Influence of tungsten on metabolic patterns in Pyrococcus furiosus,a hyperthermophilic archaeon

Pyrococcus furiosus is a strictly anaerobic heterotroph that grows optimally around 100 °C. It can be cultured in an artificial seawater-based medium with either peptides or maltose as the carbon source. Significant stimulation of cell yields were observed when trace levels of tungsten (as tungstate) were added to an energy-limited chemostat culture of P. furiosus when maltose is present, but not when peptides were the sole carbon source. The addition of tungsten also led to dramatic increases in the specific activities within cell-free extracts of aldehyde ferredoxin oxidoreductase (AOR), which is a tungsten-iron-sulfur protein. Moreover, the addition of tungsten to cells growing in maltose/peptide medium dramatically reduced the specific activity of intracellular proteases, suggesting a preference for the utilization of maltose over peptides as the carbon and energy source in the presence of tungsten.Non-standard abbreviations EPPS N-[2-Hydroxyethyl]-piperazine-N-[3-propane-sulfonic acid] - VFA volatile fatty acids - LNA 1-Lys-p-nitroaniline - MeOSAPTNA MeO-Suc-Arg-Pro-Tyr-p-nitroaniline - AOR aldehyde ferredoxin oxidoreductase     

Operon prediction in Pyrococcus furiosus

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.     

Preservation of the hyperthermophile Pyrococcus furiosus

Methods are evaluated for the preservation of the hyperthermophile Pyrococcus furiosus . The use of glass capillary tubes stored over liquid nitrogen with dimethyl sulphoxide appears to be the preferred method of preservation. Lyophilization resulted in loss of viability and storage at room temperature and +4°C resulted in considerable loss of viability within 4 weeks.     

Truncated prolyl oligopeptidase from Pyrococcus furiosus

The peptidase domain of prolyl oligopeptidase is covered by a propeller domain, which excludes large peptides and proteins from the catalytic triad. Previous studies indicated that some amino acids of the N-terminal region constitute a part of the substrate entrance to the active site. To investigate the catalytic role of the N-terminus, we removed the residues 1-32 from the enzyme and examined the kinetic, thermodynamic, and structural consequences of the deletion, using the thermophile Pyrococcus furiosus prolyl oligopeptidase. An about threefold decrease in the catalytic activity along with a 20 degrees C reduction in the temperature optimum was observed. The pH-rate profile, the rate-limiting step, and the activation parameters did not change significantly. However, a substantial decrease was observed in the stability of the protein as demonstrated by circular dichroism and differential scanning calorimetry measurements, and by denaturation with guanidinium chloride. It was concluded that the N-terminal segment did not facilitate the substrate binding, independent of the size of the substrate, but contributed principally to the protein stability required for the formation of the proper active site.     

Redox chemistry of biological tungsten: an EPR study of the aldehyde oxidoreductase from Pyrococcus furiosus

 Aldehyde:ferredoxin oxidoreductase (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus is a homodimeric protein. Each subunit carries one [4Fe-4S] cubane and a novel tungsten cofactor containing two pterins. A single iron atom bridges between the subunits. AOR has previously been studied with EPR spectroscopy in an inactive form known as the red tungsten protein (RTP): reduced RTP exhibits complex EPR interaction signals. We have now investigated the active enzyme AOR with EPR, and we have found an S = 1/2 plus S = 3/2 spin mixture from a non-interacting [4Fe-4S]¹⁺ cluster in the reduced enzyme. Oxidized AOR affords EPR signals typical for W(V) with g–values of 1.982, 1.953, and 1.885. The W(V) signals disappear at a reduction potential E _m,7.5 of +180 mV. This unexpectedly high value indicates that the active-site redox chemistry is based on the pterin part of the cofactor. Received: 18 December 1995 / Accepted: 26 March 1996     

X-ray absorption spectroscopy of Pyrococcus furiosus rubredoxin

X-ray absorption spectroscopy has been used to probe the frozen solution structure of the metal site in Pyrococcus furiosus rubredoxin in the native, iron-containing protein and in zinc- and mercury-substituted proteins. For all samples studied, the spectra have been interpreted in terms of a single shell of coordinated sulfur, with approximately tetrahedral coordination. For the native protein we obtain Fe-S bond-lengths of 2.29 and 2.33?Å for oxidized and reduced proteins, respectively. These values are in excellent agreement with those previously obtained from X-ray crystallography. The metal-substituted rubredoxins possess metal-sulfur bond lengths of 2.34 and 2.54?Å for the zinc- and mercury-substituted proteins, respectively.     

Identification of Pyrococcus furiosus amylopullulanase catalytic residues

Pyrococcus furiosus amylopullulanase (PfAPU) belongs to glycosyl hydrolase family 57. Using sequence alignments of the known family 57 enzymes and site-directed mutagenesis, E291, D394, and E396 were identified as PfAPU putative catalytic residues. The apparent catalytic efficiencies (k_cat/K_m) of PfAPU mutants E291Q and D394N on pullulan were 123.0 and 24.4 times lower, respectively, than that of PfAPU. The activity of mutant E396Q on pullulan was too low to allow reliable determination of its catalytic efficiency. The apparent specific activities of these enzymes on starch also decreased 91.0 times (E291Q), 11.7 times (D394N), and 37.2 times (E396Q). The hydrolytic patterns for pullulan and starch were the same, while the hydrolysis rates differed as reported. Based on sequence alignment and a previous report, E291 is proposed as the catalytic nucleophile.     

Structure of the prolidase from Pyrococcus furiosus

The structure of prolidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pfprol) has been solved and refined at 2.0 A resolution. This is the first structure of a prolidase, i.e., a peptidase specific for dipeptides having proline as the second residue. The asymmetric unit of the crystals contains a homodimer of the enzyme. Each of the two protein subunits has two domains. The C-terminal domain includes the catalytic site, which is centered on a dinuclear metal cluster. In the as-isolated form of Pfprol, the active-site metal atoms are Co(II) [Ghosh, M., et al. (1998) J. Bacteriol. 180, 4781-9]. An unexpected finding is that in the crystalline enzyme the active-site metal atoms are Zn(II), presumably as a result of metal exchange during crystallization. Both of the Zn(II) atoms are five-coordinate. The ligands include a bridging water molecule or hydroxide ion, which is likely to act as a nucleophile in the catalytic reaction. The two-domain polypeptide fold of Pfprol is similar to the folds of two functionally related enzymes, aminopeptidase P (APPro) and creatinase. In addition, the catalytic C-terminal domain of Pfprol has a polypeptide fold resembling that of the sole domain of a fourth enzyme, methionine aminopeptidase (MetAP). The active sites of APPro and MetAP, like that of Pfprol, include a dinuclear metal center. The metal ligands in the three enzymes are homologous. Comparisons with the molecular structures of APPro and MetAP suggest how Pfprol discriminates against oligopeptides and in favor of Xaa-Pro substrates. The crystal structure of Pfprol was solved by multiple-wavelength anomalous dispersion. The crystals yielded diffraction data of relatively high quality and resolution, despite the fact that one of the two protein subunits in the asymmetric unit was found to be significantly disordered. The final R and R(free) values are 0.24 and 0.28, respectively.     

MutS2 Family Protein from Pyrococcus furiosus

MutS2 protein of Pyrococcus furiosus has been cloned and over-expressed. Initial characterization reveals that PfuMutS2 possesses a thermostable ATPase activity and a thermostable, nonspecific DNA binding activity. However, PfuMutS2 does not have any detectable mismatch-specific DNA binding activity. It is the first in vitro characterization of an MutS2 family protein. Received: 23 April 2001 / Accepted: 27 August 2001     

Salvaging Pyrococcus furiosus Protein Targets at SECSG

Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.     

Pyrococcus furiosus ferredoxin is a functional dimer

Pyrococcus furiosus ferredoxin is subject to a monomer/dimer equilibrium as a function of ionic strength. At physiological ionic strength, approximately 0.35 M NaCl, the protein is very predominantly homodimer. The monomeric form exhibits impaired electron transfer on glassy carbon; it also has a decreased S=3/2 over S=1/2 ratio as shown by electron paramagnetic resonance spectroscopy. Even following sterilization at 121 degrees C the dimer is stable in denaturing gel electrophoresis.     

Pyruvate metabolism of the hyperthermophilic archaebacterium Pyrococcus furiosus

The hyperthermophilic anaerobe Pyrococcus furiosus was found to grow on pyruvate as energy and carbon source. Growth was dependent on yeast extract (0.1%). The organism grew with doublings times of about 1 h up to cell densities of 1–2×10⁸ cells/ml. During growth 0.6–0.8 mol acetate and 1.2–1.5 mol CO₂ and 0.8 mol H₂ were formed per mol of pyruvate consumed. The molar growth yield was 10–11 g cells(dry weight)/mol pyruvate. Cell suspensions catalyzed the conversion of 1 mol of pyruvate to 0.6–0.8 mol acetate, 1.2–1.5 mol CO₂, 1.2 mol H₂ and 0.03 mol acetoin. After fermentation of [3-¹⁴C]pyruvate the specific radioactivities of pyruvate, CO₂ and acetate were equal to 1:0.01:1. Cellfree extracts contained the following enzymatic activities: pyruvate: ferredoxin (methyl viologen) oxidoreductase (0.2 U mg^-1, T=60°C, with Clostridium pasteurianum ferredoxin as electron acceptor; 1.4 U mg^-1 at 90°C, with methyl viologen as electron acceptor); acetyl-CoA synthetase (ADP forming) [acetyl-CoA+ADP+Piacetate+ATP+CoA] (0.34 U mg^-1, T=90°C), and hydrogen: methyl viologen oxidoreductase (1.75 U mg^-1). Phosphate acetyl-transferase activity, acetate kinase activity, and carbon monoxide:methyl viologen oxidoreductase activity could not be detected. These findings indicate that the archaebacterium P. furiosus ferments pyruvate to acetate, CO₂ and H₂ involving only three enzymes, a pyruvate:ferredoxin oxidoreductase, a hydrogenase and an acetyl-CoA synthetase (ADP forming).Non-standard abbreviations DTE dithioerythritol - MV methyl viologen - MOPS morpholinopropane sulfonic acid - Tricine N-tris(hydroxymethyl)-methylglycine Part of the work was performed at the Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karlvon-Frisch-Strasse, W-3550 Marburg/Lahn, Federal Republic of Germany     

Continuous culture of the hyperthermophilic archaeum Pyrococcus furiosus

Summary A system for continuous culture of the hyperthermophilic archaeum Pyrococcus furiosus in the absence of elemental sulphur has been developed. An all-glass gas-lift bioreactor was used to provide high mass transfer at low shear forces, whilst eliminating the potential for corrosion. Steady-state cell densities of P. furiosus were found to increase with higher inert gas flow rates, reaching a maximum in this system with 0.5 vol. vol^–1 min^–1 of nitrogen (N₂). N₂ permitted higher cell densities than the other inert gases tested (argon, helium and sulphur hexafluoride) under equivalent conditions. At 0.5 vol. vol^–1 min^–1 of N₂ a cell density in excess of 3 × 10⁹ ml^–1 could be maintained indefinitely at a dilution rate of 0.2 h^–1. Higher dilution rates gave progressively lower steady-state cell densities. Teh biomass production was maximal, however, at a dilution rate of 0.4 h^–1. At this dilution rate the bioreactor was able to generate more than 1.5 g wet weight of cells h^–1 l^–1 culture volume.Correspondence to: N. Raven     

Enzymes of hydrogen metabolism in Pyrococcus furiosus.

The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni-Fe hydrogenase. Ten ORFs (mbhA-I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni-Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe-4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe-4S] protein. With P. furiosus soluble [4Fe-4S] ferredoxin as the electron donor the membranes produce H2, and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H2 evolution to H2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with Em approximately -0.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni-Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H2) connected by devices for transduction, transfer, recovery and safety-valving of energy.     

超高温古生菌强烈炽热球菌(Pyrococcus furiosus)的生理代谢

本文述及Ｐｙｒｏｃｏｃｃｕｓ ｆｕｒｉｏｓｕｓ的丙酮酸代谢、麦芽糖发酵（高温糖酵解途径）、由丙酮酸糖原异生途径、还原性末端产物－－Ｌ－丙氨酸的形成和钨对代谢类型的影响等。     

The closed structure of an archaeal DNA ligase from Pyrococcus furiosus

DNA ligases join single-strand breaks in double-stranded DNA, and are essential to maintain genome integrity in DNA metabolism. Here, we report the 1.8 A resolution structure of Pyrococcus furiosus DNA ligase (PfuLig), which represents the first full-length atomic view of an ATP-dependent eukaryotic-type DNA ligase. The enzyme comprises the N-terminal DNA-binding domain, the middle adenylation domain, and the C-terminal OB-fold domain. The architecture of each domain resembles those of human DNA ligase I, but the domain arrangements differ strikingly between the two enzymes. The closed conformation of the two "catalytic core" domains at the carboxyl terminus in PfuLig creates a small compartment, which holds a non-covalently bound AMP molecule. This domain rearrangement results from the "domain-connecting" role of the helical extension conserved at the C termini in archaeal and eukaryotic DNA ligases. The DNA substrate in the human open-ligase is replaced by motif VI in the Pfu closed-ligase. Both the shapes and electrostatic distributions are similar between motif VI and the DNA substrate, suggesting that motif VI in the closed state mimics the incoming substrate DNA. Two basic residues (R531 and K534) in motif VI reside within the active site pocket and interact with the phosphate group of the bound AMP. The crystallographic and functional analyses of mutant enzymes revealed that these two residues within the RxDK sequence play essential and complementary roles in ATP processing. This sequence is also conserved exclusively among the covalent nucleotidyltransferases, even including mRNA-capping enzymes with similar helical extensions at the C termini.     

Dps-like protein from the hyperthermophilic archaeon Pyrococcus furiosus

Oxidative stress is a universal phenomenon experienced by organisms in all domains of life. Proteins like those in the ferritin-like di-iron carboxylate superfamily have evolved to manage this stress. Here we describe the cloning, isolation, and characterization of a Dps-like protein from the hyperthermophilic archaeon Pyrococcus furiosus (PfDps-like). Phylogenetic analysis, primary structure alignments and higher order structural predictions all suggest that the P. furiosus protein is related to proteins within the broad superfamily of ferritin-like di-iron carboxylate proteins. The recombinant PfDps protein self-assembles into a 12 subunit quaternary structure with an outer shell diameter of approximately 10nm and an interior diameter of approximately 5 nm. Dps proteins functionally manage the toxicity of oxidative stress by sequestering intracellular ferrous iron and using it to reduce H(2)O(2) in a two electron process to form water. The iron is converted to a benign form as Fe(III) within the protein cage. This Dps-mediated reduction of hydrogen peroxide, coupled with the protein's capacity to sequester iron, contributes to its service as a multifunctional antioxidant.     

Phosphoenolpyruvate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus

Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690 ± 20 kDa and is assumed to be octomeric, while approximately 30% of the protein is purified as a large (~1.6 MDa) complex that is not active. The apparent K_m values and catalytic efficiencies for the substrates pyruvate and ATP (at 80°C, pH 8.4) were 0.11 mM and 1.43 × 10⁴ mM⁻¹ · s⁻¹ and 0.39 mM and 3.40 × 10³ mM⁻¹ · s⁻¹, respectively. Maximal activity was measured at pH 9.0 (at 80°C) and at 90°C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [k_cat/K_m = 32 (mM · s)⁻¹]. In contrast to several other nucleotide-dependent enzymes from P. furiosus, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration (~5% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.     

The GINS complex from Pyrococcus furiosus stimulates the MCM helicase activity

Pyrococcus furiosus, a hyperthermophilic Archaea, has homologs of the eukaryotic MCM (mini-chromosome maintenance) helicase and GINS complex. The MCM and GINS proteins are both essential factors to initiate DNA replication in eukaryotic cells. Many biochemical characterizations of the replication-related proteins have been reported, but it has not been proved that the homologs of each protein are also essential for replication in archaeal cells. Here, we demonstrated that the P. furiosus GINS complex interacts with P. furiosus MCM. A chromatin immunoprecipitation assay revealed that the GINS complex is detected preferentially at the oriC region on Pyrococcus chromosomal DNA during the exponential growth phase but not in the stationary phase. Furthermore, the GINS complex stimulates both the ATPase and DNA helicase activities of MCM in vitro. These results strongly suggest that the archaeal GINS is involved in both the initiation and elongation processes of DNA replication in P. furiosus, as observed in eukaryotic cells.     

Preparation and properties of gelatin-immobilized beta-glucosidase from Pyrococcus furiosus

Hyperthermostable beta-glucosidase from Pyrococcus furiosus was enclosed in gelatin gel by cross-linking with transglutaminase. Gelatin-immobilized beta-glucosidase was considerably more thermostable than the native enzyme. Lyophilized immobilisate was stored at 90 degrees C for 1 month without loss of activity. The immobilized beta-glucosidase catalyzed transglucosylation of 5-phenylpentanol with 10.0 equivalent of cellobiose at pH 5.0 and 70 degrees C for 12 h to afford 5-phenylpentyl beta-D-glucopyranoside in 41% yield. The immobilized enzyme was more effective than the native one in transglucosylation. The gelatin-immobilized Pfu-beta-glucosidase recovered from the first run of the reaction was reusable on successive runs.