Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Pyrococcus”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 505 records · Page 28Linked to original sources
PubMed1996

The 3-phosphoglycerate kinase of the hyperthermophilic archaeum Pyrococcus woesei produced in Escherichia coli: loss of heat resistance due to internal translation initiation and its restoration by site-directed mutagenesis.

Heterologous expression of the gene coding for 3-phosphoglycerate kinase (PGK) of the hyperthermophilic archaeum, Pyrococcus woesei (Pw), in Escherichia coli (Ec) yielded only low recovery of recombinant PGK (re-PGK) in heat-precipitated crude extracts. Moreover, we noticed contamination with a 28-kDa protein, from which PGK could hardly be separated, even under stringent conditions after tagging the re-PGK with a His6-tag. The preparations contaminated with the 28-kDa protein showed an unexpectedly low thermal stability. Under the same conditions (85 degrees C, 30 min), however, the enzyme from the original organism was completely resistant to heat inactivation. As shown by size-exclusion chromatography, re-PGK forms tight associations with the 28-kDa protein, which was found to represent a C-terminal fragment of PGK and to arise as a product of internal translation initiation within the pgk gene. Mutations changing the internal ribosome-binding site effectively suppressed the production of the 28-kDa protein and restored the thermal stability of the Pw re-PGK.

Archaea↗
PubMed1995

The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures.

BACKGROUND: The hyperthermophile Pyrococcus furiosus is one of the most thermostable organisms known, with an optimum growth temperature of 100 degrees C. The proteins from this organism display extreme thermostability. We have undertaken the structure determination of glutamate dehydrogenase from P. furiosus in order to gain further insights into the relationship between molecular structure and thermal stability. RESULTS: The structure of P. furiosus glutamate dehydrogenase, a homohexameric enzyme, has been determined at 2.2 A resolution and compared with the structure of glutamate dehydrogenase from the mesophile Clostridium symbiosum. CONCLUSIONS: Comparison of the structures of these two enzymes has revealed one major difference: the structure of the hyperthermophilic enzyme contains a striking series of ion-pair networks on the surface of the protein subunits and buried at both interdomain and intersubunit interfaces. We propose that the formation of such extended networks may represent a major stabilizing feature associated with the adaptation of enzymes to extreme temperatures.

Amino Acid Sequence↗
PubMed2000

Characterization of FtsZ homolog from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1.

The gene of bacterial type ftsZ homolog in hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1 (Pk-ftsZ), was identified. The gene product of the Pk-ftsZ gene is composed of 380 amino acids with a molecular mass of 41,354 Da. In the deduced amino acid sequence of the Pk-ftsZ gene, a glycine-rich sequence (Gly-Gly-Gly-Thr-Gly-Ala-Gly) implicated in GTP binding was well conserved. The Pk-ftsZ gene was overexpressed using Escherichia coli as a host and the recombinant protein was purified. The purified Pk-FtsZ protein exhibited GTPase activity with optimum temperatures higher than 80 degrees C. However, the protein showed little GTPase activity at 40 degrees C, indicating that a high reaction temperature is required for the GTPase activity in accordance with the thermophilic nature of P. kodakaraensis KOD1. The GTP-binding ability of Pk-FtsZ protein could also be detected by UV-induced cross-linking of a protein to [alpha-32P] GTP. The Pk-ftsZ gene was expressed in E. coli cells with a temperature-sensitive ftsZ mutation, E. coli ftsZ84 (ts), but its mutant phenotype of elongated cell form at a nonpermissive temperature (42 degrees C) could not be compensated, possibly because of the thermophilic nature of the Pk-FtsZ. Pk-FtsZ could form protofilaments in a GTP-dependent manner at 90 degrees C. Results of phylogenetic analysis suggest that there might be additional factors required for formation of the Z ring in P. kodakaraensis KOD1.

Journal Article↗
PubMed2000

Acceptor specificity of 4-alpha-glucanotransferase from Pyrococcus kodakaraensis KOD1, and synthesis of cycloamylose.

4-Alpha-glucanotransferase from a hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 showed a broad acceptor specificity to various saccharides in an intermolecular transglycosylation reaction. In particular, the enzyme produced large amounts of transfer products of various acceptors such as D-glucose, methyl-alpha-D-glucoside, phenyl-alpha-D-glucoside, and D-xylose. It is suggested that the requirement for an effective acceptor in the intermolecular transglycosylation reaction catalyzed by this enzyme is the pyranose structure with the same configurations of the free C2-, C3-, and C4-hydroxyl groups as d-glucopyranose, like cyclomaltodextrin glucanotransferase (CGTase). However, the enzyme showed some acceptor specificities unlike those of CGTase. Analysis of the action of 4-alpha-glucanotransferase indicated that the enzyme catalyzes an intramolecular trans-glycosylation (cyclization) reaction of amylose to produce cyclic alpha-1,4-glucan (cycloamylose). The yield of cycloamylose reached 67%, and the degree of polymerization was found to range from 16 to above 55.

Journal Article↗
PubMed1999

Gene analysis and enzymatic properties of thermostable beta-glycosidase from Pyrococcus kodakaraensis KOD1.

A beta-glycosidase with broad substrate specificity was identified from a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoding beta-glycosidase (Pk-gly) consists of 1449 nucleotides corresponding to a polypeptide of 483 amino acids. The protein showed similarity with other beta-glycosidases from family-1 glycosyl hydrolases, in particular, it showed high identity to beta-mannosidase from P. furiosus (55.7%), beta-glycosidase from Sulfolobus solfataricus (42.7%) and beta-glucosidase from P. furiosus (41.9%). The cloned gene was expressed in Escherichia coli and the recombinant protein was purified. The beta-glycosidase showed optimal activity at pH 6.5 and at an extremely high temperature of 100 degrees C, and had a half-life of 18 h at 90 degrees C. The beta-glycosidase hydrolyzed various pNp-beta-glycopyranosides, with kcat K(m) values in the order of pNp-beta-glucopyranoside = pNp-beta-mannopyranoside > pNp-beta-galactopyranoside > pNp-beta-xylopyranoside. pNp-beta-mannopyranoside was the substrate exhibiting the lowest K(m) value [0.254 mM] with a kcat K(m) ratio comparable to that of pNp-beta-glucopyranoside. This substrate specificity was distinct from previously reported beta-glycosidases. We observed that the region in PK-Gly corresponding to the fifth alpha-helix and beta-strand region of beta-glycosidase from S. solfataricus, which constitutes a large portion of the channel for substrate incorporation, displayed a chimeric structure, with the N-terminal region similar to beta-glycosidases and the C-terminal region similar to beta-mannosidases. An exo-type hydrolytic activity and transglycosylation activity were also observed towards cellooligomers.

Journal Article↗
PubMed1999

Inhibitory effect of acetic acid on growth of hyperthermophilic archaeon Pyrococcus furiosus.

The growth inhibition of Pyrococcus furiosus by acetic acid was stronger than that by hydrogen and could be described by a non-competitive inhibition model in which the inhibition constants of undissociated acetic acid, K(p) and n, were estimated to be 0.69 mM (25 mM total acetic acid at pH 6.5; pKa=4.96; 98 degrees C) and 1.0, respectively. In order to reduce the acetic acid inhibition, repeated-batch culturing was performed using a filtration module. This yielded 0.49 g of dry cells l(-1) after growth for 12 h after inoculation. It became impossible, however, to continue repeated-batch culturing manually because the time intervals for medium replacement became too short. In order to automatically maintain a low concentration of acetic acid, a perfusion culture was carried out in which medium feeding coupled to a pH-auxostat was performed. In this perfusion culture, it was possible to maintain the acetic acid concentration below 7.6 mM during exponential growth of P. furiosus, resulting in 1.8 g of dry cells l(-1) at 15 h after inoculation.

Journal Article↗
PubMed1995

1H NMR investigation of the paramagnetic cluster environment in Pyrococcus furiosus three-iron ferredoxin: sequence-specific assignment of ligated cysteines independent of tertiary structure.

One- and two-dimensional 1H NMR data tailored to detect paramagnetically relaxed protons near the S = 1/2, three-iron-sulfur cluster of the ferredoxin from the hyperthermophile Pyrococcus furiosus are analyzed to sequence specifically assign the hyperfine shifted ligated cysteine signals, to determine the nature of the secondary structural elements on which these cysteines reside, and to define the tertiary contacts of the cluster with the remainder of the previously characterized secondary structure remote from the cluster [Teng, Q., Zhou, Z.-H., Busse, S.C., Howard, J.B., Adams, M. W. W., & La Mar, G. N. (1994) Biochemistry 33, 6316-6326]. Inspection of the geometry of the cluster ligating cysteines in the six structurally characterized cubane ferredoxin (Fd) clusters reveals a pattern of distances from the cluster iron(s) that indicate that each Cys will exhibit one backbone proton that will allow the detection of dipolar connectivities to the backbone of adjacent residues. It is expected that the first and last of the Cys in the cluster consensus binding sequence will exhibit weakly relaxed peptide NH and strongly relaxed C alpha H signals, while the two central Cys in that sequence will exhibit strongly relaxed peptide NH but weakly relaxed C alpha H peaks. These dipolar contacts are clearly observed for the three ligated Cys in 3Fe P. furiosus Fd, providing the first sequence specific assignment of ligated cysteines which do not explicitly require knowledge of the tertiary structure of the protein. This approach is proposed to have very general application to cubane ferredoxins. A combination of steady-state NOEs and short mixing time NOESY experiments demonstrate that Cys17 is on a short helix through Leu20 and that Cys56 likely initiates a type I turn, as observed in the crystal structure of the 3Fe Fd for Desulfovibrio gigas [Kissinger, C. R., Sieker, L. C., Adman, E. T., & Jensen, L. H. (1991) J. Mol. Biol. 219, 693-715]. The observed relaxation rates of resolved or partially resolved signals are shown to correlate with their proximity to the various iron in the cluster, as determined for the homologous residues in D. gigas Fd, providing additional qualitative information on tertiary contacts of the cluster.

Amino Acid Sequence↗
PubMed1995

Participation of the disulfide bridge in the redox cycle of the ferredoxin from the hyperthermophile Pyrococcus furiosus: 1H nuclear magnetic resonance time resolution of the four redox states at ambient temperature.

The oxidized and reduced forms of the [4Fe-4S]-containing ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus, Pf, have been investigated by 1H nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy and thiol titrations. We have identified and isolated at Ambient temperature four distinct redox states for the [4Fe-4S] form of the ferredoxin. These states differ in the redox state of the cluster, which is coordinated by Cys 11, Asp 14, Cys 17, and Cys 56, and of a disulfide bridge between Cys 21 and Cys 48. The protein, as isolated under anaerobic conditions, designated 4Fe FdBred, contains the reduced cluster and two free thiols. The cluster, but not the thiols, is readily oxidized by brief exposure to O2 to yield 4Fe FdBOX. Prolonged O2 treatment (> 24 h at 30 degrees C) is required to generate the protein with a disulfide (4Fe FdAOX) while this fully oxidized form is readily converted by brief reduction with sodium dithionite to the protein with a reduced cluster and a disulfide (4Fe FdAred). Analyses of the magnitude and the number of hyperfine-shifted resonances in each of the four redox states are discussed.

Archaea↗
PubMed1995

Response of rubredoxin from Pyrococcus furiosus to environmental changes: implications for the origin of hyperthermostability.

The bases of the hyperthermostability of rubredoxin from Pyrococcus furiosus (RdPf) have been probed by structural perturbations induced by solution pH and ionic strength changes. Comparison of the solution behavior at pH 7 and pH 2, as probed by far- and near-UV circular dichroism, Trp fluorescence emission, 1-anilinonaphthalene-8-sulfonate (ANS) binding, and NMR spectroscopy, reveals the presence of only minimal structural variations at room temperature. At pH 2, the protein displays a surprising nearly native-like behavior at high ionic strength while, at low ionic strength, it is capable of strongly binding the hydrophobic probe ANS. All the secondary and tertiary structural features, including the environment of the hydrophobic core, appear to be intact regardless of pH and ionic strength. The apparent "melting" or denaturation temperature at pH 2, however, is 42 degrees C lower than at pH 7. This is attributed to the perturbation of many electrostatic interactions, including the disruption of all the ion pairs, which is complete at pH 2, as indicated by electrometric pH titrations. The implications of these findings for the origins of the hyperthermostability of rubredoxin are discussed.

Anilino Naphthalenesulfonates↗
PubMed1995

1H NMR investigation of the electronic and molecular structure of the four-iron cluster ferredoxin from the hyperthermophile Pyrococcus furiosus. Identification of Asp 14 as a cluster ligand in each of the four redox states.

The molecular and electronic structure of the four-iron cluster of the ferredoxin (Fd) from the hyperthermophilic archaeon, Pyrococcus furiosus, Pf (which has only three Cys in the cluster binding consensus sequence), has been investigated by 1H NMR in order to determine the identity of the noncysteinyl cluster ligand in each of the four redox states [Gorst, C. M., Zhou, Z. H., Ma, K., Teng, Q., Howard, J. B., Adams, M. W., & La Mar, G. N. (1995) Biochemistry 34, 8788-8795], and to characterize the electron spin ground state for the reduced cluster which at 10 K exhibits an unusual predominant S = 3/2 ground state [Conover, R. C., Kowal, A. T., Fu, W., Park, J. -B., Aono, S., Adams, M. W. W., & Johnson, M. K. (1990) J. Biol. Chem. 265, 8533-8541]. It is demonstrated that a combination of 1D and 2D NMR tailored to relaxed resonances allows the location of four hyperfine shifted and paramagnetically relaxed spin systems which dictates that all four cluster ligands are amino acid side chains, rather than a solvent water/hydroxide at the unique non-Cys ligation site. Three of the ligands could be sequence-specifically assigned to the three Cys residues (positions 11, 17, and 56) in the consensus sequence for cluster binding, hence identifying the fourth ligand as Asp 14. It is concluded that the identification of Asp ligation to a 4Fe cluster is readily achieved in the reduced, but not in the oxidized cluster of Fd. Analysis of the relaxation properties and pattern of the hyperfine shifts in Pf Fd reveals very strong similarities to other Fds with S = 1/2 ground states, leading to the conclusion that the S = 3/2 ground state is not detected in solution at ambient temperatures, and this in independent of the redox state of the two remaining Cys residues in the protein (positions 21 and 48). However, the electron self-exchange rate for 4Fe Pf Fd is significantly slower than for other 4Fe Fd with complete Cys ligation. Changes in the pattern of hyperfine shifts between oxidized and reduced clusters for the four ligands in Pf Fd reveal that the most significant variation occurs for the Asp 14 orientation, suggesting that the altered Asp orientation may "gate" the electron transfer.

Archaea↗
PubMed1993

Structures of the modified folates in the thermophilic archaebacteria Pyrococcus furiosus.

The structures of the modified folates present in Pyrococcus furiosus have been determined. This was accomplished largely by the characterization of the arylamines resulting from the air oxidative cleavage of the reduced modified folates present in these cells, using both chemical and enzymatic methods. Cell extracts separated on DEAE-Sephadex columns showed one major peak containing the arylamines derived from the modified folates. These arylamines were not retained on the DEAE-Sephadex columns, indicating that they contained no net negative charge. Purification of the azo dye derivatives of these arylamines on a Bio-Gel P-6 column showed the presence of three different compounds (compounds 1, 2, and 3) in an average amount of 4.1, 7.6, and 22 nmol/g dry weight of cells, respectively. Each of these compounds readily underwent mild acid hydrolysis (0.1 M HCl, 110 degrees C, 1 min) to produce the azo dye derivative of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane (pAPT). The structure and stereochemistry (ribo) of the pAPT was the same as the pAPT present in methanopterin. In addition, compounds 1, 2, and 3 were each shown to contain 1 mol equiv of ribose and 1, 2, and 3 mol equiv of N-acetylglucosamine (gluNAc), respectively, and were designated as the azo dye derivatives of pAPT-ribose-gluNAc, pAPT-ribose-(gluNAc)2, and pAPT-ribose-(gluNAc)3. Each of these compounds was readily cleaved to the azo dye derivative of pAPT-ribose by the enzymatic action of beta-N-acetylglucosaminidase, indicating that all the gluNAc residues were beta-linked.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosamine↗
PubMed1991

Determinants of protein hyperthermostability: purification and amino acid sequence of rubredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus and secondary structure of the zinc adduct by NMR.

The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. The molecular mass (5397 Da), iron content (1.2 +/- 0.2 g-atom of Fe/mol), UV-vis spectrophotometric properties, and amino acid sequence (60% sequence identity with Clostridium pasteurianum Rd) are found to be typical of this class of redox protein. However, P. furiosus Rd is remarkably thermostable, being unaffected after incubation for 24 h at 95 degrees C. One- and two-dimensional 1H nuclear magnetic resonance spectra of the oxidized [Fe(III)Rd] and reduced [Fe(II)Rd] forms of P. furiosus Rd exhibited substantial paramagnetic line broadening, and this precluded detailed 3D structural studies. The apoprotein was not readily amenable to NMR studies due to apparent protein oxidation involving the free cysteine sulfhydryls. However, high-quality NMR spectra were obtained for the Zn-substituted protein, Zn(Rd), enabling detailed NMR signal assignment for all backbone amide and alpha and most side-chain protons. Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra. Residues A1-K6, Y10-E14, and F48-E51 form a three-strand antiparallel beta-sheet, which comprises ca. 30% of the primary sequence. Residues C5-Y10 and C38-A43 form types I and II amide-sulfur tight turns common to iron-sulfur proteins. These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C. pasteurianum. However, the beta-sheet domain in P. furiosus Rd is larger than that in C. pasteurianum Rd and appears to begin at the N-terminal residue. From analysis of the secondary structure, potentially stabilizing electrostatic interactions involving the charged groups of residues Ala(1), Glu(14), and Glu(52) are proposed. These interactions, which are not present in rubredoxins from mesophilic organisms, may prevent the beta-sheet from "unzipping" at elevated temperatures.

Amino Acid Sequence↗
PubMed1992

Proton NMR investigation of the oxidized three-iron clusters in the ferredoxins from the hyperthermophilic archae Pyrococcus furiosus and Thermococcus litoralis.

The 3Fe forms of ferredoxins (Fd) from the hyperthermophilic archaebacteria Pyrococcus furiosus (Pf) and Thermococcus litoralis (Tl) have been investigated by 1H NMR. A combination of one-dimensional nuclear Overhauser and two-dimensional NOESY and bond correlation spectroscopy provides the assignment of the aromatic residues, one conserved valine, and the location of the signals for each of the three cysteines coordinated to the clusters. Dipolar contacts between the Trp 2 and Tyr 46 in Pf Fd and from an invariant phenylalanine to an invariant valine and a cluster cysteine in both Fd confirm a folding pattern for these proteins that is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibro gigas. The sequence-specific assignment of the buried cysteine near the invariant phenylalanine has been made. The temperature dependence of the contact-shifted cysteinyl residues reveals a distinct 2:1 asymmetry in the magnetic coupling among the three high-spin ferric ions, in that one cysteine exhibits Curie behavior, while the other two cysteines display anti-Curie behavior. These magnetic properties are rationalized qualitatively on the basis of a magnetic coupling scheme where two iron couple to yield an intermediate spin of 2 which couples to the remaining S = 5/2 iron to yield the total cluster spin 1/2. This magnetic asymmetry appears to be a characteristic feature of oxidized 3 Fe clusters. Pf Fd also undergoes a dynamic equilibrium between two alternate forms that differ slightly in the environment of two of the coordinated cysteines. Analysis of the pattern of the contact shifts for the three cysteines in the two ferredoxins suggests that the cysteine coordinated to the unique iron does not have the same sequence origin.

Amino Acid Sequence↗
PubMed1994

Pyruvate ferredoxin oxidoreductases of the hyperthermophilic archaeon, Pyrococcus furiosus, and the hyperthermophilic bacterium, Thermotoga maritima, have different catalytic mechanisms.

Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from two hyperthermophiles, the archaeon Pyrococcus furiosus (Pf, Topt = 100 degrees C) and the bacterium Thermotoga maritima (Tm, Topt = 80 degrees C). Each catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 near the optimal growth temperature of the organism and are virtually inactive at 25 degrees C. Both PORs contain a thiamine pyrophosphate (TPP) cofactor and at least two [4Fe-4S] ferredoxin-type clusters. We have now shown, using EPR spectroscopy and metal analyses, that PfPOR also contains an unusual copper center that is not present in Tm POR. In addition, distinct catalytic intermediates were generated in both enzymes by the addition, separately and in combination, of the substrates pyruvate and CoASH, and these were examined by EPR spectroscopy. The addition of pyruvate to oxidized Pf POR produced an isotropic signal centered at g = 2.01, which was measurably broader in the presence of pyruvate-2(13)C. This signal, which was assigned to a (hydroxyethyl)thiamine pyrophosphate radical intermediate, was not observed in Tm POR under the same experimental conditions. Incubation of the oxidized enzymes with CoASH resulted in the partial reduction of the copper site in Pf POR and the partial reduction of a novel iron-sulfur center in Tm POR, which was not seen in the dithionite-reduced enzyme. The addition of both pyruvate and CoASH to the PORs in their oxidized states resulted in the reduction of the same iron-sulfur centers that are reduced by sodium dithionite.(ABSTRACT TRUNCATED AT 250 WORDS)

Archaea↗
PubMed1994

Solution 1H NMR determination of secondary structure for the three-iron form of ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.

Two-dimensional 1H NMR data have been used to make sequence-specific assignments and define the secondary structure of the three-iron form of the oxidized ferredoxin, Fd, from the hyperthermophilic archaeon Pyrococcus furiosus, Pf. Signals for at least some protons were located for 65 of the 66 amino acids in the sequence, in spite of the paramagnetic (S = 1/2) ground state, but not all could be assigned. Unassigned and missing signals could be qualitatively correlated with the expected proximity of the protons to the paramagnetic cluster. The secondary structure was deduced from qualitative analysis of the 2D nuclear Overhauser effect, which identified two antiparallel beta-sheets, one triple-stranded including Ala1-Ser5, Val39-Glu41, and Thr62-Ala66, and one double-stranded consisting of Glu26-Asn28 and Lys32-Glu34, as well as an alpha-helix involving Glu43-Glu54. Three tight type I turns are located at residues Asp7-Thr10, Pro22-Phe25, and Asp29-Gly31. Comparison with the crystal structure of Desulfovibrio gigas, Dg, Fd (Kissinger et al., 1991) reveals a very similar folding topology, although several secondary structural elements are extended in Pf relative to Dg Fd. Thus the beta-sheet involving the two termini is expanded to include the two terminal residues and incorporates a third strand from the internal loop that is lengthened by several insertions in Pf relative to Dg Fd. The double-stranded beta-sheet in the interior of Pf Fd is lengthened slightly due to a much tighter type I turn between the two strands. The helix near the C-terminus is three residues longer in Pf than in Dg Fd, as well as being shifted toward the N-terminus. The disulfide link between the two nonligating Cys residues (Cys21 and Cys48) is conserved in Pf Fd, but the link near the C-terminus is in the middle of the long alpha-helix in Pf Fd, instead of at the N-terminus of the helix as in Dg Fd. The extensions of the beta-sheets and alpha-helix increase the number of main-chain hydrogen bonds in Pf Fd by approximately 8 relative to those in Dg Fd and likely contribute to its remarkable thermostability (it is unaffected by anaerobic incubation at 95 degrees C for 24 h).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1996

Temperature dependence of the redox potential of rubredoxin from Pyrococcus furiosus: a molecular dynamics study.

Molecular dynamics simulations are used to evaluate the temperature dependent differences in structure, solvation, and energies for the iron-sulfur protein rubredoxin from the hyperthermophilic archebacterium Pyrococcus furiosus to understand the unusual temperature dependence of its redox potential [Adams, M. W. W. (1992) Adv. Inorg. Chem. 38, 341-396]. Simulations of both redox states performed at 295 and 363 K reveal that almost no backbone structure alteration occurs at the higher temperature and that the radius of gyration of the protein is temperature and redox state independent. The most striking change is that the penetration of the redox site by solvent molecules in the reduced from at 295 K, which was also seen in simulations of the reduced form of the mesophilic Clostridium pasteurianum rubredoxin at 295 K (Yelle, R. B., et al. (1995) Proteins 22, 154-167], is no longer seen to a significant extent in either redox state at 363 K. Comparing 295 to 363 K, the calculated change in the electrostatic potential of about -300 mV and in the negative of the potential energy of about -550 meV is consistent with the observed change in redox potential of -160 mV. Moreover, the calculated change is in the wrong direction if the penetrating water is excluded. These results show that changing solvent accessibility may be responsible for the temperature dependence of the redox potential of P. furiosus rubredoxin.

Archaea↗
PubMed1997

Dissecting contributions to the thermostability of Pyrococcus furiosus rubredoxin: beta-sheet chimeras.

The contributions to thermostability of interactions within the beta-sheet region of rubredoxins (Rds) were investigated by examining proteins in which beta-strand sequences of Rds from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) and the mesophilic bacterium Clostridium pasteurianum (Cp) were interchanged. The thermostabilities of the chimeric Rds were assessed by monitoring the decay of the visible absorbance at 490 nm and of the far-UV CD vs time at 92 degrees C. The chimeric Rds Pf15 Cp47 Pf (Pf Rd residues 2-15 and 48-54 and Cp Rd residues 16-47) and Cp15 Pf47 Cp were both found to be far less thermostable than wild-type Pf Rd, indicating that neither the beta-sheet residues (2-7, 10-15, and 48-53) nor the "core residues" (16-47) of Pf Rd independently confer Pf Rd-like thermostability. However, the chimeric Rd Pf47 Cp exhibits thermostability close to that of wild-type Pf Rd, suggesting that Pf Rd-like thermostability is conferred by interactions of beta-sheet strands 1 and 2 (residues 2-15) together with Pf core residues. In contrast, Cp Rd beta-sheet strands 1 and 2 connecting to Pf Rd core residues are thermodestabilizing in the chimera Cp15 Pf Rd. These results suggest that a global alignment which optimizes both main chain and side chain interactions between beta-sheet strands and core residues is more important than a few localized interactions within the beta-sheet in conferring Pf Rd-like thermostability.

Amino Acid Sequence↗
PubMed1997

Site-directed mutations of the 4Fe-ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus: role of the cluster-coordinating aspartate in physiological electron transfer reactions.

Ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus is a monomeric protein (7.5 kDa) that contains a single [4Fe-4S]1+, 2+ cluster. The protein is unusual in that its cluster is coordinated by three Cys and one Asp residue, rather than by the typical four Cys residues. Site-directed mutagenesis has been used to obtain mutant forms in which the cluster-coordinating Asp was replaced by Cys (D14C) and also by Ser (D14S), together with a third mutant (A1K) which contained N-Met-Lys at the N-terminus instead of N-Ala. Analyses using UV-visible absorption, far-UV circular dichroism, and EPR spectroscopy showed that there were no gross structural differences between the native and the three mutant forms and that they each contained a [4Fe-4S] cluster. The reduction potentials, determined by direct electrochemistry (at 23 degrees C, pH 8.0), of the D14S, D14C, and A1K mutants were -490, -422, and -382 mV, respectively, which compare with values of -375 mV for native [4Fe-4S]-containing ferredoxin and -160 mV for the [3Fe-4S]-containing form. The native, D14C, and A1K proteins functioned as electron acceptors in vitroat 80 degrees C for pyruvate ferredoxin oxidoreductase (POR) and aldehyde ferredoxin oxidoreductase (AOR) from P. furiosus using pyruvate and crotonaldehyde as substrates, respectively. The calculated kcat/Km values were similar for the three proteins when ferredoxin reduction was measured either directly by visible absorption or indirectly by coupling ferredoxin reoxidation to the reduction of metronidazole. In contrast, using the D14S mutant and the 3Fe-form of the native ferredoxin as electron acceptors, the activity with AOR was virtually undetectable, and with POR the calculated kcat/Km values were at least 3-fold lower than those obtained with the native (4Fe-), D14C, and A1K proteins. The ability of this 4Fe-ferredoxin to accept electrons from two oxidoreductases of the same organism is therefore not absolutely dependent upon Asp14, as this residue can be effectively replaced by Cys. However, the efficiency of electron transfer is compromised if Asp14 is replaced by Ser, or if the 4Fe-cluster is converted to the 3Fe-form, but Asp14 does not appear to offer any kinetic advantage over the expected Cys.

Archaea↗