over a wider range of sulfide concentrations, temperature and pressure, relative to lower pH conditions. T. fumicolans and Pyrococcus strain GB-D grew under very high pressures that exceeded in-situ pressures typical of hydrothermal vent depths, and included deep subsurface pressures. However, under the same conditions, but in the absence of carbon substrates and electron acceptors, survival was generally lower, and decreased rapidly when low pH stress was combined with high pressure and high temperature.

  20. Characterization of the TrmB-like protein, PF0124, a TGM-recognizing global transcriptional regulator of the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed

    Lee, Sung-Jae; Surma, Melanie; Seitz, Sabine; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2007-07-01

    The characterization of the transcriptional regulator TrmBL1 of the hyperthermophilic archaeon Pyrococcus furiosus, homologous to TrmB (transcriptional regulator of the maltose system), was studied. The genome of P. furiosus contains three TrmB paralogues. One of the TrmB-like proteins (TrmBL), PF0124 (TrmBL1), was analysed in more detail. It regulated the expression of the genes encoding enzymes of the glycolytic pathway as well as the maltodextrin (MD) ABC transporter. By molecular sieve chromatography, purified TrmBL1 behaved at ambient temperature as a tetramer of 148.8 kDa. In the presence of 1 mM maltotriose or 5 mM maltose TrmBL1 formed octamers. As shown by electrophoretic mobility shift assay (EMSA) TrmBL1 was found to bind the MD (maltodextrin ABC transport genes) promoter DNA with sixfold higher binding affinity (K(d) 0.2 microM) than to the trehalose/maltose ABC transporter (TM) promoter (K(d) 1.2 microM). Maltotriose and maltose interfered in these assays indicating inducer function. In vitro transcription assays using purified transcription components corroborated the data obtained with EMSA and showed inhibition of transcription of the MD promoter by TrmBL1. Recently, van de Werken et al. (FEMS Microbiol Lett 2006; 260: 69-76) identified TGM, a conserved sequence (Thermococcales-Glycolytic-Motif) upstream of genes encoding glycolytic enzymes and the MD ABC transporter. The position of TGM is invariably located downstream of the BRE-TATA box and overlapping the transcription start site on each promoter. By footprint analysis TrmBL1 was found to recognize the TGM sequence in several TGM-containing promoter sequences. We identified the recognition helix in TrmBL1 revealing tyrosine (Y49) to be essential for target DNA binding. However, the TGM motif was not essential for TrmBL1 binding. We conclude that TrmBL1 is a global sugar-sensing transcriptional regulator controlling the genes of transport systems and of sugar-metabolizing enzymes.

  1. Genome Evolution at the Genus Level: Comparison of Three Complete Genomes of Hyperthermophilic Archaea

    PubMed Central

    Lecompte, Odile; Ripp, Raymond; Puzos-Barbe, Valérie; Duprat, Simone; Heilig, Roland; Dietrich, Jacques; Thierry, Jean-Claude; Poch, Olivier

    2001-01-01

    We have compared three complete genomes of closely related hyperthermophilic species of Archaea belonging to the Pyrococcus genus: Pyrococcus abyssi, Pyrococcus horikoshii, and Pyrococcus furiosus. At the genomic level, the comparison reveals a differential conservation among four regions of the Pyrococcus chromosomes correlated with the location of genetic elements mediating DNA reorganization. This discloses the relative contribution of the major mechanisms that promote genomic plasticity in these Archaea, namely rearrangements linked to the replication terminus, insertion sequence-mediated recombinations, and DNA integration within tRNA genes. The combination of these mechanisms leads to a high level of genomic plasticity in these hyperthermophilic Archaea, at least comparable to the plasticity observed between closely related bacteria. At the proteomic level, the comparison of the three Pyrococcus species sheds light on specific selection pressures acting both on their coding capacities and evolutionary rates. Indeed, thanks to two independent methods, the “reciprocal best hits“ approach and a new distance ratio analysis, we detect the false orthology relationships within the Pyrococcus lineage. This reveals a high amount of differential gains and losses of genes since the divergence of the three closely related species. The resulting polymorphism is probably linked to an adaptation of these free-living organisms to differential environmental constraints. As a corollary, we delineate the set of orthologous genes shared by the three species, that is, the genes that may characterize the Pyrococcus genus. In this conserved core, the amino acid substitution rate is equal between P. abyssi and P. horikoshii for most of their shared proteins, even for fast-evolving ones. In contrast, strong discrepancies exist among the substitution rates observed in P. furiosus relative to the two other species, which is in disagreement with the molecular clock hypothesis. PMID

  2. 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

  3. 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

  4. Characterization of an extremely thermostable but cold-adaptive β-galactosidase from the hyperthermophilic archaeon Pyrococcus furiosus for use as a recombinant aggregation for batch lactose degradation at high temperature.

    PubMed

    Dong, Qing; Yan, Xufan; Zheng, Minhui; Yang, Ziwen

    2014-06-01

    β-Galactosidase (lactase), which catalyzes the hydrolysis of lactose into glucose and galactose, is one of the most important enzymes used in dairy processing. In this study, a gene that encoded an extremely thermostable β-galactosidase from Pyrococcus furiosus (Pflactase) was cloned and expressed in Escherichia coli BL21. The recombinant enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The enzyme displayed optimal activity at 90°C and pH 7.0 in phosphate buffer. The specific activity of the recombinant enzyme on o-nitrophenyl-β-d-galactopyranoside was 10.2 U/mg at 0°C and 130.0dU/mg at 90°C. The half-lives of the enzyme were 31423.4, 8168.3, 4017.7, 547.4, 309.6, and 203.5 min at 70°C, 80°C, 85°C, 90°C, 95°C, and 100°C, respectively. The recombinant enzyme exhibited both β-galactosidase and β-glucosidase activity. The active inclusion bodies of β-galactosidase were easily isolated by nonionic detergent treatment and directly used for lactose conversion in a repetitive batch mode. More than 54% (90°C) or 88% (10°C) of the original enzyme activity was retained after 10 conversion cycles under optimum conditions. These results suggest that the recombinant thermostable β-galactosidase may be suitable for the hydrolysis of lactose in milk processing.

  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. 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

  7. Reconstitution of Diphthine Synthase Activity In Vitro

    PubMed Central

    Zhu, Xuling; Kim, Jungwoo; Su, Xiaoyang; Lin, Hening

    2010-01-01

    Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). Although diphthamide modification was discovered three decades ago, in vitro reconstitution of diphthamide biosynthesis using purified proteins has not been reported. The proposed biosynthesis pathway of diphthamide involves three steps. Our laboratory has recently showed that in Pyrococcus horikoshii (P. horikoshii), the first step uses an [4Fe-4S] enzyme PhDph2 to generate a 3-amino-3-carboxypropyl radical from S-adenosyl-L-methionine (SAM) to form a C-C bond. The second step is the trimethylation of an amino group to form the diphthine intermediate. This step is catalyzed by a methyltransferase called diphthine synthase or Dph5. Here we report the in vitro reconstitution of the second step using P. horikoshii Dph5 (PhDph5). Our results demonstrate that PhDph5 is sufficient to catalyze the mono-, di-, and trimethylation of P. horikoshii EF2 (PhEF2). Interestingly, the trimethylated product from PhDph5-catalyzed reaction can easily eliminate the trimethylamino group. The potential implication of this unexpected finding on the diphthamide biosynthesis pathway is discussed. PMID:20873788

  8. Purification and crystallization of the entire recombinant subunit E of the energy producer A(1)A(o) ATP synthase.

    PubMed

    Balakrishna, Asha Manikkoth; Hunke, Cornelia; Grüber, Gerhard

    2010-03-01

    A(1)A(o) ATP synthases are the major energy producers in archaea. Subunit E of the stator domain of the ATP synthase from Pyrococcus horikoshii OT3 was cloned, expressed and purified to homogeneity. The monodispersed protein was crystallized by vapour diffusion. A complete diffraction data set was collected to 3.3 A resolution with 99.4% completeness using a synchrotron-radiation source. The crystals belonged to space group I4, with unit-cell parameters a = 112.51, b = 112.51, c = 96.25 A, and contained three molecules in the asymmetric unit.

  9. Combining biomass wet disk milling and endoglucanase/β-glucosidase hydrolysis for the production of cellulose nanocrystals.

    PubMed

    Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant'Ana; Jang, Jae-Hyuk; Kim, Han-Woo; Ishikawa, Kazuhiko; Endo, Takashi; Lee, Seung-Hwan; Bon, Elba P S

    2015-09-01

    Cellulose nanocrystals (CNCs), a biomaterial with high added value, were obtained from pure cellulose, Eucalyptus holocellulose, unbleached Kraft pulp, and sugarcane bagasse, by fibrillating these biomass substrates using wet disk milling (WDM) followed by enzymatic hydrolysis using endoglucanase/β-glucosidase. The hydrolysis experiments were conducted using the commercial enzyme OptimashBG or a blend of Pyrococcus horikoshii endoglucanase and Pyrococcus furiosus β-glucosidase. The fibrillated materials and CNCs were analyzed by X-ray diffraction, atomic force microscopy, scanning electron microscopy, and the specific surface area (SSA) was measured. WDM resulted in the formation of long and twisted microfibers of 1000-5000 nm in length and 4-35 nm in diameter, which were hydrolyzed into shorter and straighter CNCs of 500-1500 nm in length and 4-12 nm in diameter, with high cellulose crystallinity. Therefore, the CNC's aspect ratio was successfully adjusted by endoglucanases under mild reaction conditions, relative to the reported acidic hydrolysis method.

  10. Use of cellobiohydrolase-free cellulase blends for the hydrolysis of microcrystalline cellulose and sugarcane bagasse pretreated by either ball milling or ionic liquid [Emim][Ac].

    PubMed

    Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant'Ana; Kim, Han-Woo; Ishikawa, Kazuhiko; Endo, Takashi; Lee, Seung-Hwan; Bon, Elba P S

    2013-12-01

    This study investigated the requirement of cellobiohydrolases (CBH) for saccharification of microcrystalline cellulose and sugarcane bagasse pretreated either by ball milling (BM) or by ionic liquid (IL) [Emim][Ac]. Hydrolysis was done using CBH-free blends of Pyrococcus horikoshii endoglucanase (EG) plus Pyrococcus furiosus β-glucosidase (EGPh/BGPf) or Optimash™ BG while Acremonium Cellulase was used as control. IL-pretreated substrates were hydrolyzed more effectively by CBH-free enzymes than were the BM-pretreated substrates. IL-treatment decreased the crystallinity and increased the specific surface area (SSA), whereas BM-treatment decreased the crystallinity without increasing the SSA. The hydrolysis of IL-treated cellulose by EGPh/BGPf showed a saccharification rate of 3.92 g/Lh and a glucose yield of 81% within 9h. These results indicate the efficiency of CBH-free enzymes for the hydrolysis of IL-treated substrates.

  11. Coevolution of RtcB and Archease created a multiple-turnover RNA ligase.

    PubMed

    Desai, Kevin K; Beltrame, Amanda L; Raines, Ronald T

    2015-11-01

    RtcB is a noncanonical RNA ligase that joins either 2',3'-cyclic phosphate or 3'-phosphate termini to 5'-hydroxyl termini. The genes encoding RtcB and Archease constitute a tRNA splicing operon in many organisms. Archease is a cofactor of RtcB that accelerates RNA ligation and alters the NTP specificity of the ligase from Pyrococcus horikoshii. Yet, not all organisms that encode RtcB also encode Archease. Here we sought to understand the differences between Archease-dependent and Archease-independent RtcBs so as to illuminate the evolution of Archease and its function. We report on the Archease-dependent RtcB from Thermus thermophilus and the Archease-independent RtcB from Thermobifida fusca. We find that RtcB from T. thermophilus can catalyze multiple turnovers only in the presence of Archease. Remarkably, Archease from P. horikoshii can activate T. thermophilus RtcB, despite low sequence identity between the Archeases from these two organisms. In contrast, RtcB from T. fusca is a single-turnover enzyme that is unable to be converted into a multiple-turnover ligase by Archease from either P. horikoshii or T. thermophilus. Thus, our data indicate that Archease likely evolved to support multiple-turnover activity of RtcB and that coevolution of the two proteins is necessary for a functional interaction.

  12. 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.

  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. 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

  15. 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

  16. A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

    PubMed Central

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

    2014-01-01

    Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2′,3′-cyclic phosphate or 3′-phosphate termini to 5′-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3′-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal structure of P. horikoshii Archease that reveals a metal-binding site consisting of conserved carboxylates located at the protein tip. Substitution of the Archease metal-binding residues drastically reduced Archease-dependent activation of RtcB. Thus, evolution has sought to co-express archease and rtcB by creating a tRNA splicing operon. PMID:24435797

  17. 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

  18. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    SciTech Connect

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  19. Pyrococcus furiosus-immobilized anodized tubular titania cathode in a hydrogen production system

    NASA Astrophysics Data System (ADS)

    Yoon, Jaekyung; Bae, Sanghyun; Shim, Eunjung; Joo, Hyunku

    Anodized tubular titania (TiO 2) electrodes (ATTEs) are prepared and used as both the photoanode and the cathode substrate in a photoelectrochemical system designed to split water into hydrogen with the assistance of an enzyme and an external bias of 1.5 V. In particular, the ATTE used as the cathode substrate for the immobilization of the enzyme is prepared by two methods-adsorption and crosslinking. Results show that the optimized amount of enzyme is 10.98 units for the slurried enzyme, 3.66 units for the adsorbed one and 7.32 units for the crosslinked one, and the corresponding hydrogen evolution rates are 33.04, 148.58 and 234.88 μmol h -1, respectively. The immobilized enzyme, specifically the chemically crosslinked one, seems to be much superior to the slurried enzyme, due to the enhanced charge-transfer process that is caused by the lower electrical resistance between the enzyme and the ATTE. This results in a greater number of accepted electrons and a larger amount of enzymes able to deal with the electrons.

  20. 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.

  1. 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

  2. Structure of the dodecamer of the aminopeptidase APDkam598 from the archaeon Desulfurococcus kamchatkensis.

    PubMed

    Petrova, T E; Slutskaya, E S; Boyko, K M; Sokolova, O S; Rakitina, T V; Korzhenevskiy, D A; Gorbacheva, M A; Bezsudnova, E Y; Popov, V O

    2015-03-01

    The crystal structure of the aminopeptidase APDkam589 from the thermophilic crenarchaeon Desulfurococcus kamchatkensis was determined at a resolution of 3.0 Å. In the crystal, the monomer of APDkam589 and its symmetry-related monomers are densely packed to form a 12-subunit complex. Single-particle electron-microscopy analysis confirms that APDkam589 is present as a compact dodecamer in solution. The APDkam589 molecule is built similarly to the molecules of the PhTET peptidases, which have the highest sequence identity to APDkam589 among known structures and were isolated from the more thermostable archaeon Pyrococcus horikoshii. A comparison of the interactions of the subunits in APDkam589 with those in PhTET1, PhTET2 and PhTET3 reveals that APDkam589 has a much lower total number of salt bridges, which correlates with the lower thermostability of APDkam589. The monomer of APDkam589 has six Trp residues, five of which are on the external surface of the dodecamer. A superposition of the structure of APDkam589 with those having a high sequence similarity to APDkam589 reveals that, although the positions of Trp45, Trp252 and Trp358 are not conserved in the sequences, the spatial locations of the Trp residues in these models are similar.

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

    PubMed

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

    2014-11-01

    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.

  4. Structural and functional analysis of the archaeal endonuclease Nob1

    PubMed Central

    Veith, Thomas; Martin, Roman; Wurm, Jan P.; Weis, Benjamin L.; Duchardt-Ferner, Elke; Safferthal, Charlotta; Hennig, Raoul; Mirus, Oliver; Bohnsack, Markus T.; Wöhnert, Jens; Schleiff, Enrico

    2012-01-01

    Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site. PMID:22156373

  5. 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

  6. 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

  7. Crystal structure of a signal recognition particle Alu domain in the elongation arrest conformation.

    PubMed

    Bousset, Luc; Mary, Camille; Brooks, Mark A; Scherrer, Anne; Strub, Katharina; Cusack, Stephen

    2014-12-01

    The signal recognition particle (SRP) is a conserved ribonucleoprotein particle that targets membrane and secreted proteins to translocation channels in membranes. In eukaryotes, the Alu domain, which comprises the 5' and 3' extremities of the SRP RNA bound to the SRP9/14 heterodimer, is thought to interact with the ribosome to pause translation elongation during membrane docking. We present the 3.2 Å resolution crystal structure of a chimeric Alu domain, comprising Alu RNA from the archaeon Pyrococcus horikoshii bound to the human Alu binding proteins SRP9/14. The structure reveals how intricate tertiary interactions stabilize the RNA 5' domain structure and how an extra, archaeal-specific, terminal stem helps constrain the Alu RNA into the active closed conformation. In this conformation, highly conserved noncanonical base pairs allow unusually tight side-by-side packing of 5' and 3' RNA stems within the SRP9/14 RNA binding surface. The biological relevance of this structure is confirmed by showing that a reconstituted full-length chimeric archaeal-human SRP is competent to elicit elongation arrest in vitro. The structure will be useful in refining our understanding of how the SRP Alu domain interacts with the ribosome.

  8. Comparison of three ionic liquid-tolerant cellulases by molecular dynamics.

    PubMed

    Jaeger, Vance; Burney, Patrick; Pfaendtner, Jim

    2015-02-17

    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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. Biochemical Characterization of the C4-Dicarboxylate Transporter DctA from Bacillus subtilis▿

    PubMed Central

    Groeneveld, Maarten; Detert Oude Weme, Ruud G. J.; Duurkens, Ria H.; Slotboom, Dirk Jan

    2010-01-01

    Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C4-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C4-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter GltPh from Pyrococcus horikoshii. PMID:20363944

  14. 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.

  15. 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

  16. Structure of RNA 3′-phosphate cyclase bound to substrate RNA

    PubMed Central

    Desai, Kevin K.; Bingman, Craig A.; Cheng, Chin L.; Phillips, George N.

    2014-01-01

    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 Nε 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

  17. 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.

  18. 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

  19. 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.

  20. Molecular dynamics simulations of the Nip7 proteins from the marine deep- and shallow-water Pyrococcus species

    PubMed Central

    2014-01-01

    Background The identification of the mechanisms of adaptation of protein structures to extreme environmental conditions is a challenging task of structural biology. We performed molecular dynamics (MD) simulations of the Nip7 protein involved in RNA processing from the shallow-water (P. furiosus) and the deep-water (P. abyssi) marine hyperthermophylic archaea at different temperatures (300 and 373 K) and pressures (0.1, 50 and 100 MPa). The aim was to disclose similarities and differences between the deep- and shallow-sea protein models at different temperatures and pressures. Results The current results demonstrate that the 3D models of the two proteins at all the examined values of pressures and temperatures are compact, stable and similar to the known crystal structure of the P. abyssi Nip7. The structural deviations and fluctuations in the polypeptide chain during the MD simulations were the most pronounced in the loop regions, their magnitude being larger for the C-terminal domain in both proteins. A number of highly mobile segments the protein globule presumably involved in protein-protein interactions were identified. Regions of the polypeptide chain with significant difference in conformational dynamics between the deep- and shallow-water proteins were identified. Conclusions The results of our analysis demonstrated that in the examined ranges of temperatures and pressures, increase in temperature has a stronger effect on change in the dynamic properties of the protein globule than the increase in pressure. The conformational changes of both the deep- and shallow-sea protein models under increasing temperature and pressure are non-uniform. Our current results indicate that amino acid substitutions between shallow- and deep-water proteins only slightly affect overall stability of two proteins. Rather, they may affect the interactions of the Nip7 protein with its protein or RNA partners. PMID:25315147

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

    PubMed

    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.

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

    PubMed

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

    2016-02-29

    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, tRNA(Leu) 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 tRNA(Leu). Purified ArcTGT from Pyrococcus horikoshii, for which the tRNA recognition mechanism and structure were previously characterized, exchanged only the G15 base in a tRNA(Leu) transcript with (14)C-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 tRNA(Leu) variants were expressed. Mass spectrometry analysis of purified tRNA(Leu) variants revealed the modifications of G(+)13 and G(+)15 in the wild-type tRNA(Leu). Thus, T. acidophilum ArcTGT has a multisite specificity and is responsible for the formation of both G(+)13 and G(+)15 modifications.

  3. Anticodon Recognition and Discrimination by the α-Helix Cage Domain of Class I Lysyl-tRNA Synthetase†

    PubMed Central

    Levengood, Jeffrey D.; Roy, Hervé; Ishitani, Ryuichiro; Söll, Dieter; Nureki, Osamu; Ibba, Michael

    2008-01-01

    Aminoacyl-tRNA synthetases are normally found in one of two mutually exclusive structural classes, the only known exception being lysyl-tRNA synthetase which exists in both classes I (LysRS1) and II (LysRS2). Differences in tRNA acceptor stem recognition between LysRS1 and LysRS2 do not drastically impact cellular aminoacylation levels, focusing attention on the mechanism of tRNA anticodon recognition by LysRS1. On the basis of structure-based sequence alignments, seven tRNALys anticodon variants and seven LysRS1 anticodon binding site variants were selected for analysis of the Pyrococcus horikoshii LysRS1–tRNALys docking model. LysRS1 specifically recognized the bases at positions 35 and 36, but not that at position 34. Aromatic residues form stacking interactions with U34 and U35, and aminoacylation kinetics also identified direct interactions between Arg502 and both U35 and U36. Tyr491 was also found to interact with U36, and the Y491E variant exhibited significant improvement compared to the wild type in aminoacylation of a tRNALysUUG mutant. Refinement of the LysRS1–tRNALys docking model based upon these data suggested that anticodon recognition by LysRS1 relies on considerably fewer interactions than that by LysRS2, providing a structural basis for the more significant role of the anticodon in tRNA recognition by the class II enzyme. To date, only glutamyl-tRNA synthetase (GluRS) has been found to contain an α-helix cage anticodon binding domain homologous to that of LysRS1, and these data now suggest that specificity for the anticodon of tRNALys could have been acquired through relatively few changes to the corresponding domain of an ancestral GluRS enzyme. PMID:17760422

  4. 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.

  5. 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

  6. Dph3 Is an Electron Donor for Dph1-Dph2 in the First Step of Eukaryotic Diphthamide Biosynthesis

    PubMed Central

    2015-01-01

    Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. The biosynthesis of diphthamide was proposed to involve three steps. The first step is the transfer of the 3-amino-3-carboxypropyl group from S-adenosyl-l-methionine (SAM) to the histidine residue of EF2, forming a C–C bond. Previous genetic studies showed this step requires four proteins in eukaryotes, Dph1–Dph4. However, the exact molecular functions for the four proteins are unknown. Previous study showed that Pyrococcus horikoshii Dph2 (PhDph2), a novel iron-sulfur cluster-containing enzyme, forms a homodimer and is sufficient for the first step of diphthamide biosynthesis in vitro. Here we demonstrate by in vitro reconstitution that yeast Dph1 and Dph2 form a complex (Dph1-Dph2) that is equivalent to the homodimer of PhDph2 and is sufficient to catalyze the first step in vitro in the presence of dithionite as the reductant. We further demonstrate that yeast Dph3 (also known as KTI11), a CSL-type zinc finger protein, can bind iron and in the reduced state can serve as an electron donor to reduce the Fe-S cluster in Dph1-Dph2. Our study thus firmly establishes the functions for three of the proteins involved in eukaryotic diphthamide biosynthesis. For most radical SAM enzymes in bacteria, flavodoxins and flavodoxin reductases are believed to serve as electron donors for the Fe-S clusters. The finding that Dph3 is an electron donor for the Fe-S clusters in Dph1-Dph2 is thus interesting and opens up new avenues of research on electron transfer to Fe-S proteins in eukaryotic cells. PMID:24422557

  7. Dph3 is an electron donor for Dph1-Dph2 in the first step of eukaryotic diphthamide biosynthesis.

    PubMed

    Dong, Min; Su, Xiaoyang; Dzikovski, Boris; Dando, Emily E; Zhu, Xuling; Du, Jintang; Freed, Jack H; Lin, Hening

    2014-02-01

    Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. The biosynthesis of diphthamide was proposed to involve three steps. The first step is the transfer of the 3-amino-3-carboxypropyl group from S-adenosyl-l-methionine (SAM) to the histidine residue of EF2, forming a C-C bond. Previous genetic studies showed this step requires four proteins in eukaryotes, Dph1-Dph4. However, the exact molecular functions for the four proteins are unknown. Previous study showed that Pyrococcus horikoshii Dph2 (PhDph2), a novel iron-sulfur cluster-containing enzyme, forms a homodimer and is sufficient for the first step of diphthamide biosynthesis in vitro. Here we demonstrate by in vitro reconstitution that yeast Dph1 and Dph2 form a complex (Dph1-Dph2) that is equivalent to the homodimer of PhDph2 and is sufficient to catalyze the first step in vitro in the presence of dithionite as the reductant. We further demonstrate that yeast Dph3 (also known as KTI11), a CSL-type zinc finger protein, can bind iron and in the reduced state can serve as an electron donor to reduce the Fe-S cluster in Dph1-Dph2. Our study thus firmly establishes the functions for three of the proteins involved in eukaryotic diphthamide biosynthesis. For most radical SAM enzymes in bacteria, flavodoxins and flavodoxin reductases are believed to serve as electron donors for the Fe-S clusters. The finding that Dph3 is an electron donor for the Fe-S clusters in Dph1-Dph2 is thus interesting and opens up new avenues of research on electron transfer to Fe-S proteins in eukaryotic cells.

  8. 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.

  9. 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

  10. The structure of the archaebacterial ribosomal protein S7 and its possible interaction with 16S rRNA.

    PubMed

    Hosaka, H; Yao, M; Kimura, M; Tanaka, I

    2001-11-01

    Ribosomal protein S7 is one of the ubiquitous components of the small subunit of the ribosome. It is a 16S rRNA-binding protein positioned close to the exit of the tRNA, and it plays a role in initiating assembly of the head of the 30S subunit. Previous structural analyses of eubacterial S7 have shown that it has a stable alpha-helix core and a flexible beta-arm. Unlike these eubacterial proteins, archaebacterial or eukaryotic S7 has an N-terminal extension of approximately 60 residues. The crystal structure of S7 from archaebacterium Pyrococcus horikoshii (PhoS7) has been determined at 2.1 A resolution. The final model of PhoS7 consists of six major alpha-helices, a short 3(10)-helix and two beta-stands. The major part (residues 18-45) of the N-terminal extension of PhoS7 reinforces the alpha-helical core by well-extended hydrophobic interactions, while the other part (residues 46-63) is not visible in the crystal and is possibly fixed only by interacting with 16S rRNA. These differences in the N-terminal extension as well as in the insertion (between alpha1 and alpha2) of the archaebacterial S7 structure from eubacterial S7 are such that they do not necessitate a major change in the structure of the currently available eubacterial 16S rRNA. Some of the inserted chains might pass through gaps formed by helices of the 16S rRNA.

  11. 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

  12. 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

  13. Transport rates of a glutamate transporter homologue are influenced by the lipid bilayer.

    PubMed

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

    2015-04-10

    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.

  14. The three-dimensional structure of TrmB, a transcriptional regulator of dual function in the hyperthermophilic archaeon Pyrococcus furiosus in complex with sucrose.

    PubMed

    Krug, Michael; Lee, Sung-Jae; Boos, Winfried; Diederichs, Kay; Welte, Wolfram

    2013-06-01

    TrmB is a repressor that binds maltose, maltotriose, and sucrose, as well as other α-glucosides. It recognizes two different operator sequences controlling the TM (Trehalose/Maltose) and the MD (Maltodextrin) operon encoding the respective ABC transporters and sugar-degrading enzymes. Binding of maltose to TrmB abrogates repression of the TM operon but maintains the repression of the MD operon. On the other hand, binding of sucrose abrogates repression of the MD operon but maintains repression of the TM operon. The three-dimensional structure of TrmB in complex with sucrose was solved and refined to a resolution of 3.0 Å. The structure shows the N-terminal DNA binding domain containing a winged-helix-turn-helix (wHTH) domain followed by an amphipathic helix with a coiled-coil motif. The latter promotes dimerization and places the symmetry mates of the putative recognition helix in the wHTH motif about 30 Å apart suggesting a canonical binding to two successive major grooves of duplex palindromic DNA. This suggests that the structure resembles the conformation of TrmB recognizing the pseudopalindromic TM promoter but not the conformation recognizing the nonpalindromic MD promoter.

  15. The three-dimensional structure of TrmB, a transcriptional regulator of dual function in the hyperthermophilic archaeon Pyrococcus furiosus in complex with sucrose

    PubMed Central

    Krug, Michael; Lee, Sung-Jae; Boos, Winfried; Diederichs, Kay; Welte, Wolfram

    2013-01-01

    TrmB is a repressor that binds maltose, maltotriose, and sucrose, as well as other α-glucosides. It recognizes two different operator sequences controlling the TM (Trehalose/Maltose) and the MD (Maltodextrin) operon encoding the respective ABC transporters and sugar-degrading enzymes. Binding of maltose to TrmB abrogates repression of the TM operon but maintains the repression of the MD operon. On the other hand, binding of sucrose abrogates repression of the MD operon but maintains repression of the TM operon. The three-dimensional structure of TrmB in complex with sucrose was solved and refined to a resolution of 3.0 Å. The structure shows the N-terminal DNA binding domain containing a winged-helix-turn-helix (wHTH) domain followed by an amphipathic helix with a coiled-coil motif. The latter promotes dimerization and places the symmetry mates of the putative recognition helix in the wHTH motif about 30 Å apart suggesting a canonical binding to two successive major grooves of duplex palindromic DNA. This suggests that the structure resembles the conformation of TrmB recognizing the pseudopalindromic TM promoter but not the conformation recognizing the nonpalindromic MD promoter. PMID:23576322

  16. 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.

  17. 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

  18. 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

  19. 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

  20. A complex standard for protein identification, designed by evolution.

    PubMed

    Vaudel, Marc; Burkhart, Julia M; Breiter, Daniela; Zahedi, René P; Sickmann, Albert; Martens, Lennart

    2012-10-01

    Shotgun proteomic investigations rely on the algorithmic assignment of mass spectra to peptides. The quality of these matches is therefore a cornerstone in the analysis and has been the subject of numerous recent developments. In order to establish the benefits of novel algorithms, they are applied to reference samples of known content. However, these were recently shown to be either too simple to resemble typical real-life samples or as leading to results of lower accuracy as the method itself. Here, we describe how to use the proteome of Pyrococcus furiosus , a hyperthermophile, as a standard to evaluate proteomics identification workflows. Indeed, we prove that the Pyrococcus furiosus proteome provides a valid method for detecting random hits, comparable to the decoy databases currently in popular use, but we also prove that the Pyrococcus furiosus proteome goes squarely beyond the decoy approach by also providing many hundreds of highly reliable true positive hits. Searching the Pyrococcus furiosus proteome can thus be used as a unique test that provides the ability to reliably detect both false positives as well as proteome-scale true positives, allowing the rigorous testing of identification algorithms at the peptide and protein level.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    SciTech Connect

    Woodward, J.; Mattingly, S.M.; Danson, M.

    1996-07-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based on 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 the continuous recycling of cofactor. 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 chemical commodity. 23 refs., 5 figs.

  8. Interpretation of nuclear resonant vibrational spectra of rubredoxin using a combined quantum mechanics and molecular mechanics approach.

    PubMed

    Paulsen, Hauke; Trautwein, Alfred X; Wegner, Patrick; Schmidt, Christian; Chumakov, Aleksandr I; Schünemann, Volker

    2011-12-01

    Nuclear resonant vibrational spectra of the reduced and oxidized form of a mutant of rubredoxin from Pyrococcus abyssii were measured and are compared with simulated spectra that were calculated by a combined quantum mechanics (QM) and molecular mechanics (MM) method. Density functional theory was used for the QM level. Calculations were performed for different models of rubredoxin. Realistic spectra were simulated with reduced models that include at least the iron center, the four cysteins coordinating it, and the residues connected to the cysteins together with a QM layer that comprises the first two coordination shells of the iron center. Larger QM layers did not lead to significant changes of the simulated spectra.

  9. 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.

  10. 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.

  11. Expression, purification, crystallization and preliminary crystallographic analysis of the PAB0955 gene product

    PubMed Central

    Gras, Stéphanie; Fernandez, Bernard; Chaumont, Valérie; Carpentier, Philippe; Armengaud, Jean; Housset, Dominique

    2005-01-01

    PAB0955 from Pyrococcus abyssi is a prototype of a new Walker-type ATPase/GTPase conserved in archaea and eukaryota but not found in bacteria. PAB0955 has been expressed, purified and crystallized, and it has been shown that this thermostable protein is dimeric in reductive conditions. Crystals have been obtained either without nucleotide or in the presence of GDP or GTPγS. Preliminary X-ray crystallographic data up to 2.08 Å resolution have been collected from these crystals. PMID:16510996

  12. In vitro replication slippage by DNA polymerases from thermophilic organisms.

    PubMed

    Viguera, E; Canceill, D; Ehrlich, S D

    2001-09-14

    Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base.

  13. 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 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

  14. Effects of Dissolved Sulfide, pH, and Temperature on Growth and Survival of Marine Hyperthermophilic Archaea

    PubMed Central

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

    2005-01-01

    The ability of metabolically diverse hyperthermophilic archaea to withstand high temperatures, low pHs, high sulfide concentrations, and the absence of carbon and energy sources was investigated. Close relatives of our study organisms, Methanocaldococcus jannaschii, Archaeoglobus profundus, Thermococcus fumicolans, and Pyrococcus sp. strain GB-D, are commonly found in hydrothermal vent chimney walls and hot sediments and possibly deeper in the subsurface, where highly dynamic hydrothermal flow patterns and steep chemical and temperature gradients provide an ever-changing mosaic of microhabitats. These organisms (with the possible exception of Pyrococcus strain GB-D) tolerated greater extremes of low pH, high sulfide concentration, and high temperature when actively growing and metabolizing than when starved of carbon sources and electron donors/acceptors. Therefore these organisms must be actively metabolizing in the hydrothermal vent chimneys, sediments, and subsurface in order to withstand at least 24 h of exposure to extremes of pH, sulfide, and temperature that occur in these environments. PMID:16204562

  15. 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

  16. Recent advances in genetic analyses of hyperthermophilic archaea and bacteria.

    PubMed

    Noll, K M; Vargas, M

    1997-08-01

    Hyperthermophilic Archaea and Bacteria are an extraordinarily important class of organisms for which genetic tools remain to be developed. Unique technological obstacles to this goal are posed by the thermophilic and, in some cases, strictly anaerobic nature of these organisms. However, recent advances in the cultivation of hyperthermophiles, in the discovery of genetic elements for vector development, and in the construction of genetic markers point toward the achievement of this goal in the near future. Transformation protocols have already been reported for Sulfolobus and Pyrococcus, and plasmid-mediated conjugation was recently found in Sulfolobus. Plasmids are available for Sulfolobus, Pyrococcus, and the bacterial hyperthermophile Thermotoga, and these provide the bases for vector construction in these hosts. A Desulfurococcus mobile intron may provide a novel means to introduce genes into a variety of archaeal hosts. With full genome sequences of several hyperthermophiles available soon, genetic tools will allow full exploitation of this information to study these organisms in depth and to utilize their unique properties in biotechnological applications.

  17. Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic Bacteria and Archaea.

    PubMed

    Leduc, D; Graziani, S; Meslet-Cladiere, L; Sodolescu, A; Liebl, U; Myllykallio, H

    2004-04-01

    The hyperthermophilic anaerobic archaeon Pyrococcus abyssi, which lacks thymidine kinase, incorporates label from extracellular uracil, but not from thymidine, into its DNA. This implies that P. abyssi must synthesize dTMP (thymidylate), an essential precursor for DNA synthesis, de novo. However, iterative similarity searches of the three completed Pyrococcus genomes fail to detect candidate genes for canonical thymidylate synthase ThyA, suggesting the presence of alternative pathways for dTMP synthesis. Indeed, by identifying a novel class of flavin-dependent thymidylate synthases, ThyX, we have recently proven that two distinct pathways for de novo synthesis of dTMP are operational in the microbial world. While both thyX and thyA can be found in hyperthermophilic micro-organisms, the phylogenetic distribution of thyX among hyperthermophiles is wider than that of thyA. In this contribution, we discuss the differences in the distinct mechanisms of dTMP synthesis, with a special emphasis on hyperthermophilic micro-organisms.

  18. 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.

  19. Antibiofilm activity of coral-associated bacteria against different clinical M serotypes of Streptococcus pyogenes.

    PubMed

    Thenmozhi, Ramalingam; Nithyanand, Paramasivam; Rathna, Janarthanam; Pandian, Shunmugiah Karutha

    2009-12-01

    Streptococcus pyogenes is the frequent cause of purulent infections in humans. Formation of a biofilm is one of the important aspects of its pathogenicity. Streptococcus pyogenes biofilm communities tend to exhibit significant tolerance to antimicrobial challenge during infections. Exploring novel targets against biofilm-forming pathogens is therefore an important alternative treatment measure. We attempted to screen marine bacteria, especially coral-associated bacteria (CAB), for antibiofilm activity against streptococcal biofilm formation. The bacterial biofilms were quantified by crystal violet staining. Of 43 CAB isolates, nine clearly demonstrated antibiofilm activity. At biofilm inhibitory concentrations (BIC), biofilm formation was reduced up to 80%, and sub-BIC (0.5 and 0.25 BIC) significantly reduced biofilm formation by up to 60% and 40-60%, respectively. Extracts of Bacillus horikoshii (E6) displayed efficient antibiofilm activity. As quorum sensing (QS) and cell surface hydrophobicity (CSH) are crucial factors for biofilm formation in S. pyogenes, the CAB were further screened for QS inhibition properties and CSH reduction properties. This study reveals the antibiofilm and QS inhibition property of CAB. PMID:19845763

  20. 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

  1. Global transcriptional regulator TrmB family members in prokaryotes.

    PubMed

    Kim, Minwook; Park, Soyoung; Lee, Sung-Jae

    2016-10-01

    Members of the TrmB family act as global transcriptional regulators for the activation or repression of sugar ABC transporters and central sugar metabolic pathways, including glycolytic, gluconeogenic, and other metabolic pathways, and also as chromosomal stabilizers in archaea. As a relatively newly classified transcriptional regulator family, there is limited experimental evidence for their role in Thermococcales, halophilic archaeon Halobacterium salinarum NRC1, and crenarchaea Sulfolobus strains, despite being one of the extending protein families in archaea. Recently, the protein structures of Pyrococcus furiosus TrmB and TrmBL2 were solved, and the transcriptomic data uncovered by microarray and ChIP-Seq were published. In the present review, recent evidence of the functional roles of TrmB family members in archaea is explained and extended to bacteria. PMID:27687225

  2. Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin.

    PubMed

    Matsunaga, Fujihiko; Norais, Cédric; Forterre, Patrick; Myllykallio, Hannu

    2003-02-01

    Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.

  3. 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.

  4. 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.

  5. Proteolysis in hyperthermophilic microorganisms

    DOE PAGES

    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

  6. Enzymatic production of hydrogen gas from glucose and cellulose

    SciTech Connect

    Mattingly, S.M.; Woodward, J.

    1996-10-01

    An enzymatic process has been used to convert glucose to molecular hydrogen with the ultimate goal of converting cellulose to hydrogen. Two enzymes from the Archae, Thermoplasma acidophilium glucose dehydrogenase (GDH) and Pyrococcus furiosus hydrogenase, were used to oxidize glucose and NADPH respectively, resulting in the formation of molecular hydrogen. The stoichiometric yield of hydrogen from glucose was close to the theoretical maximum expected. Further, the molar amount of hydrogen produced was greater than the molar equivalent of NADP{sup +} present in the reaction mixture indicating that this GDH cofactor was regenerated throughout the course of the reaction. Hydrogen was also shown to be produced from cellulose if cellulase was included in the reaction mixture.

  7. Electroanalytical determination of tungsten and molybdenum in proteins.

    PubMed

    Hagedoorn, P L; van't Slot, P; van Leeuwen, H P; Hagen, W R

    2001-10-01

    Recent crystal structure determinations accelerated the progress in the biochemistry of tungsten-containing enzymes. In order to characterize these enzymes, a sensitive determination of this metal in protein-containing samples is necessary. An electroanalytical tungsten determination has successfully been adapted to determine the tungsten and molybdenum content in enzymes. The tungsten and molybdenum content can be measured simultaneously from 1 to 10 microg of purified protein with little or no sample handling. More crude protein samples require precipitation of interfering surface active material with 10% perchloric acid. This method affords the isolation of novel molybdenum- and tungsten-containing proteins via molybdenum and tungsten monitoring of column fractions, without using radioactive isotopes. A screening of soluble proteins from Pyrococcus furiosus for tungsten, using anion-exchange column chromatography to separate the proteins, has been performed. The three known tungsten-containing enzymes from P. furiosus were recovered with this screening.

  8. 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

  9. Thermostable tag (TST) protein expression system: engineering thermotolerant recombinant proteins and vaccines.

    PubMed

    Luke, Jeremy M; Carnes, Aaron E; Sun, Ping; Hodgson, Clague P; Waugh, David S; Williams, James A

    2011-02-10

    Methods to increase temperature stability of vaccines and adjuvants are needed to reduce dependence on cold chain storage. We report herein creation and application of pVEX expression vectors to improve vaccine and adjuvant manufacture and thermostability. Defined media fermentation yields of 6g/L thermostable toll-like receptor 5 agonist flagellin were obtained using an IPTG inducible pVEX-flagellin expression vector. Alternative pVEX vectors encoding Pyrococcus furiosus maltodextrin-binding protein (pfMBP) as a fusion partner improved Influenza hemagglutinin antigen vaccine solubility and thermostability. A pfMBP hemagglutinin HA2 domain fusion protein was a potent immunogen. Manufacturing processes that combined up to 5 g/L defined media fermentation yields with rapid, selective, thermostable pfMBP fusion protein purification were developed. The pVEX pfMBP-based thermostable tag (TST) platform is a generic protein engineering approach to enable high yield manufacture of thermostable recombinant protein vaccine components.

  10. Global transcriptional regulator TrmB family members in prokaryotes.

    PubMed

    Kim, Minwook; Park, Soyoung; Lee, Sung-Jae

    2016-10-01

    Members of the TrmB family act as global transcriptional regulators for the activation or repression of sugar ABC transporters and central sugar metabolic pathways, including glycolytic, gluconeogenic, and other metabolic pathways, and also as chromosomal stabilizers in archaea. As a relatively newly classified transcriptional regulator family, there is limited experimental evidence for their role in Thermococcales, halophilic archaeon Halobacterium salinarum NRC1, and crenarchaea Sulfolobus strains, despite being one of the extending protein families in archaea. Recently, the protein structures of Pyrococcus furiosus TrmB and TrmBL2 were solved, and the transcriptomic data uncovered by microarray and ChIP-Seq were published. In the present review, recent evidence of the functional roles of TrmB family members in archaea is explained and extended to bacteria.

  11. Evolution of translational elongation factor (EF) sequences: reliability of global phylogenies inferred from EF-1 alpha(Tu) and EF-2(G) proteins.

    PubMed Central

    Creti, R; Ceccarelli, E; Bocchetta, M; Sanangelantoni, A M; Tiboni, O; Palm, P; Cammarano, P

    1994-01-01

    The EF-2 coding genes of the Archaea Pyrococcus woesei and Desulfurococcus mobilis were cloned and sequenced. Global phylogenies were inferred by alternative tree-making methods from available EF-2(G) sequence data and contrasted with phylogenies constructed from the more conserved but shorter EF-1 alpha(Tu) sequences. Both the monophyly (sensu Hennig) of Archaea and their subdivision into the kingdoms Crenarchaeota and Euryarchaeota are consistently inferred by analysis of EF-2(G) sequences, usually at a high bootstrap confidence level. In contrast, EF-1 alpha(Tu) phylogenies tend to be inconsistent with one another and show low bootstrap confidence levels. While evolutionary distance and DNA maximum parsimony analyses of EF-1 alpha(Tu) sequences do show archaeal monophyly, protein parsimony and DNA maximum-likelihood analyses of these data do not. In no case, however, do any of the tree topologies inferred from EF-1 alpha(Tu) sequence analyses receive significant bootstrap support. PMID:8159735

  12. The Lrp family of transcription regulators in archaea.

    PubMed

    Peeters, Eveline; Charlier, Daniel

    2010-11-30

    Archaea possess a eukaryotic-type basal transcription apparatus that is regulated by bacteria-like transcription regulators. A universal and abundant family of transcription regulators are the bacterial/archaeal Lrp-like regulators. The Lrp family is one of the best studied regulator families in archaea, illustrated by investigations of proteins from the archaeal model organisms: Sulfolobus, Pyrococcus, Methanocaldococcus, and Halobacterium. These regulators are extremely versatile in their DNA-binding properties, response to effector molecules, and molecular regulatory mechanisms. Besides being involved in the regulation of the amino acid metabolism, they also regulate central metabolic processes. It appears that these regulatory proteins are also involved in large regulatory networks, because of hierarchical regulations and the possible combinatorial use of different Lrp-like proteins. Here, we discuss the recent developments in our understanding of this important class of regulators.

  13. DJ-1 family Maillard deglycases prevent acrylamide formation.

    PubMed

    Richarme, Gilbert; Marguet, Evelyne; Forterre, Patrick; Ishino, Sonoko; Ishino, Yoshizumi

    2016-09-23

    The presence of acrylamide in food is a worldwide concern because it is carcinogenic, reprotoxic and neurotoxic. Acrylamide is generated in the Maillard reaction via condensation of reducing sugars and glyoxals arising from their decomposition, with asparagine, the amino acid forming the backbone of the acrylamide molecule. We reported recently the discovery of the Maillard deglycases (DJ-1/Park7 and its prokaryotic homologs) which degrade Maillard adducts formed between glyoxals and lysine or arginine amino groups, and prevent glycation damage in proteins. Here, we show that these deglycases prevent acrylamide formation, likely by degrading asparagine/glyoxal Maillard adducts. We also report the discovery of a deglycase from the hyperthermophilic archaea Pyrococcus furiosus, which prevents acrylamide formation at 100 °C. Thus, Maillard deglycases constitute a unique enzymatic method to prevent acrylamide formation in food without depleting the components (asparagine and sugars) responsible for its formation. PMID:27530919

  14. 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

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

    PubMed Central

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

    2015-01-01

    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

  16. 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

  17. 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.

  18. Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea.

    PubMed Central

    Heider, J; Mai, X; Adams, M W

    1996-01-01

    Cell extracts of the proteolytic and hyperthermophilic archaea Thermococcus litoralis, Thermococcus sp. strain ES-1, Pyrococcus furiosus, and Pyrococcus sp. strain ES-4 contain an enzyme which catalyzes the coenzyme A-dependent oxidation of branched-chain 2-ketoacids coupled to the reduction of viologen dyes or ferredoxin. This enzyme, termed VOR (for keto-valine-ferredoxin oxidoreductase), has been purified from all four organisms. All four VORs comprise four different subunits and show amino-terminal sequence homology. T. litoralis VOR has an M(r) of ca. 230,000, with subunit M(r) values of 47,000 (alpha), 34,000 (beta), 23,000 (gamma), and 13,000 (delta). It contains about 11 iron and 12 acid-labile sulfide atoms and 13 cysteine residues per heterotetramer (alpha beta gamma delta), but thiamine pyrophosphate, which is required for catalytic activity, was lost during purification. The most efficient substrates (kcat/Km > 1.0 microM-1 s-1; Km < 100 microM) for the enzyme were the 2-ketoacid derivatives of valine, leucine, isoleucine, and methionine, while pyruvate and aryl pyruvates were very poor substrates (kcat/Km < 0.2 microM-1 s-1) and 2-ketoglutarate was not utilized. T. litoralis VOR also functioned as a 2-ketoisovalerate synthase at 85 degrees C, producing 2-ketoisovalerate and coenzyme A from isobutyryl-coenzyme A (apparent Km, 250 microM) and CO2 (apparent Km, 48 mM) with reduced viologen as the electron donor. The rate of 2-ketoisovalerate synthesis was about 5% of the rate of 2-ketoisovalerate oxidation. The optimum pH for both reactions was 7.0. A mechanism for 2-ketoisovalerate oxidation based on data from substrate-induced electron paramagnetic resonance spectra is proposed, and the physiological role of VOR is discussed. PMID:8550513

  19. Novel type of ADP-forming acetyl coenzyme A synthetase in hyperthermophilic archaea: heterologous expression and characterization of isoenzymes from the sulfate reducer Archaeoglobus fulgidus and the methanogen Methanococcus jannaschii.

    PubMed

    Musfeldt, Meike; Schönheit, Peter

    2002-02-01

    Acetyl coenzyme A (CoA) synthetase (ADP forming) (ACD) represents a novel enzyme of acetate formation and energy conservation (acetyl-CoA + ADP + P(i) right harpoon over left harpoon acetate + ATP + CoA) in Archaea and eukaryotic protists. The only characterized ACD in archaea, two isoenzymes from the hyperthermophile Pyrococcus furiosus, constitute 145-kDa heterotetramers (alpha(2), beta(2)). The coding genes for the alpha and beta subunits are located at different sites in the P. furiosus chromosome. Based on significant sequence similarity of the P. furiosus genes, five open reading frames (ORFs) encoding putative ACD were identified in the genome of the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus and one ORF was identified in the hyperthermophilic methanogen Methanococcus jannaschii. The ORFs constitute fusions of the homologous P. furiosus genes encoding the alpha and beta subunits. Two ORFs, AF1211 and AF1938, of A. fulgidus and ORF MJ0590 of M. jannaschii were cloned and functionally overexpressed in Escherichia coli. The purified recombinant proteins were characterized as distinctive isoenzymes of ACD with different substrate specificities. In contrast to the Pyrococcus ACD, the ACDs of Archaeoglobus and Methanococcus constitute homodimers of about 140 kDa composed of two identical 70-kDa subunits, which represent fusions of the homologous P. furiosus alpha and beta subunits in an alphabeta (AF1211 and MJ0590) or betaalpha (AF1938) orientation. The data indicate that A. fulgidus and M. jannaschii contains a novel type of ADP-forming acetyl-CoA synthetase in Archaea, in which the subunit polypeptides and their coding genes are fused.

  20. Holo-Ni(II)HpNikR is an asymmetric tetramer containing two different nickel binding sites

    PubMed Central

    West, Abby L.; John, Franz St.; Lopes, Pedro E. M.; MacKerell, Alexander D.; Pozharski, Ed

    2010-01-01

    The metalloregulatory protein NikR from Helicobacter pylori (HpNikR) is a master regulator of gene expression which both activates and represses specific genes in response to nickel availability. Here, we report the first crystal structure (at 2.37Å resolution) of Ni(II)HpNikR prepared directly from the holo-protein. The protein contains four nickel ions located in two distinct coordination environments. Two nickel ions are bound to sites in a four-coordinate square planar geometry as predicted based on the structures of NikR from E. coli and P. horikoshii. The remaining two nickel ions are bound to sites with unexpected 5 or 6 coordination geometries which were previously thought to be involved in nickel incorporation into the protein. The nickel with 5/6 coordination geometry utilizes three histidines from two separate monomeric HpNikR units along with two or three water molecules as ligands. The spatial location of the nickel in the 5/6 coordinate site is within approximately 5 Å of the expected site if a 4- coordinate square planar geometry occurred. Two of the histidines that participate as ligands in 5/6 coordinate site would also participate as ligands if the 4-coordinate site was occupied making it impossible for both sites to be occupied simultaneously. DFT calculations show that the 5/6 coordinate geometries are energetically favorable when the local protein environment is included in the calculations. The presence of two distinct coordination environments in HpNikR is suggested to be related to the specificity and binding affinity of this transcription factor for DNA. PMID:20863122

  1. 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)).

  2. Bacillus tianshenii sp. nov., isolated from a marine sediment sample.

    PubMed

    Jiang, Zhao; Zhang, Dao-Feng; Khieu, Thi-Nhan; Son, Chu Ky; Zhang, Xiao-Mei; Cheng, Juan; Tian, Xin-Peng; Zhang, Si; Li, Wen-Jun

    2014-06-01

    A novel Gram-stain-positive, motile, catalase- and oxidase-positive, aerobic, endospore-forming, peritrichous, rod-shaped bacterium, designated YIM M13235(T), was isolated from a marine sediment sample collected from the South China Sea. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM M13235(T) belonged to the genus Bacillus. The strain grew optimally at 30 °C, pH 7.0 and in the presence of 2-4% (w/v) NaCl. meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. Strain YIM M13235(T) exhibited a menaquinone system with MK-7, and the major polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unknown phospholipids and one unknown glycolipid. The major fatty acids (>5%) were iso-C(15 : 0), anteiso-C(15 : 0), anteiso-C(17 : 0), iso-C(17 : 1)ω10c and summed feature 4 (anteiso-C(17 : 1) and/or iso-C(17 : 1)). The genomic DNA G+C content was 42.1 mol%. The DNA-DNA relatedness values between strain YIM M13235(T) and its close relatives (16S rRNA gene sequence similarities >97%) including Bacillus halmapalus DSM 8723(T), Bacillus horikoshii DSM 8719(T) and Bacillus zhanjiangensis JSM 099021(T) were 41%, 44% and 44%, respectively. On the basis of genotypic, phenotypic and DNA-DNA relatedness data, it is apparent that strain YIM M13235(T) represents a novel species of the genus Bacillus, for which the name Bacillus tianshenii sp. nov. is proposed. The type strain is YIM M13235(T) ( = DSM 25879(T) = KCTC 33044(T)).

  3. Bacillus zhanjiangensis sp. nov., isolated from an oyster in South China Sea.

    PubMed

    Chen, Yi-Guang; Hu, Song-Ping; Tang, Shu-Kun; He, Jian-Wu; Xiao, Jian-Qing; Zhu, Hong-Yi; Li, Wen-Jun

    2011-03-01

    A novel Gram-stain-positive, motile, catalase- and oxidase-positive, endospore-forming, aerobic, rod-shaped bacterium, designated strain JSM 099021(T), was isolated from an oyster collected from Naozhou Island in the South China Sea. Growth occurred with 0-15% (w/v) NaCl (optimum 2-4%) and at pH 6.0-10.0 (optimum pH 7.5) and at 10-45°C (optimum 30-35°C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The predominant respiratory quinone was menaquinone 7 (MK-7) and the major polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major cellular fatty acids were anteiso-C15:0, anteiso-C17:0, iso-C15:0 and iso-C16:0. The genomic DNA G + C content was 39.5 mol%. A phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JSM 099021(T) belongs to the genus Bacillus, and was most closely related to the type strains of Bacillus halmapalus (sequence similarity 99.0%), Bacillus horikoshii (98.4%) and Bacillus cohnii (98.0%). The combination of phylogenetic analysis, DNA-DNA hybridization, phenotypic characteristics and chemotaxonomic data supported the proposal that strain JSM 099021(T) represents a new species of the genus Bacillus, for which the name Bacillus zhanjiangensis sp. nov. is proposed. The type strain was JSM 099021(T) (=DSM 23010(T) = KCTC 13713(T)).

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Processivity and Subcellular Localization of Glycogen Synthase Depend on a Non-catalytic High Affinity Glycogen-binding Site*

    PubMed Central

    Díaz, Adelaida; Martínez-Pons, Carlos; Fita, Ignacio; Ferrer, Juan C.; Guinovart, Joan J.

    2011-01-01

    Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS. PMID:21464127

  10. Novel energy metabolism in anaerobic hyperthermophilic archaea: a modified Embden-Meyerhof pathway.

    PubMed

    Sakuraba, Haruhiko; Ohshima, Toshihisa

    2002-01-01

    Hyperthermophiles, a group of microorganisms whose optimum growth temperatures are above 80 degrees C, have been isolated mainly from marine and continental volcanic environments. They are viewed as potential sources of extraordinarily stable biomolecules with applications in novel industrial processes. Most hyperthermophiles belong to the domain Archaea, the third domain of life, and are considered to be the most ancient of all extant life forms. Recent studies have revealed unusual energy metabolic processes in hyperthermophilic archaea, e.g. a modified Embden-Meyerhof pathway, that have not been observed so far in organisms belonging to the Bacteria and Eucarya domains. Several novel enzymes--ADP-dependent glucokinase, ADP-dependent phosphofruktokinase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase, phosphoenolpyruvate synthase, pyruvate: ferredoxin oxidoreductase, and ADP-forming acetyl-CoA synthetase--have been found to be involved in the modified Embden-Meyerhof pathway of the hyperthermophilic archaeon Pyrococcus furiosus. In addition, a novel regulation site for energy metabolism and a unique mode of ATP regeneration have been postulated to exist in the pathway of P. furiosus. The metabolic design observed in this microorganism might reflect the situation at an early stage of evolution. This review focuses mainly on the unique energy metabolism and related enzymes of P. furiosus that have recently been described.

  11. 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

  12. Homology modeling of phosphoryl thymidine kinase of enterohemorrhagic Escherichia coli OH: 157

    PubMed Central

    Kaistha, Shilpa Deshpande; Sinha, Ranjana

    2009-01-01

    Enterohemorrhagic Escherichia coli (EHEC) are source of emerging infectious disease in India. Escherichia coli O157:H7 is an EHEC strain showing multiple antibiotic resistances and the cause of infantile diarrhea and hemolytic uremic syndrome worldwide. A novel strategy to counteract multiple antibiotic resistant organisms is to design drugs which specifically target metabolic pathways such as thiamine biosynthetic pathways found exclusively in prokaryotes. Homology modeling was used for model building of a terminal thiamine biosynthesis enzyme phosphoryl thymidine kinase (Thi E) using Geno3D, Swiss Model and Modeller. The best model was selected based on overall stereochemical quality. The potential ligand binding sites in the model were identified by CASTp server. The validated theoretical model of the 3D structure of the thiE protein of E. coli O157:H7 was predicted using a thiamine phosphate pyrophosphatase from Pyrococcus furiosus (PDB ID: 1X13_A) as template. The active pockets of ligand binding sites in the enzyme were identified. In this study, phosphoryl thymidine kinase (thi E), a terminal enzyme in the thiamine biosynthesis pathway in the pathogen has been modeled to be used in future as a potential drug target by the design of suitable inhibitors. PMID:19255642

  13. 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

  14. Enzymatic catalysis in organic solvents: Polyethylene glycol modified hydrogenase retains sulfhydrogenase activity in toluene

    SciTech Connect

    Woodward, C.A.; Kaufman, E.N.

    1996-11-05

    Naturally occurring enzymes may be modified by covalently attaching hydrophobic groups that render the enzyme soluble and active in organic solvents, and have the potential to greatly expand applications of enzymatic catalysis. The reduction of elemental sulfur to hydrogen sulfide by a hydrogenase isolated from Pyrococcus furiosus has been investigated as a model system for organic biocatalysis. While the native hydrogenase catalyzed the reduction of sulfur to H{sub 2}S in aqueous solution, no activity was observed when the aqueous solvent was replaced with anhydrous toluene. Hydrogenase modified with PEG p-nitrophenyl carbonate demonstrated its native biocatalytic ability in toluene when the reducing dye, benzyl viologen, was also present. Neither benzyl viologen or PEG p-nitrophenyl carbonate alone demonstrated reducing capability. PEG modified cellulase and benzyl viologen were also incapable of reducing sulfur to H{sub 2}S, indicating that the enzyme itself, and not the modification procedure, is responsible for the conversion in the nonpolar organic solvent. Sulfide production in toluene was tenfold higher than that produced in an aqueous system with equal enzyme activity, demonstrating the advantages of organic biocatalysis. Applications of bioprocessing in nonaqueous media are expected to provide significant advances in the areas of fossil fuels, renewable feedstocks, organic synthesis, and environmental control technology.

  15. 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.

  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. The biochemical properties and phylogenies of phosphofructokinases from extremophiles.

    PubMed

    Ronimus, R S; Morgan, H W

    2001-12-01

    The enzyme phosphofructokinase (PFK) is a defining activity of the highly conserved glycolytic pathway, and is present in the domains Bacteria, Eukarya, and Archaea. PFK subtypes are now known that utilize either ATP, ADP, or pyrophosphate as the primary phosphoryl donor and share the ability to catalyze the transfer of phosphate to the 1-position of fructose-6-phosphate. Because of the crucial position in the glycolytic pathway of PFKs, their biochemical characteristics and phylogenies may play a significant role in elucidating the origins of glycolysis and, indeed, of metabolism itself. Despite the shared ability to phosphorylate fructose-6-phosphate, PFKs that have been characterized to date now fall into three sequence families: the PFKA family, consisting of the well-known higher eukaryotic ATP-dependent PFKs together with their ATP- and pyrophosphate-dependent bacterial cousins (including the crenarchaeal pyrophosphate-dependent PFK of Thermoprotetus tenax) and plant pyrophosphate-dependent phosphofructokinases; the PFKB family, exemplified by the minor ATP-dependent PFK activity of Escherichia coli (PFK 2), but which also includes at least one crenarchaeal enzyme in Aeropyrum pernix; and the tentatively named PFKC family, which contains the unique ADP-dependent PFKs from the euryarchaeal genera of Pyrococcus and Thermococcus, which are indicated by sequence analysis to be present also in the methanogenic species Methanococcus jannaschii and Methanosarcina mazei.

  18. 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.

  19. 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.

  20. 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.

  1. A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon.

    PubMed

    Rashid, N; Morikawa, M; Kanaya, S; Atomi, H; Imanaka, T

    1999-02-19

    A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein.

  2. Coated-wall microreactor for continuous biocatalytic transformations using immobilized enzymes.

    PubMed

    Thomsen, Malene S; Nidetzky, Bernd

    2009-01-01

    Microstructured flow reactors are emerging tools for biocatalytic process development. A compelling design is that of the coated-wall reactor where enzyme is present as a surface layer attached to microchannel walls. However, preparation of a highly active wall biocatalyst remains a problem. Here, a stainless steel microreactor was developed where covalent immobilization of the enzyme in multiple linear flow channels of the reaction plate was supported by a macroporous wash-coat layer of gamma-aluminum oxide. Using surface functionalization with aminopropyl triethoxysilane followed by activation with glutardialdehyde, the thermophilic beta-glycosidase CelB from Pyrococcus furiosus was bound with retention of half of the specific activity of the free enzyme (800 U/mg), yielding a high catalyst loading of about 500 U/mL. This microreactor was employed for the continuous hydrolysis of lactose (100 mM) at 80 degrees C, providing a space-time yield of 500 mg glucose/(mL h) at a stable conversion of > or =70%. The immobilized enzyme displayed a half-life of 15 days under the operational conditions. Due to the absence of hydrophobic solute-material interactions, which limit the scope of microstructures fabricated from poly(dimethylsiloxane) for biocatalytic applications, the new microreactor was fully compatible with the alternate enzyme substrate 2-nitro-phenyl-beta-D-galactoside and the 2-nitro-phenol product resulting from its hydrolysis catalyzed by CelB. PMID:18618472

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. Probing the mechanism of rubredoxin thermal unfolding in the absence of salt bridges by temperature jump experiments

    SciTech Connect

    Henriques, Barbara J.; Saraiva, Ligia M.; Gomes, Claudio M. . E-mail: gomes@itqb.unl.pt

    2005-08-05

    Rubredoxins are the simplest type of iron-sulphur proteins and in recent years they have been used as model systems in protein folding and stability studies, especially the proteins from thermophilic sources. Here, we report our studies on the rubredoxin from the hyperthermophile Methanococcus jannaschii (T {sub opt} = 85 deg C), which was investigated in respect to its thermal unfolding kinetics by temperature jump experiments. Different spectroscopic probes were used to monitor distinct structural protein features during the thermal transition: the integrity of the iron-sulphur centre was monitored by visible absorption spectroscopy, whereas tertiary structure was followed by intrinsic tryptophan fluorescence and exposure of protein hydrophobic patches was sensed by 1-anilinonaphthalene-8-sulphonate fluorescence. The studies were performed at acidic pH conditions in which any stabilising contributions from salt bridges are annulled due to protonation of protein side chain groups. In these conditions, M. jannaschii rubredoxin assumes a native-like, albeit more flexible and open conformation, as indicated by a red shift in the tryptophan emission maximum and 1-anilinonaphthalene-8-sulphonate binding. Temperature jumps were monitored by the three distinct techniques and showed that the protein undergoes thermal denaturation via a simple two step mechanism, as loss of tertiary structure, hydrophobic collapse, and disintegration of the iron-sulphur centre are concomitant processes. The proposed mechanism is framed with the multiphasic one proposed for Pyrococcus furiosus rubredoxin, showing that a common thermal unfolding mechanism is not observed between these two closely related thermophilic rubredoxins.

  10. 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.

  11. 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).

  12. 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

  13. Crystal Structure of the First Eubacterial Mre11 Nuclease Reveals Novel Features that may Discriminate Substrates During DNA Repair

    PubMed Central

    Das, Debanu; Moiani, Davide; Axelrod, Herbert L.; Miller, Mitchell D.; McMullan, Daniel; Jin, Kevin K.; Abdubek, Polat; Astakhova, Tamara; Burra, Prasad; Carlton, Dennis; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ernst, Dustin; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Grzechnik, Slawomir K.; Han, Gye Won; Jaroszewski, Lukasz; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Paulsen, Jessica; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; van den Bedem, Henry; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Tainer, John A.; Wilson, Ian A.

    2010-01-01

    Mre11 nuclease plays a central role in the repair of cytotoxic and mutagenic DNA double-strand breaks (DSBs). As x-ray structural information has only been available for the Pyrococcus furiosus enzyme (PfMre11), the conserved and variable features of this nuclease across the domains of life have not been experimentally defined. Our crystal structure and biochemical studies demonstrate that TM1635 from Thermotoga maritima, originally annotated as a putative nuclease, is the Mre11 endo/exonuclease from T. maritima (TmMre11) and the first such structure from eubacteria. TmMre11 and PfMre11 display similar overall structures, despite sequence identity in the twilight zone of only ∼20%. However, they differ substantially in their DNA specificity domains and in their dimeric organization. Residues in the nuclease domain are highly conserved, but those in the DNA specificity domain are not. The structural differences likely affect how Mre11s from different organisms recognize and interact with single-stranded DNA, double-stranded DNA and DNA hairpin structures during DNA repair. The TmMre11 nuclease active site has no bound metal ions, but is conserved in sequence and structure with exception of a histidine that is important in PfMre11 nuclease activity. Nevertheless, biochemical characterization confirms that TmMre11 possesses both endonuclease and exonuclease activities on ssDNA and dsDNA substrates, respectively. PMID:20122942

  14. Production of chitooligosaccharides from Rhizopus oligosporus NRRL2710 cells by chitosanase digestion.

    PubMed

    Mahata, Maria; Shinya, Shoko; Masaki, Eiko; Yamamoto, Takashi; Ohnuma, Takayuki; Brzezinski, Ryszard; Mazumder, Tapan K; Yamashita, Kazuhiko; Narihiro, Kazue; Fukamizo, Tamo

    2014-01-13

    The intact cells of Rhizopus oligosporus NRRL2710, whose cell walls are abundant source of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN), were digested with three chitinolytic enzymes, a GH-46 chitosanase from Streptomyces sp. N174 (CsnN174), a chitinase from Pyrococcus furiosus, and a chitinase from Trichoderma viride, respectively. Solubilization of the intact cells by CsnN174 was found to be the most efficient from solid state CP/MAS (13)C NMR spectroscopy. Chitosanase products from Rhizopus cells were purified by cation exchange chromatography on CM-Sephadex C-25 and gel-filtration on Cellulofine Gcl-25m. NMR and MALDI-TOF-MS analyses of the purified products revealed that GlcN-GlcNAc, (GlcN)2-GlcNAc, and (GlcN)2 were produced by the enzymatic digestion of the intact cells. The chitosanase digestion of Rhizopus cells was found to be an excellent system for the conversion of fungal biomass without any environmental impact.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. Crystal structures of the bifunctional tRNA methyltransferase Trm5a

    PubMed Central

    Wang, Caiyan; Jia, Qian; Chen, Ran; Wei, Yuming; Li, Juntao; Ma, Jie; Xie, Wei

    2016-01-01

    tRNA methyltransferase Trm5 catalyses the transfer of a methyl group from S-adenosyl-L-methionine to G37 in eukaryotes and archaea. The N1-methylated guanosine is the product of the initial step of the wyosine hypermodification, which is essential for the maintenance of the reading frame during translation. As a unique member of this enzyme family, Trm5a from Pyrococcus abyssi (PaTrm5a) catalyses not only the methylation of N1, but also the further methylation of C7 on 4-demethylwyosine at position 37 to produce isowyosine, but the mechanism for the double methylation is poorly understood. Here we report four crystal structures of PaTrm5a ranging from 1.7- to 2.3-Å, in the apo form or in complex with various SAM analogues. These structures reveal that Asp243 specifically recognises the base moiety of SAM at the active site. Interestingly, the protein in our structures all displays an extended conformation, quite different from the well-folded conformation of Trm5b from Methanocaldococcus jannaschii reported previously, despite their similar overall architectures. To rule out the possibilities of crystallisation artefacts, we conducted the fluorescence resonance energy transfer (FRET) experiments. The FRET data suggested that PaTrm5a adopts a naturally extended conformation in solution, and therefore the open conformation is a genuine state of PaTrm5a. PMID:27629654

  2. 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

  3. A SELEX study of the DNA-binding specificity of archaeal FFRPs: 2. FL4 (pot1613368)

    PubMed Central

    Yokoyama, Katsushi; Ihara, Michiko; Ebihara, Sonomi; Suzuki, Masashi

    2006-01-01

    The DNA-binding specificity of a transcription factor, the FFRP FL4 (pot1613368) from Pyrococcus sp. OT3, was studied. Using SELEX (systematic evolution of ligands by exponential environment) experiments, from a set of fragments, ∼150 bps, of the genomic DNA of P. OT3, seven were selected as containing binding sites. Thirteen bases identified as shared by the seven selected fragments with the least mismatches, 2.71 on average, was ATGAAAAAGTCAT. This sequence was closely related with another sequence, ATGAA[AAA/TTT]TTCAT, in the 5-3-5 arrangement, i.e. NANBNCNDNE[AAA/TTT]NENDNCNBNA, where, e.g. NA was the base complementary to NA. The average number of mismatches found between this sequence and the seven fragments was 3.14. A sequence, TTGAAATTTACAA, resembling the sequence ATGAA[AAA/TTT]TTCAT and also another 5-3-5 sequence, TTGAA[AAA/TTT]TTCAA, was found upstream of the fl4 gene, which is potentially recognized by FL4 for auto-regulation. Thus it is likely that an ideal binding-site of FL4 is ATGAA[AAA/TTT]TTCAT or TTGAA[AAA/TTT]TTCAA. In this abstract, the sequences were highlighted in Italic at 3, and with bold characters at 5 and 5. When two sequences compared were the same at some positions, there they were underlined. PMID:25792768

  4. Crystal structure of the sugar binding domain of the archaeal transcriptional regulator TrmB.

    PubMed

    Krug, Michael; Lee, Sung-Jae; Diederichs, Kay; Boos, Winfried; Welte, Wolfram

    2006-04-21

    TrmB is an alpha-glucoside-sensing transcriptional regulator controlling two operons encoding maltose/trehalose and maltodextrin ABC transporters of Pyrococcus furiosus. The crystal structure of an N-terminal truncated derivative of TrmB (amino acids 2-109 deleted; TrmB(delta2-109)) was solved at 1.5 A resolution. This protein has lost its DNA binding domain but has retained its sugar recognition site. The structure represents a novel sugar-binding fold. TrmB(delta2-109) bound maltose, glucose, sucrose, and maltotriose, exhibiting Kd values of 6.8, 25, 34, and 160 microM, respectively. TrmB(delta2-109) behaved as a monomer in dilute buffer solution in contrast to the full-length protein, which is a dimer. Co-crystallization with bound maltose identified a binding site involving seven amino acid residues: Ser229, Asn305, Gly320, Met321, Val324, Ile325, and Glu326. Six of these residues interact with the nonreducing glucosyl residue of maltose. The nonreducing glucosyl residue is shared by all substrates bound to TrmB, suggesting it as a common recognition motif.

  5. TrmB, a sugar-specific transcriptional regulator of the trehalose/maltose ABC transporter from the hyperthermophilic archaeon Thermococcus litoralis.

    PubMed

    Lee, Sung-Jae; Engelmann, Afra; Horlacher, Reinhold; Qu, Qiuhao; Vierke, Gudrun; Hebbeln, Carina; Thomm, Michael; Boos, Winfried

    2003-01-10

    We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 microm and 0.5 mm sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80 degrees C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

  6. Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca2+-Binding Site

    PubMed Central

    Zeng, Jing; Gao, Xiaowei; Dai, Zheng; Tang, Bing

    2014-01-01

    Pyrolysin is an extracellular subtilase produced by the marine hyperthermophilic archaeon Pyrococcus furiosus. This enzyme functions at high temperatures in seawater, but little is known about the effects of metal ions on the properties of pyrolysin. Here, we report that the supplementation of Na+, Ca2+, or Mg2+ salts at concentrations similar to those in seawater destabilizes recombinant pyrolysin but leads to an increase in enzyme activity. The destabilizing effect of metal ions on pyrolysin appears to be related to the disturbance of surface electrostatic interactions of the enzyme. In addition, mutational analysis of two predicted high-affinity Ca2+-binding sites (Ca1 and Ca2) revealed that the binding of Ca2+ is important for the stabilization of this enzyme. Interestingly, Asn substitutions at residues Asp818 and Asp820 of the Ca2 site, which is located in the C-terminal extension of pyrolysin, resulted in improvements in both enzyme thermostability and activity without affecting Ca2+-binding affinity. These effects were most likely due to the elimination of unfavorable electrostatic repulsion at the Ca2 site. Together, these results suggest that metal ions play important roles in modulating the stability and activity of pyrolysin. PMID:24561589

  7. Role of Mn2+ and Compatible Solutes in the Radiation Resistance of Thermophilic Bacteria and Archaea

    PubMed Central

    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 Mn2+-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

  8. Proton Inventory and Dynamics in the Nia-S to Nia-C Transition of a [NiFe] Hydrogenase.

    PubMed

    Greene, Brandon L; Wu, Chang-Hao; Vansuch, Gregory E; Adams, Michael W W; Dyer, R Brian

    2016-03-29

    Hydrogenases (H2ases) represent one of the most striking examples of biological proton-coupled electron transfer (PCET) chemistry, functioning in facile proton reduction and H2 oxidation involving long-range proton and electron transport. Spectroscopic and electrochemical studies of the [NiFe] H2ases have identified several catalytic intermediates, but the details of their interconversion are still a matter of debate. Here we use steady state and time-resolved infrared spectroscopy, sensitive to the CO ligand of the active site iron, as a probe of the proton inventory as well as electron and proton transfer dynamics in the soluble hydrogenase I from Pyrococcus furiosus. Subtle shifts in infrared signatures associated with the Nia-C and Nia-S states as a function of pH revealed an acid-base equilibrium associated with an ionizable amino acid near the active site. Protonation of this residue was found to correlate with the photoproduct distribution that results from hydride photolysis of the Nia-C state, in which one of the two photoproduct states becomes inaccessible at low pH. Additionally, the ability to generate Nia-S via PCET from Nia-C was weakened at low pH, suggesting prior protonation of the proton acceptor. Kinetic and thermodynamic analysis of electron and proton transfer with respect to the various proton inventories was utilized to develop a chemical model for reversible hydride oxidation involving two intermediates differing in their hydrogen bonding character.

  9. 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.

  10. Transcription Start Site Associated RNAs (TSSaRNAs) Are Ubiquitous in All Domains of Life

    PubMed Central

    ten-Caten, Felipe; Baliga, Nitin S.; Koide, Tie

    2014-01-01

    A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs) are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq) and a primary-transcript enriched (dRNA-seq) strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome). Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life. PMID:25238539

  11. Structural Features of a Hyperthermostable Endo-β-1,3-glucanase in Solution and Adsorbed on “Invisible” Particles

    PubMed Central

    Koutsopoulos, Sotirios; van der Oost, John; Norde, Willem

    2005-01-01

    Conformational characteristics and the adsorption behavior of endo-β-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus were studied by circular dichroism, steady-state and time-resolved fluorescence spectroscopy, and calorimetry in solution and in the adsorbed state. The adsorption isotherms were determined on two types of surfaces: hydrophobic Teflon and hydrophilic silica particles were specially designed so that they do not interact with light and therefore do not interfere with spectroscopic measurements. We present the most straightforward method to study structural features of adsorbed macromolecules in situ using common spectroscopic techniques. The enzyme was irreversibly adsorbed and immobilized in the adsorbed state even at high temperatures. Adsorption offered further stabilization to the heat-stable enzyme and in the case of adsorption on Teflon its denaturation temperature was measured at 133°C, i.e., the highest experimentally determined for a protein. The maintenance of the active conformation and biological function particularly at high temperatures is important for applications in biocatalysis and biotechnology. With this study we also suggest that nature may employ adsorption as a complementary mode to maintain structural integrity of essential biomolecules at extreme conditions of temperature. PMID:15516527

  12. Production of chitooligosaccharides from Rhizopus oligosporus NRRL2710 cells by chitosanase digestion.

    PubMed

    Mahata, Maria; Shinya, Shoko; Masaki, Eiko; Yamamoto, Takashi; Ohnuma, Takayuki; Brzezinski, Ryszard; Mazumder, Tapan K; Yamashita, Kazuhiko; Narihiro, Kazue; Fukamizo, Tamo

    2014-01-13

    The intact cells of Rhizopus oligosporus NRRL2710, whose cell walls are abundant source of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN), were digested with three chitinolytic enzymes, a GH-46 chitosanase from Streptomyces sp. N174 (CsnN174), a chitinase from Pyrococcus furiosus, and a chitinase from Trichoderma viride, respectively. Solubilization of the intact cells by CsnN174 was found to be the most efficient from solid state CP/MAS (13)C NMR spectroscopy. Chitosanase products from Rhizopus cells were purified by cation exchange chromatography on CM-Sephadex C-25 and gel-filtration on Cellulofine Gcl-25m. NMR and MALDI-TOF-MS analyses of the purified products revealed that GlcN-GlcNAc, (GlcN)2-GlcNAc, and (GlcN)2 were produced by the enzymatic digestion of the intact cells. The chitosanase digestion of Rhizopus cells was found to be an excellent system for the conversion of fungal biomass without any environmental impact. PMID:24252625

  13. 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.

  14. Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex.

    PubMed

    Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki

    2015-06-01

    Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR-Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR-Cas Cmr complex, composed of the six Cas proteins (Cmr1-Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (CmrΔ1) complex composed of Pyrococcus furiosus Cmr2-Cmr3, Archaeoglobus fulgidus Cmr4-Cmr5-Cmr6 and the 39-mer P. furiosus 7.01-crRNA was prepared. The CmrΔ1 complex was cocrystallized with single-stranded DNA (ssDNA) complementary to the crRNA guide by the vapour-diffusion method. The crystals diffracted to 2.1 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 75.5, b = 76.2, c = 139.2 Å, α = 90.3, β = 104.8, γ = 118.6°. The asymmetric unit of the crystals is expected to contain one CmrΔ1-ssDNA complex, with a Matthews coefficient of 2.03 Å(3) Da(-1) and a solvent content of 39.5%.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

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

    PubMed

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

    1994-08-01

    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.

  1. 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.

  2. 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

  3. Structural and functional analyses of an archaeal XPF/Rad1/Mus81 nuclease: asymmetric DNA binding and cleavage mechanisms.

    PubMed

    Nishino, Tatsuya; Komori, Kayoko; Ishino, Yoshizumi; Morikawa, Kosuke

    2005-08-01

    XPF/Rad1/Mus81/Hef proteins recognize and cleave branched DNA structures. XPF and Rad1 proteins cleave the 5' side of nucleotide excision repair bubble, while Mus81 and Hef cleave similar sites of the nicked Holliday junction, fork, or flap structure. These proteins all function as dimers and consist of catalytic and helix-hairpin-helix DNA binding (HhH) domains. We have determined the crystal structure of the HhH domain of Pyrococcus furiosus Hef nuclease (HefHhH), which revealed the distinct mode of protein dimerization. Our structural and biochemical analyses also showed that each of the catalytic and HhH domains binds to distinct regions within the fork-structured DNA: each HhH domain from two separate subunits asymmetrically binds to the arm region, while the catalytic domain binds near the junction center. Upon binding to DNA, Hef nuclease disrupts base pairs near the cleavage site. It is most likely that this bipartite binding mode is conserved in the XPF/Rad1/Mus81 nuclease family. PMID:16084390

  4. Crystal structures of the bifunctional tRNA methyltransferase Trm5a.

    PubMed

    Wang, Caiyan; Jia, Qian; Chen, Ran; Wei, Yuming; Li, Juntao; Ma, Jie; Xie, Wei

    2016-01-01

    tRNA methyltransferase Trm5 catalyses the transfer of a methyl group from S-adenosyl-L-methionine to G37 in eukaryotes and archaea. The N1-methylated guanosine is the product of the initial step of the wyosine hypermodification, which is essential for the maintenance of the reading frame during translation. As a unique member of this enzyme family, Trm5a from Pyrococcus abyssi (PaTrm5a) catalyses not only the methylation of N1, but also the further methylation of C7 on 4-demethylwyosine at position 37 to produce isowyosine, but the mechanism for the double methylation is poorly understood. Here we report four crystal structures of PaTrm5a ranging from 1.7- to 2.3-Å, in the apo form or in complex with various SAM analogues. These structures reveal that Asp243 specifically recognises the base moiety of SAM at the active site. Interestingly, the protein in our structures all displays an extended conformation, quite different from the well-folded conformation of Trm5b from Methanocaldococcus jannaschii reported previously, despite their similar overall architectures. To rule out the possibilities of crystallisation artefacts, we conducted the fluorescence resonance energy transfer (FRET) experiments. The FRET data suggested that PaTrm5a adopts a naturally extended conformation in solution, and therefore the open conformation is a genuine state of PaTrm5a. PMID:27629654

  5. Formation of the conserved pseudouridine at position 55 in archaeal tRNA.

    PubMed

    Roovers, Martine; Hale, Caryn; Tricot, Catherine; Terns, Michael P; Terns, Rebecca M; Grosjean, Henri; Droogmans, Louis

    2006-01-01

    Pseudouridine (Psi) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Psi55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Psi55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.

  6. 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

  7. ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling.

    PubMed

    Deshpande, Rajashree A; Williams, Gareth J; Limbo, Oliver; Williams, R Scott; Kuhnlein, Jeff; Lee, Ji-Hoon; Classen, Scott; Guenther, Grant; Russell, Paul; Tainer, John A; Paull, Tanya T

    2014-03-01

    The Mre11-Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP-driven states regulate the sensing, processing and signaling of DNA double-strand breaks are largely unknown. Here we design structure-based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP-driven movements within the catalytic domains. With this strategy we identify Rad50 separation-of-function mutants that either promote or destabilize the ATP-bound state. Crystal structures, X-ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP-induced 'closed' conformation promotes DNA end binding and end tethering, while hydrolysis-induced opening is essential for DNA resection. Reducing the stability of the ATP-bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double-strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11-Rad50-Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure-based mutational analyses. These collective results suggest that ATP-dependent Rad50 conformations switch the Mre11-Rad50 complex between DNA tethering, ATM signaling, and 5' strand resection, revealing molecular mechanisms regulating responses to DNA double-strand breaks.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. Structure of the Archaeal Pab87 Peptidase Reveals a Novel Self-Compartmentalizing Protease Family

    PubMed Central

    Delfosse, Vanessa; Girard, Eric; Birck, Catherine; Delmarcelle, Michaël; Delarue, Marc; Poch, Olivier; Schultz, Patrick; Mayer, Claudine

    2009-01-01

    Self-compartmentalizing proteases orchestrate protein turnover through an original architecture characterized by a central catalytic chamber. Here we report the first structure of an archaeal member of a new self-compartmentalizing protease family forming a cubic-shaped octamer with D4 symmetry and referred to as CubicO. We solved the structure of the Pyrococcus abyssi Pab87 protein at 2.2 Å resolution using the anomalous signal of the high-phasing-power lanthanide derivative Lu-HPDO3A. A 20 Å wide channel runs through this supramolecular assembly of 0.4 MDa, giving access to a 60 Å wide central chamber holding the eight active sites. Surprisingly, activity assays revealed that Pab87 degrades specifically d-amino acid containing peptides, which have never been observed in archaea. Genomic context of the Pab87 gene showed that it is surrounded by genes involved in the amino acid/peptide transport or metabolism. We propose that CubicO proteases are involved in the processing of d-peptides from environmental origins. PMID:19266066

  14. 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.

  15. 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).

  16. Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

    PubMed Central

    Elmore, Joshua R.; Sheppard, Nolan F.; Ramia, Nancy; Deighan, Trace; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

    2016-01-01

    CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM. PMID:26848045

  17. 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

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

    DOE PAGES

    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

  19. 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

  20. RNA size is a critical factor for U-containing substrate selectivity and permanent pseudouridylated product release during the RNA:Ψ-synthase reaction catalyzed by box H/ACA sRNP enzyme at high temperature.

    PubMed

    Tillault, Anne-Sophie; Fourmann, Jean-Baptiste; Loegler, Christine; Blaud, Magali; Branlant, Christiane; Charpentier, Bruno

    2015-06-01

    The box H/ACA small ribonucleoprotein particles (H/ACA sRNPs) are RNP enzymes that isomerize uridines (U) into pseudouridines (Ψ) in archaeal RNAs. The RNA component acts as a guide by forming base-pair interactions with the substrate RNA to specify the target nucleotide of the modification to the catalytic subunit Cbf5. Here, we have analyzed association of an H/ACA sRNP enzyme from the hyperthermophilic archaeon Pyrococcus abyssi with synthetic substrate RNAs of different length and with target nucleotide variants, and estimated their turnover at high temperature. In these conditions, we found that a short substrate, which length is restricted to the interaction with RNA guide sequence, has higher turnover rate. However, the longer substrate with additional 5' and 3' sequences non-complementary to the guide RNA is better discriminated by the U to Ψ conversion allowing the RNP enzyme to distinguish the modified product from the substrate. In addition, we identified that the conserved residue Y179 in the catalytic center of Cbf5 is crucial for substrate selectivity.

  1. 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.

  2. Bioinformatic and biochemical analysis of a novel maltose-forming α-amylase of the GH57 family in the hyperthermophilic archaeon Thermococcus sp. CL1.

    PubMed

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

    2014-06-10

    Maltose-forming α-amylase is a glycoside hydrolase family 57 (GH57) member that is unique because it displays dual hydrolysis activity toward α-1,4- and α-1,6-glycosidic linkages and only recognizes maltose. This enzyme was previously identified only in Pyrococcus sp. ST04 (PSMA); however, we recently found two homologs subgroups in Thermococcus species. One subgroup (subgroup A) showed relatively high amino acid sequence similarity to PSMA (>71%), while the other subgroup (subgroup B) showed lower homology with PSMA (<59%). To characterize the subgroup B maltose-forming α-amylase from Thermococcus species (TCMA), we cloned the CL1_0868 gene from Thermococcus sp. CL1 and then successfully expressed the gene in Escherichia coli. Although TCMA has a different oligomeric state relative to PSMA, TCMA showed similar substrate specificity. However, TCMA was shown to hydrolyze maltooligosaccharides more easily than PSMA. Also, TCMA displayed different optimum conditions depending on the glycosidic linkage of the substrate. TCMA had the highest activity at 85°C and at pH 5.0 for α-1,4-glycosidic linkage hydrolysis whereas it showed its maximal activity to cleave α-1,6-glycosidic linkages at 98°C and pH 6.0. PMID:24835094

  3. 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

  4. 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

  5. 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.

  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. Molecular recognition of canonical and deaminated bases by P. abyssi family B DNA polymerase.

    PubMed

    Gouge, Jérôme; Ralec, Céline; Henneke, Ghislaine; Delarue, Marc

    2012-10-26

    Euryarchaeal polymerase B can recognize deaminated bases on the template strand, effectively stalling the replication fork 4nt downstream the modified base. Using Pyrococcus abyssi DNA B family polymerase (PabPolB), we investigated the discrimination between deaminated and natural nucleotide(s) by primer extension assays, electrophoretic mobility shift assays, and X-ray crystallography. Structures of complexes between the protein and DNA duplexes with either a dU or a dH in position +4 were solved at 2.3Å and 2.9Å resolution, respectively. The PabPolB is found in the editing mode. A new metal binding site has been uncovered below the base-checking cavity where the +4 base is flipped out; it is fully hydrated in an octahedral fashion and helps guide the strongly kinked template strand. Four other crystal structures with each of the canonical bases were also solved in the editing mode, and the presence of three nucleotides in the exonuclease site caused a shift in the coordination state of its metal A from octahedral to tetrahedral. Surprisingly, we find that all canonical bases also enter the base-checking pocket with very small differences in the binding geometry and in the calculated binding free energy compared to deaminated ones. To explain how this can lead to stalling of the replication fork, the full catalytic pathway and its branches must be taken into account, during which the base is checked several times. Our results strongly suggest a switch from elongation to editing modes right after nucleotide insertion when the modified base is at position +5.

  8. 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

  9. Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals.

    PubMed

    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

  10. Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior.

    PubMed

    Campbell, Elliot; Wheeldon, Ian R; Banta, Scott

    2010-12-01

    Cofactor specificity in the aldo-keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site-directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild-type. Results of previous pre-steady-state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2'-phosphate of the cofactor. Pre-steady-state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2'-phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. The complete genome sequence of Staphylothermus marinus reveals differences in sulfur metabolism among heterotrophic Crenarchaeota

    SciTech Connect

    Anderson, Iain; Lakshmi, Lakshmi Dharmarajan; Rodriquez, Jason; Hooper, Sean; Porat, I.; Ulrich, Luke; Mavromatis, K; Sun, Hui; Land, Miriam L; Lapidus, Alla L.; Lucas, Susan; Barry, Kerrie; Huber, Harald; Zhulin, Igor B; Whitman, W. B.; Mukhopadhyay, Biswarup; Woese, Carl; Bristow, James; Kyrpides, Nikos C

    2009-01-01

    Background 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. Results 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. Conclusion 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.

  17. 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.

  18. 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.

  19. 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

  20. Activation of Archaeal Transcription Mediated by Recruitment of Transcription Factor B*

    PubMed Central

    Ochs, Simon M.; Thumann, Sybille; Richau, Renate; Weirauch, Matt T.; Lowe, Todd M.; Thomm, Michael; Hausner, Winfried

    2012-01-01

    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

  1. A global transcriptional regulator in Thermococcus kodakaraensis controls the expression levels of both glycolytic and gluconeogenic enzyme-encoding genes.

    PubMed

    Kanai, Tamotsu; Akerboom, Jasper; Takedomi, Shogo; van de Werken, Harmen J G; Blombach, Fabian; van der Oost, John; Murakami, Taira; Atomi, Haruyuki; Imanaka, Tadayuki

    2007-11-16

    We identified a novel regulator, Thermococcales glycolytic regulator (Tgr), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Tgr (TK1769) displays similarity (28% identical) to Pyrococcus furiosus TrmB (PF1743), a transcriptional repressor regulating the trehalose/maltose ATP-binding cassette transporter genes, but is more closely related (67%) to a TrmB paralog in P. furiosus (PF0124). Growth of a tgr disruption strain (Deltatgr) displayed a significant decrease in growth rate under gluconeogenic conditions compared with the wild-type strain, whereas comparable growth rates were observed under glycolytic conditions. A whole genome microarray analysis revealed that transcript levels of almost all genes related to glycolysis and maltodextrin metabolism were at relatively high levels in the Deltatgr mutant even under gluconeogenic conditions. The Deltatgr mutant also displayed defects in the transcriptional activation of gluconeogenic genes under these conditions, indicating that Tgr functions as both an activator and a repressor. Genes regulated by Tgr contain a previously identified sequence motif, the Thermococcales glycolytic motif (TGM). The TGM was positioned upstream of the Transcription factor B-responsive element (BRE)/TATA sequence in gluconeogenic promoters and downstream of it in glycolytic promoters. Electrophoretic mobility shift assay indicated that recombinant Tgr protein specifically binds to promoter regions containing a TGM. Tgr was released from the DNA when maltotriose was added, suggesting that this sugar is most likely the physiological effector. Our results strongly suggest that Tgr is a global transcriptional regulator that simultaneously controls, in response to sugar availability, both glycolytic and gluconeogenic metabolism in T. kodakaraensis via its direct binding to the TGM.

  2. 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

  3. 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

  4. 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

  5. Enzymatic and structural characterization of an archaeal thiamin phosphate synthase.

    PubMed

    Hayashi, Maria; Kobayashi, Kazuya; Esaki, Hiroyoshi; Konno, Hiroyuki; Akaji, Kenichi; Tazuya, Keiko; Yamada, Kazuko; Nakabayashi, Toshikatsu; Nosaka, Kazuto

    2014-04-01

    Studies on thiamin biosynthesis have so far been achieved in eubacteria, yeast and plants, in which the thiamin structure is formed as thiamin phosphate from a thiazole and a pyrimidine moiety. This condensation reaction is catalyzed by thiamin phosphate synthase, which is encoded by the thiE gene or its orthologs. On the other hand, most archaea do not seem to have the thiE gene, but instead their thiD gene, coding for a 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) kinase/HMP phosphate kinase, possesses an additional C-terminal domain designated thiN. These two proteins, ThiE and ThiN, do not share sequence similarity. In this study, using recombinant protein from the hyperthermophile archaea Pyrobaculum calidifontis, we demonstrated that the ThiN protein is an analog of the ThiE protein, catalyzing the formation of thiamin phosphate with the release of inorganic pyrophosphate from HMP pyrophosphate and 4-methyl-5-β-hydroxyethylthiazole phosphate (HET-P). In addition, we found that the ThiN protein can liberate an inorganic pyrophosphate from HMP pyrophosphate in the absence of HET-P. A structure model of the enzyme-product complex of P. calidifontis ThiN domain was proposed on the basis of the known three-dimensional structure of the ortholog of Pyrococcus furiosus. The significance of Arg320 and His341 residues for thiN-coded thiamin phosphate synthase activity was confirmed by site-directed mutagenesis. This is the first report of the experimental analysis of an archaeal thiamin synthesis enzyme.

  6. 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

  7. 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

  8. 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

  9. Introduction of a thermophile-sourced ion pair network in the fourth beta/alpha unit of a psychophile-derived triosephosphate isomerase from Methanococcoides burtonii significantly increases its kinetic thermal stability.

    PubMed

    Dhaunta, Neeraj; Arora, Kanika; Chandrayan, Sanjeev K; Guptasarma, Purnananda

    2013-06-01

    Hyperthermophile proteins commonly have higher numbers of surface ionic interactions than homologous proteins from other domains of life. PfuTIM, a triosephosphate isomerase (TIM) from the hyperthermophile archaeon, Pyrococcus furiosus, contains an intricate network of 4 ion pairs in its 4th beta/alpha unit, (β/α)4, whereas MbuTIM, a triosephosphate isomerase from a psychrophile archaeon, Methanococcoides burtonii, lacks this network. Notably, (β/α)4 is the first element of the structure formed during folding of certain TIM-type (beta/alpha)8 barrel proteins. Previously, we have shown that elimination of PfuTIM's ion pair network in PfuTIM significantly decreases its kinetic structural stability. Here, we describe the reciprocal experiment in which this ion pair network is introduced into MbuTIM, to produce MutMbuTIM. Recombinant MbuTIM displays multi-state unfolding with apparent Tm values of autonomous structural elements approaching, or above, 70°C, when a temperature scanning rate of 90°C/h is used. The protein displays significant intrinsic kinetic stability, i.e., there is a marked temperature scan rate-dependence of the Tm values associated with unfolding transitions. The Tm values drop by as much as ~10°C when the temperature scanning rate is lowered to 5°C/h. MutMbuTIM, incorporating PfuTIM's ion pair network, shows significantly higher apparent Tm values (raised by 4-6°C over those displayed by MbuTIM). MutMbuTIM also displays significantly higher kinetic thermal stability. Thus, it appears that the thermal stability of triosephosphate isomerase can be increased, or decreased, by either enhancing, or reducing, the strength of ion pair interactions stabilizing (β/α)4, presumably through reduced cooperativity (and increased autonomy) in unfolding transitions.

  10. Self-assembly is prerequisite for catalysis of Fe(II) oxidation by catalytically active subunits of ferritin.

    PubMed

    Ebrahimi, Kourosh Honarmand; Hagedoorn, Peter-Leon; Hagen, Wilfred R

    2015-10-30

    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.

  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.

  12. Development of an ultra-high-temperature process for the enzymatic hydrolysis of lactose: II. Oligosaccharide formation by two thermostable beta-glycosidases.

    PubMed

    Petzelbauer, I; Zeleny, R; Reiter, A; Kulbe, K D; Nidetzky, B

    2000-07-20

    During lactose conversion at 70 degrees C, when catalyzed by beta-glycosidases from the archea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB), galactosyl transfer to acceptors other than water competes efficiently with complete hydrolysis of substrate. This process leads to transient formation of a range of new products, mainly disaccharides and trisaccharides, and shows a marked dependence on initial substrate concentration and lactose conversion. Oligosaccharides have been analyzed quantitatively by using capillary electrophoresis and high performance anion-exchange chromatography. At 270 g/L initial lactose, they accumulate at a maximum concentration of 86 g/L at 80% lactose conversion. With both enzymes, the molar ratio of trisaccharides to disaccharides is maximal at an early stage of reaction and decreases directly proportional to increasing substrate conversion. Overall, CelB produces about 6% more hydrolysis byproducts than SsbetaGly. However, the product spectrum of SsbetaGly is richer in trisaccharides, and this agrees with results obtained from the steady-state kinetics analyses of galactosyl transfer catalyzed by SsbetaGly and CelB. The major transgalactosylation products of SsbetaGly and CelB have been identified. They are beta-D-Galp-(1-->3)-Glc and beta-D-Galp-(1-->6)-Glc, and beta-D-Galp-(1-->3)-lactose and beta-D-Galp-(1-->6)-lactose, and their formation and degradation have been shown to be dependent upon lactose conversion. Both enzymes accumulate beta(1-->6)-linked glycosides, particularly allolactose, at a late stage of reaction. Because a high oligosaccharide concentration prevails until about 80% lactose conversion, thermostable beta-glycosidases are efficient for oligosaccharide production from lactose. Therefore, they prove to be stable and versatile catalysts for lactose utilization. PMID:10861393

  13. 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

  14. Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide.

    PubMed

    Keller, Matthew W; Schut, Gerrit J; Lipscomb, Gina L; Menon, Angeli L; Iwuchukwu, Ifeyinwa J; Leuko, Therese T; Thorgersen, Michael P; Nixon, William J; Hawkins, Aaron S; Kelly, Robert M; Adams, Michael W W

    2013-04-01

    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°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°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°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.

  15. Leucine 41 is a gate for water entry in the reduction of Clostridium pasteurianum rubredoxin.

    PubMed

    Min, T; Ergenekan, C E; Eidsness, M K; Ichiye, T; Kang, C

    2001-03-01

    Biological electron transfer is an efficient process even though the distances between the redox moieties are often quite large. It is therefore of great interest to gain an understanding of the physical basis of the rates and driving forces of these reactions. The structural relaxation of the protein that occurs upon change in redox state gives rise to the reorganizational energy, which is important in the rates and the driving forces of the proteins involved. To determine the structural relaxation in a redox protein, we have developed methods to hold a redox protein in its final oxidation state during crystallization while maintaining the same pH and salt conditions of the crystallization of the protein in its initial oxidation state. Based on 1.5 A resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins (Rd) from Clostridium pasteurianum (Cp), the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated. First, expansion of the [Fe-S] cluster and concomitant contraction of the NH...S hydrogen bonds lead to greater electrostatic stabilization of the extra negative charge. Second, a gating mechanism caused by the conformational change of Leucine 41, a nonpolar side chain, allows transient penetration of water molecules, which greatly increases the polarity of the redox site environment and also provides a source of protons. Our method of producing crystals of Cp Rd from a reducing solution leads to a distribution of water molecules not observed in the crystal structure of the reduced Rd from Pyrococcus furiosus. How general this correlation is among redox proteins must be determined in future work. The combination of our high-resolution crystal structures and molecular dynamics simulations provides a molecular picture of the structural rearrangement that occurs upon reduction in Cp rubredoxin.

  16. Differential roles of archaeal box H/ACA proteins in guide RNA-dependent and independent pseudouridine formation.

    PubMed

    Gurha, Priyatansh; Joardar, Archi; Chaurasia, Priyasri; Gupta, Ramesh

    2007-10-01

    RNA-guided pseudouridine (Psi) synthesis in Archaea and Eukarya requires a four-protein one-RNA containing box H/ACA ribonucleoprotein (RNP) complex. The proteins in the archaeal RNP are aCbf5, aNop10, aGar1 and L7Ae. Pyrococcus aCbf5-aNop10 is suggested to be the minimal catalytic core in this synthesis and the activity is enhanced by L7Ae and aGar1. The protein aCbf5 is homologous to eukaryal Cbf5 (dyskerin, NAP57) as well as to bacterial TruB and eukaryal Pus4; the last two produce YPsi55 in tRNAs in a guide RNA-independent manner. Here, using recombinant Methanocaldococcus jannaschii proteins, we report that aCbf5 and aGar1 together can function as a tRNA Psi55 synthase in a guide RNA-independent manner. This activity is enhanced by aNop10, but not by L7Ae. The aCbf5 alone can also produce Psi55 in tRNAs that contain the canonical 3'-CCA sequence and this activity is stimulated by aGar1. These results suggest that the roles of accessory proteins are different in guide RNA-dependent and independent Psi synthesis by aCbf5. The presence of conserved C (or U) and A at tRNA positions 56 and 58, respectively, which are required for TruB/Pus4 activity, is not essential for aCbf5-mediated Psi55 formation. Conserved A58 in tRNA normally forms a tertiary reverse Hoogstein base pair with an equally conserved U54. This base pair is recognized by TruB. Apparently aCbf5 does not require this base pair to recognize U55 for conversion to Psi55.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. Evolution of mal ABC transporter operons in the Thermococcales and Thermotogales

    PubMed Central

    2008-01-01

    Background The mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea Thermococcus litoralis and Pyrococcus furiosus. Bacterial hyperthermophiles of the order Thermotogales live among these archaea and so may have shared in these transfers. The genome sequence of Thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. We examined deep phylogenetic relationships among the mal genes of these hyperthermophiles and their close relatives to look for evidence of shared ancestry. Results We demonstrate that the two maltose ATP binding cassette (ABC) transporter operons now found in Tc. litoralis and P. furiosus (termed mal and mdx genes, respectively) are not closely related to one another. The Tc. litoralis and P. furiosus mal genes are most closely related to bacterial mal genes while their respective mdx genes are archaeal. The genes of the two mal operons in Tt. maritima are not related to genes in either of these archaeal operons. They are highly similar to one another and belong to a phylogenetic lineage that includes mal genes from the enteric bacteria. A unique domain of the enteric MalF membrane spanning proteins found also in these Thermotogales MalF homologs supports their relatively close relationship with these enteric proteins. Analyses of genome sequence data from other Thermotogales species, Fervidobacterium nodosum, Thermosipho melanesiensis, Thermotoga petrophila, Thermotoga lettingae, and Thermotoga neapolitana, revealed a third apparent mal operon, absent from the published genome sequence of Tt. maritima strain MSB8. This third operon, mal3, is more closely related to the Thermococcales' bacteria-derived mal genes than are mal1 and mal2. F. nodosum, Ts. melanesiensis, and Tt. lettingae have only one of the mal1-mal2 paralogs. The mal2 operon from an unknown species of Thermotoga appears to have been horizontally

  10. 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

  11. 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

  12. NMR and X-ray analysis of structural additivity in metal binding site-swapped hybrids of rubredoxin

    PubMed Central

    LeMaster, David M; Anderson, Janet S; Wang, Limin; Guo, Yi; Li, Hongmin; Hernández, Griselda

    2007-01-01

    Background Chimeric hybrids derived from the rubredoxins of Pyrococcus furiosus (Pf) and Clostridium pasteurianum (Cp) provide a robust system for the characterization of protein conformational stability and dynamics in a differential mode. Interchange of the seven nonconserved residues of the metal binding site between the Pf and Cp rubredoxins yields a complementary pair of hybrids, for which the sum of the thermodynamic stabilities is equal to the sum for the parental proteins. Furthermore, the increase in amide hydrogen exchange rates for the hyperthermophile-derived metal binding site hybrid is faithfully mirrored by a corresponding decrease for the complementary hybrid that is derived from the less thermostable rubredoxin, indicating a degree of additivity in the conformational fluctuations that underlie these exchange reactions. Results Initial NMR studies indicated that the structures of the two complementary hybrids closely resemble "cut-and-paste" models derived from the parental Pf and Cp rubredoxins. This protein system offers a robust opportunity to characterize differences in solution structure, permitting the quantitative NMR chemical shift and NOE peak intensity data to be analyzed without recourse to the conventional conversion of experimental NOE peak intensities into distance restraints. The intensities for 1573 of the 1652 well-resolved NOE crosspeaks from the hybrid rubredoxins were statistically indistinguishable from the intensities of the corresponding parental crosspeaks, to within the baseplane noise level of these high sensitivity data sets. The differences in intensity for the remaining 79 NOE crosspeaks were directly ascribable to localized dynamical processes. Subsequent X-ray analysis of the metal binding site-swapped hybrids, to resolution limits of 0.79 Å and 1.04 Å, demonstrated that the backbone and sidechain heavy atoms in the NMR-derived structures lie within the range of structural variability exhibited among the individual

  13. 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

  14. 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

  15. 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

  16. Comparative study of two box H/ACA ribonucleoprotein pseudouridine-synthases: relation between conformational dynamics of the guide RNA, enzyme assembly and activity.

    PubMed

    Fourmann, Jean-Baptiste; Tillault, Anne-Sophie; Blaud, Magali; 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.

  17. 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

  18. 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.

  19. 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

  20. Mössbauer characterization of an unusual high-spin side-on peroxo-Fe3+ species in the active site of superoxide reductase from Desulfoarculus Baarsii. Density functional calculations on related models.

    PubMed

    Horner, Olivier; Mouesca, Jean-Marie; Oddou, Jean-Louis; Jeandey, Claudine; Nivière, Vincent; Mattioli, Tony A; Mathé, Christelle; Fontecave, Marc; Maldivi, Pascale; Bonville, Pierre; Halfen, Jason A; Latour, Jean-Marc

    2004-07-13

    Superoxide reductase (SOR) is an Fe protein that catalyzes the reduction of superoxide to give H(2)O(2). Recently, the mutation of the Glu47 residue into alanine (E47A) in the active site of SOR from Desulfoarculus baarsii has allowed the stabilization of an iron-peroxo species when quickly reacted with H(2)O(2) [Mathé et al. (2002) J. Am. Chem. Soc. 124, 4966-4967]. To further investigate this non-heme peroxo-iron species, we have carried out a Mössbauer study of the (57)Fe-enriched E47A SOR from D. baarsii reacted quickly with H(2)O(2). Considering the Mössbauer data, we conclude, in conjunction with the other spectroscopic data available and with the results of density functional calculations on related models, that this species corresponds to a high-spin side-on peroxo-Fe(3+) complex. This is one of the first examples of such a species in a biological system for which Mössbauer parameters are now available: delta(/Fe) = 0.54 (1) mm/s, DeltaE(Q) = -0.80 (5) mm/s, and the asymmetry parameter eta = 0.60 (5) mm/s. The Mössbauer and spin Hamiltonian parameters have been evaluated on a model from the side-on peroxo complex (model 2) issued from the oxidized iron center in SOR from Pyrococcus furiosus, for which structural data are available in the literature [Yeh et al. (2000) Biochemistry 39, 2499-2508]. For comparison, similar calculations have been carried out on a model derived from 2 (model 3), where the [CH(3)-S](1)(-) group has been replaced by the neutral [NH(3)](0) group [Neese and Solomon (1998) J. Am. Chem. Soc. 120, 12829-12848]. Both models 2 and 3 contain a formally high-spin Fe(3+) ion (i.e., with empty minority spin orbitals). We found, however, a significant fraction ( approximately 0.6 for 2, approximately 0.8 for 3) of spin (equivalently charge) spread over two occupied (minority spin) orbitals. The quadrupole splitting value for 2 is found to be negative and matches quite well the experimental value. The computed quadrupole tensors are

  1. 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

  2. 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

  3. 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

  4. 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