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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↗
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↗
PubMed2002

Overexpression in Escherichia coli of the AT-rich trpA and trpB genes from the hyperthermophilic archaeon Pyrococcus furiosus.

Expression of AT-rich genes from microorganisms such as archaea is often inefficient in Escherichia coli. The trpA and trpB genes encoding the tryptophan synthase subunits were cloned from the hyperthermophilic archaeon Pyrococcus furiosus. No apparent difference in codon bias was found between the genes. However, using a conventional cloning vector having the lac promoter, the trpB gene was expressed poorly in E. coli, whereas the trpA gene was overexpressed. The expression of the trpB gene was remarkably enhanced (>12-fold) by the introduction of an overlapping leader open reading frame. The expression of the trpA gene was also improved ( approximately 1.5-fold). This approach may be useful for overexpressing various kinds of AT-rich genes.

AT Rich Sequence↗
PubMed1997

Purification and Characterization of a Proteasome from the Hyperthermophilic Archaeon Pyrococcus furiosus.

A 640-kDa proteasome consisting of (alpha) (25-kDa) and (beta) (22-kDa) subunits, and with a temperature optimum of 95(deg)C, was purified from crude cell extracts of a hyperthermophilic archaeon, Pyrococcus furiosus. Although this is the fourth member of the kingdom Euryarchaeota (and the first hyperthermophile) found to contain a proteasome, none has been identified among the members of the kingdom Crenarchaeota.

Journal Article↗
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↗
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↗
PubMed1998

A topA intein in Pyrococcus furiosus and its relatedness to the r-gyr intein of Methanococcus jannaschii.

A new intein coding sequence was found in a topA (DNA topoisomerase I) gene by cloning and sequencing this gene from the hyperthermophilic Archaeon Pyrococcus furiosus. The predicted Pfu topA intein sequence is 373 amino acids long and located two residues away from the catalytic tyrosine of the topoisomerase. It contains putative intein sequence blocks (C, E, and H) associated with intein endonuclease activity, in addition to intein sequence blocks (A, B, F, and G) that are necessary for protein splicing. This DNA topoisomerase I intein is most related to a reverse gyrase intein from the methanogenic Archaeon Methanococcus jannaschii. These two inteins share 31% amino acid sequence identity and, more importantly, have the same insertion sites in their respective host proteins. It is suggested that these two inteins are homologous inteins present in structurally related, but functionally distinct, proteins, with implications on intein evolution and intein homing.

Amino Acid Sequence↗
PubMed1998

The tRNA(guanine-26,N2-N2) methyltransferase (Trm1) from the hyperthermophilic archaeon Pyrococcus furiosus: cloning, sequencing of the gene and its expression in Escherichia coli.

The structural gene pfTRM1 (GenBank accession no. AF051912), encoding tRNA(guanine-26, N 2- N 2) methyltransferase (EC 2.1.1.32) of the strictly anaerobic hyperthermophilic archaeon Pyrococcus furiosus, has been identified by sequence similarity to the TRM1 gene of Saccharomyces cerevisiae (YDR120c). The pfTRM1 gene in a 3.0 kb restriction DNA fragment of P.furiosus genomic DNA has been cloned by library screening using a PCR probe to the 5'-part of the corresponding ORF. Sequence analysis revealed an entire ORF of 1143 bp encoding a polypeptide of 381 residues (calculated molecular mass 43.3 kDa). The deduced amino acid sequence of this newly identified gene shares significant similarity with the TRM1- like genes of three other archaea (Methanococcus jannaschii, Methanobacterium thermoautotrophicum and Archaeoglobus fulgidus), one eukaryon (Caenorhabditis elegans) and one hyperthermophilic eubacterium (Aquifex aeolicus). Two short consensus motifs for S-adenosyl-l-methionine binding are detected in the sequence of pfTrm1p. Cloning of the P.furiosus TRM1 gene in an Escherichia coli expression vector allowed expression of the recombinant protein (pfTrm1p) with an apparent molecular mass of 42 kDa. A protein extract from the transformed E.coli cells shows enzymatic activity for the quantitative formation of N 2, N 2-dimethylguanosine at position 26 in a transcript of yeast tRNAPhe used as substrate. The recombinant enzyme was also shown to modify bulk E.coli tRNAs in vivo.

Amino Acid Sequence↗
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

A novel DNA polymerase in the hyperthermophilic archaeon, Pyrococcus furiosus: gene cloning, expression, and characterization.

BACKGROUND: In many respects Archaea are much more like eukaryotes than prokaryotes with respect to the conservation of many of the components involved in transcription, translation and DNA replication. So far, only a few DNA polymerases with structures similar to those of eukaryotic DNA polymerase a have been found in Archaea. The identification and characterization of all the DNA polymerases of one archaeon would add considerably to our knowledge of the basic mechanisms of DNA replication in these organisms. RESULTS: We have identified a novel DNA polymerase composed of two proteins, DP1 and DP2, with molecular weights of 69294 Da and 143161 Da, respectively, in the hyperthermophilic archaeon, Pyrococcus furiosus, and have cloned the corresponding genes which are tandemly arranged on the Pyrococcus genome. No significant sequence homology was found between these two proteins and other known DNA polymerases. The pol genes were transcribed as part of a single operon that additionally contained genes homologous to the cdc18+/CDC6 and Dmc1/Rad51 family of proteins. We purified the Pyrococcus DNA polymerase from Escherichia coli strains expressing the cloned genes and characterized its activity. It possesses strong 3' --> 5' exonucleolytic activity and has a template-primer preference which is characteristic of a replicative DNA polymerase. CONCLUSION: In P. furiosus, we identified a second DNA polymerase encoded by two genes, neither of which display significant homology to any other known DNA polymerase. Both the enzymatic properties of the enzyme and the gene organization raise the possibility that this enzyme might be the replicative DNA polymerase of P. furiosus.

Antigens, CD↗
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↗
PubMed1997

Methionine aminopeptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: molecular cloning and overexpression in Escherichia coli of the gene, and characteristics of the enzyme.

A gene for a methionine aminopeptidase (MAP; EC 3.4.11.18), which catalyzes the removal of amino-terminal methionine from the growing peptide chain on the ribosome, has been cloned from the hyperthermophilic Archaeon, Pyrococcus furiosus, by a novel method effectively using its cosmid protein library, sequenced and expressed in Escherichia coli. The DNA sequence encodes a protein containing 295 amino acid residues with methionine at the N-terminus. From protein analyses of the recombinant protein expressed in E. coli, by using both amino acid sequence analysis from the N-terminus by automated Edman degradation and analyses of molecular masses of the peptides generated by two enzymatic cleavages performed independently, digestions with lysylendopeptidase and Endoproteinase Asp-N, with ionspray mass spectrometry, the primary structure of the protein has been elucidated to be completely identical with that deduced from its DNA sequence. Comparison of the amino acid sequence of P. furiosus MAP (P.f. MAP) with those of other MAPs from Eukarya and Bacteria showed that the protein has a high degree of sequence homology in the stretches surrounding the five cobalt-binding residues fully preserved in all of MAPs determined so far, but P.f. MAP belongs to Type II because it has an extra long insertion of about 60 amino acid residues between the fourth and fifth cobalt-binding ligands, similar to MAPs from human and rat, and to Met-AP2 from Saccharomyces cerevisiae, in comparison to Type I MAPs from Bacteria. Therefore, P.f. MAP seems to be rather close to those from Eukarya, although it is distinct in lacking the N-terminal extension of about 90-150 residues universally found in MAPs from Eukarya. These findings suggest that P.f. MAP is evolutionally located at the Eukarya-Bacteria boundary. The enzyme expressed in E. coli exhibits a considerable thermostability, with a half-life of approximately 4.5 h at 90 degrees C and an optimum temperature of around 90 degrees C.

Amino Acid Sequence↗
PubMed1995

Insights into thermal stability from a comparison of the glutamate dehydrogenases from Pyrococcus furiosus and Thermococcus litoralis.

In the light of the solution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from the mesophile Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequences for the thermophilic glutamate dehydrogenases from Thermococcus litoralis and Pyrococcus furiosus against the sequence and the molecular structure of the glutamate dehydrogenase from C. symbiosum, to provide insights into the molecular basis of their thermostability. This homology-based modelling is simplified by the relatively small number of amino acid substitutions between the two thermophilic glutamate dehydrogenase sequences. The most frequent amino acid exchanges involve substitutions which increase the hydrophobicity and sidechain branching in the more thermostable enzyme; particularly common is the substitution of valine to isoleucine. Examination of the sequence differences suggests that enhanced packing within the buried core of the protein plays an important role in maintaining stability at extreme temperatures. One hot spot for the accumulation of exchanges lies close to a region of the molecule involved in its conformational flexibility and these changes may modulate the dynamics of this enzyme and thereby contribute to increased stability.

Amino Acid Sequence↗
PubMed1998

A protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus contains two thioredoxin fold units.

Protein disulfide bond formation is a rate limiting step in protein folding and is catalyzed by enzymes belonging to the protein disulfide oxidoreductase superfamily, including protein disulfide isomerase (PDI) in eucarya and DsbA in bacteria. The first high resolution X-ray crystal structure of a protein disulfide oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus reveals structural details that suggest a relation to eukaryotic PDI. The protein consists of two homologous structural units with low sequence identity. Each unit contains a thioredoxin fold with a distinct CXXC active site motif. The accessibilities of both active sites are rather different as are, very likely, their redox properties. The protein shows the ability to catalyze the oxidation of dithiols as well as the reduction of disulfide bridges.

Amino Acid Sequence↗
PubMed1992

Quantitative measurement of small through-hydrogen-bond and 'through-space' 1H-113Cd and 1H-199Hg J couplings in metal-substituted rubredoxin from Pyrococcus furiosus.

A method is described for measurement of small unresolvable heteronuclear J couplings. The method is based on quantitative analysis of a phase-purged heteronuclear spin-echo difference spectrum, and is demonstrated for measuring 1H-113Cd and 1H-199Hg J couplings in metal-substituted rubredoxin (M(r) approximately 5.4 kDa) from Pyrococcus furiosus. Couplings from cadmium to backbone amide protons that are hydrogen bonded to the Cys-S atoms directly bonded to Cd vary from smaller than 0.3 to 1.8 Hz; a 'through-space' coupling between Cd and the protons of an alanine methyl group was measured to be 0.3 Hz. Couplings to 199Hg are significantly larger and fall in the 0.4-4 Hz range.

Amino Acid Sequence↗
PubMed1997

Purification and properties of acetyl-CoA synthetase (ADP-forming), an archaeal enzyme of acetate formation and ATP synthesis, from the hyperthermophile Pyrococcus furiosus.

Acetyl-CoA synthetase (ADP-forming) is an enzyme in Archaea that catalyzes the formation of acetate from acetyl-CoA and couples this reaction with the synthesis of ATP from ADP and Pi (acetyl-CoA + ADP + Pi --> acetate + ATP + CoA) [Schifer, T., Selig, M. & Schonheit, P. (1993) Arch. Microbiol. 159, 72-83]. The enzyme from the anaerobic hyperthermophile Pyrococcus furiosus was purified 96-fold with a yield of 20% to apparent electrophoretic homogeneity. The oxygen-stable enzyme had an apparent molecular mass of 145 kDa and was composed of two subunits with apparent molecular masses of 47 kDa and 25 kDa, indicating an alpha2beta2 structure. The N-terminal amino acid sequences of both subunits were determined; they do not show significant identity to other proteins in databases. The purified enzyme catalyzed the reversible conversion of acetyl-CoA, ADP and Pi to acetate, ATP and CoA. The apparent Vmax value in the direction of acetate formation was 18 U/mg (55 degrees C), the apparent Km values for acetyl-CoA, ADP and Pi were 17 microM, 60 microM and 200 microM, respectively. ADP and Pi could not be replaced by AMP and PPi, defining the enzyme as an ADP-forming rather than an AMP-forming acetyl-CoA synthetase. The apparent Vmax value in the direction of acetyl-CoA formation was about 40 U/mg (55 degrees C), and the apparent Km values for acetate, ATP and CoA were 660 microM, 80 microM and 30 microM, respectively. The purified enzyme was not specific for acetyl-CoA or acetate, in addition to acetyl-CoA (100%), the enzyme accepts propionyl-CoA (110%) and butyryl-CoA (92%), and in addition to acetate (100%), the enzyme accepts propionate (100%), butyrate (92%), isobutyrate (79%), valerate (36%) and isovalerate (34%), indicating that the enzyme functions as an acyl-CoA synthetase (ADP-forming) with a broad substrate spectrum. Succinate, phenylacetate and indoleacetate did not serve as substrates for the enzyme (<3%). In addition to ADP (100%), GDP (220%) and IDP (250%) were used, and in addition to ATP (100%), GTP (210%) and ITP (320%) were used. Pyrimidine nucleotides were not accepted. The enzyme was dependent on Mg2+, which could be partly substituted by Mn2+ and Co2+. The pH optimum was pH 7. The enzyme has a temperature optimum at 90 degrees C, which is in accordance with its physiological function under hyperthermophilic conditions. The enzyme was stabilized against heat inactivation by salts. In the presence of KCI (1 M), which was most effective, the enzyme did not loose activity after 2 h incubation at 100 degrees C.

Acetic Acid↗
PubMed1997

The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus, a missing link in the evolution of carbamoyl phosphate biosynthesis.

Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitrogen donor and are composed of two subunits (or domains), one exhibiting glutaminase activity, the other able to synthesize carbamoyl phosphate (CP) from bicarbonate, ATP, and ammonia. The pseudodimeric organization of this synthetase suggested that it has evolved by duplication of a smaller kinase, possibly a carbamate kinase (CK). In contrast to other prokaryotes the hyperthermophilic archaeon Pyrococcus furiosus was found to synthesize CP by using ammonia and not glutamine. We have purified the cognate enzyme and found it to be a dimer of two identical subunits of Mr 32,000. Its thermostability is considerable, 50% activity being retained after 1 h at 100 degrees C or 3 h at 95 degrees C. The corresponding gene was cloned by PCR and found to present about 50% amino acid identity with known CKs. The stoichiometry of the reaction (two ATP consumed per CP synthesized) and the ability of the enzyme to catalyze at high rate a bicarbonate-dependent ATPase reaction however clearly distinguish P. furiosus CPS from ordinary CKs. Thus the CPS of P. furiosus could represent a primeval step in the evolution of CPS from CK. Our results suggest that the first event in this evolution was the emergence of a primeval synthetase composed of subunits able to synthesize both carboxyphosphate and CP; this step would have preceded the duplication assumed to have generated the two subdomains of modern CPSs. The gene coding for this CK-like CPS was called cpkA.

Adenine Nucleotides↗