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PubMed1998

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

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.

Japan↗
PubMed2006

Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi.

Four open reading frames encoding putative nitrilases were identified in the genomes of the hyperthermophilic archaea Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus furiosus, and Aeropyrum pernix (growth temperature 90-100 degrees C). The nitrilase encoding genes were cloned and overexpressed in Escherichia coli. Enzymatic activity could only be detected in the case of Py. abyssi. This recombinant nitrilase was purified by heat treatment of E. coli crude extract followed by anion-exchange chromatography with a yield of 88% and a specific activity of 0.14 U/mg. The recombinant enzyme, which represents the first archaeal nitrilase, is a dimer (29.8 kDa/subunit) with an isoelectric point of pI 5.3. The nitrilase is active at a broad temperature (60-90 degrees C) and neutral pH range (pH 6.0-8.0). The recombinant enzyme is highly thermostable with a half-life of 25 h at 70 degrees C, 9 h at 80 degrees C, and 6 h at 90 degrees C. Thermostability measurements by employing circular dichroism spectroscopy and differential scanning microcalorimetry, at neutral pH, have shown that the enzyme unfolds up to 90 degrees C reversibly and has a T(m) of 112.7 degrees C. An inhibition of the enzymatic activity was observed in the presence of acetone and metal ions such as Ag(2+) and Hg(2+). The nitrilase hydrolyzes preferentially aliphatic substrates and the best substrate is malononitrile with a K(m) value of 3.47 mM.

Aeropyrum↗
PubMed2002

A solution NMR molecular model for the aspartate-ligated, cubane cluster containing ferredoxin from the hyperthermophilic archeaon Pyrococcus furiosus.

A solution molecular model for the conformationally dynamically heterogeneous Pyrococcus furiosus ferredoxin with an intact disulfide bond has been constructed on the basis of reported (1)H NMR spectral parameters using distance geometry and simulated annealing protocols. Conventional long-mixing time NOESY and H-bonding constraints have been augmented by previously reported short-mixing time NOESY, steady-state NOE, and cluster paramagnetism-induced relaxation. The family of 15 structures with inconsequential violations exhibited low rms deviations for backbone atoms for the overwhelming majority of the residues, including the cluster ligating loop with the unprecedented ligated Asp14. Larger rms deviations were observed across the disulfide bond, but closer inspection revealed that the 15 structures can be factored into 10 substructures exhibiting an "S" or right-handed disulfide orientation and 5 exhibiting an "R" or left-handed disulfide orientation. The remainder of the structure is indistinguishable for the two disulfide orientations but confirms stabilizing extensions of secondary structural elements in the lengthening of the long helix and both the lengthening and incorporation of a third strand into the beta-sheet involving the termini, with these extensions interacting strongly in a modular fashion through the rings of Tyr46 and Trp2. These extensions of stabilizing interactions in Pyrococcus furiosus Fd, however, lead to strong destabilization of the disulfide bond and destabilization of the highly conserved first and last beta-turns in the sequence. It is concluded that the structural alternations in Pyrococcus Fd relative to other hyperthermostable Fds are not to increase thermostability but to place "stress" on the disulfide bond and render it more reducible. The possible physiological implications of this unique reducible disulfide bond are discussed.

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↗
PubMed2004

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus. Mechanism of the reaction and assignment of disulfide bonds.

The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5'-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins.

Amino Acid Sequence↗
PubMed2002

Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi.

Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (10(2) to 10(3) transformants per micro g of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.

BH3 Interacting Domain Death Agonist Protein↗
PubMed1995

Chromosomal organization and nucleotide sequence of the fus-gene encoding elongation factor 2 (EF-2) of the hyperthermophilic archaeum Pyrococcus woesei.

A Pyrococcus woesei EcoRI DNA fragment (3400 bp) harbouring the gene fus for elongation factor 2 (EF-2) was cloned and almost completely sequenced. Unlike Methanococcus vannielii (which displays the 'str operon'-like fus and tuf gene context, 5'-rps12-rps7-fus-tuf-3'), and similar to Sulfolobus acidocaldarius and Desulfurococcus mobilis, the Pyrococcus fus gene (732 codons) is unlinked to the rps and tuf genes, and is immediately followed (57 bp intergenic spacing) by an ORF of 106 codons. Both ORFs are preceded by potential archaeal promoters located 52 bp (for fus) and 37 bp (for ORF106) upstream of the putative start codons. The Pyrococcus EF-2(G) equivalent factor is somewhat closer to the eukaryal than to the bacterial homolog, and also shares with the former the C-terminal sequence required for ADP ribosylation of EF-2 by Diphtheria toxin.

Amino Acid Sequence↗
PubMed2005

S-adenosylhomocysteine hydrolase from the archaeon Pyrococcus furiosus: biochemical characterization and analysis of protein structure by comparative molecular modeling.

S-adenosylhomocysteine hydrolase (AdoHcyHD) is an ubiquitous enzyme that catalyzes the breakdown of S-adenosylhomocysteine, a powerful inhibitor of most transmethylation reactions, to adenosine and L-homocysteine. AdoHcyHD from the hyperthermophilic archaeon Pyrococcus furiosus (PfAdoHcyHD) was cloned, expressed in Escherichia coli, and purified. The enzyme is thermoactive with an optimum temperature of 95 degrees C, and thermostable retaining 100% residual activity after 1 h at 90 degrees C and showing an apparent melting temperature of 98 degrees C. The enzyme is a homotetramer of 190 kDa and contains four cysteine residues per subunit. Thiol groups are not involved in the catalytic process whereas disulfide bond(s) could be present since incubation with 0.8 M dithiothreitol reduces enzyme activity. Multiple sequence alignment of hyperthermophilic AdoHcyHD reveals the presence of two cysteine residues in the N-terminus of the enzyme conserved only in members of Pyrococcus species, and shows that hyperthermophilic AdoHcyHD lack eight C-terminal residues, thought to be important for structural and functional properties of the eukaryotic enzyme. The homology-modeled structure of PfAdoHcyHD shows that Trp220, Tyr181, Tyr184, and Leu185 of each subunit and Ile244 from a different subunit form a network of hydrophobic and aromatic interactions in the central channel formed at the subunits interface. These contacts partially replace the interactions of the C-terminal tail of the eukaryotic enzyme required for tetramer stability. Moreover, Cys221 and Lys245 substitute for Thr430 and Lys426, respectively, of the human enzyme in NAD-binding. Interestingly, all these residues are fairly well conserved in hyperthermophilic AdoHcyHDs but not in mesophilic ones, thus suggesting a common adaptation mechanism at high temperatures.

Adenosine↗
PubMed2004

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

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

Adenosine Triphosphatases↗
PubMed2003

A hyperthermostable novel protein-disulfide oxidoreductase is reduced by thioredoxin reductase from hyperthermophilic archaeon Pyrococcus horikoshii.

A redox protein gene (PH0178) with high sequence homology to a glutaredoxin from Pyrococcus furiosus and a thioredoxin reductase homologue gene (PH1426) were found in the genome sequence of Pyrococcus horikoshii. These two genes were cloned and the corresponding expressed proteins were characterized. The redox protein from PH0178 had strong thioredoxin-like activity, but no glutaredoxin activity. The protein from PH1426 had some reductase activity against thioredoxin from Escherichia coli as well as the redox protein (PH0178). The protein from PH1426 was a typical, homodimeric flavoprotein. These results indicate that the redox protein (PH0178) is not a glutaredoxin but, rather, a new protein-disulfide oxidoreductase that is involved in a thioredoxin-like system with thioredoxin reductase (PH1426) in P. horikoshii. The redox protein and thioredoxin reductase retained their full activities for over 1h at 100 degrees C. The redox potential of the redox protein was similar to that of thioredoxin from E. coli and lower than that of glutathione. Site-directed mutagenesis studies revealed that the active site of the redox protein corresponds to a CPYC sequence, located in the middle of the sequence.

Amino Acid Sequence↗
PubMed2002

The X-ray crystal structure of pyrrolidone-carboxylate peptidase from hyperthermophilic archaea Pyrococcus horikoshii.

The crystal structure of pyrrolidone-carboxylate peptidase (PCP) from hyperthermophilic archaea Pyrococcus horikoshii (PhoPCP) has been determined at 1.6-A resolution by X-ray crystallography. PCP belongs to the C15 family of cysteine protease, and specifically removes the amino terminal pyroglutamate residue from a wide range of N-terminal-blocking peptides. The crystal structure is very similar to that of other hyperthermophiles, Pyrococcus furiosus and Thermococcus litoralis, and even that from the mesophile, Bacillus amyloliquefaciens. The inter-subunit disulfide bonds, which have been proposed as one of the thermostabilizing factors of the PCP from such hyperthermophiles, was not present in PhoPCP. The result suggests that the thermostability of PhoPCP may be obtained by the accumulation of many weak factors.

Amino Acid Sequence↗
PubMed2002

Pyrococcus genome comparison evidences chromosome shuffling-driven evolution.

The genomes of three Pyrococcus species, P.abyssi, P.furiosus and P.horikoshii, were compared at the DNA level, taking advantage of our identification of their replication origins. Three types of rearrangements have been identified: (i) inversion and translation across the replication axis (origin/terminus), (ii) inversion and translocation restricted to a replichore (the half chromosome divided by the replication axis) and (iii) apparent mobility of long clusters of repeated sequences. Rearrangements restricted within a replichore were more common between P.furiosus and the two other Pyrococcus species than between P.horikoshii and P.abyssi. A strong correlation was found between 23 homologous insertion sequence elements, present only in P.furiosus, and recombined segment boundaries, suggesting that transposition events have been a major cause of genomic disruption in this species. Moreover, gene orientation bias was much more disrupted than strand composition biases in fragments that switched their orientation within a replichore upon recombination. This allowed us to conclude that one reversion and one translation occurred in P.abyssi after its divergence from P.horikoshii, and that a smaller segment has specifically recombined in P.furiosus. Whereas a majority of genes are transcribed in the same direction as DNA replication in P.horikoshii and P.abyssi, the colinearity of transcription and replication is only maintained for highly transcribed genes in P.furiosus. We discuss the implications of genomic rearrangements on gene orientation and composition biases, and their consequences on sequence evolution.

Chromosome Inversion↗
PubMed2005

Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5'-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi.

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction-modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5'-GTAC and leave a 3'-TA overhang (5'-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90 degrees C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure.

Base Sequence↗
PubMed2003

Physiological responses of the hyperthermophilic archaeon "Pyrococcus abyssi" to DNA damage caused by ionizing radiation.

The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of "P. abyssi" chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated "P. abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated "P. abyssi" cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that "P. abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.

Blotting, Western↗
PubMed2002

Pyrococcus prefoldin stabilizes protein-folding intermediates and transfers them to chaperonins for correct folding.

A molecular chaperone prefoldin/GimC from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 was characterized. Pyrococcus prefoldin protected porcine heart citrate synthase from thermal aggregation whereas each subunit alone afforded little protection. It also arrested the spontaneous refolding of acid-denatured green fluorescent protein and then transferred it not only to a group II chaperonin from the hyperthermophilic archaeum Thermococcus sp. strain KS-1, but also to a group I chaperonin from the thermophilic bacterium Thermus thermophilus HB8 for subsequent ATP dependent refolding.

Animals↗
PubMed2000

Cloning, expression, and purification of the His(6)-tagged thermostable beta-galactosidase from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme.

In the previous study we cloned Pyrococcus woesei gene coding thermostable beta-galactosidase into pET30-LIC expression plasmid. The nucleotide sequence revealed that beta-galactosidase of P. woesei consists of 510 amino acids and has a molecular weight of 59, 056 kDa (GenBank Accession No. AF043283). It shows 99.9% nucleotide identity to the nucleotide sequence of beta-galactosidase from Pyrococcus furiosus. We also demonstrated that thermostable beta-galactosidase can be produced with high yield by Escherichia coli strain and can be easy separated by thermal precipitation of other bacterial proteins at 85 degrees C (S. D $$;abrowski, J. Maciuńska, and J. Synowiecki, 1998, Mol. Biotechnol. 10, 217-222). In this study we presented a new expression system for producing P. woesei beta-galactosidase in Escherichia coli and one-step chromatography purification procedure for obtaining pure enzyme (His(6)-tagged beta-galactosidase). The recombinant beta-galactosidase contained a polyhistidine tag at the N-terminus (20 additional amino acids) that allowed single-step isolation by Ni affinity chromatography. The enzyme was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Ni(2+)-TED-Sepharose columns. The enzyme was characterized and displayed high activity and thermostability. This bacterial expression system appears to be a good method for production of the thermostable beta-galactosidase.

Amino Acid Sequence↗
PubMed1998

Cloning and nucleotide sequence of the thermostable beta-galactosidase gene from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme.

Pyrococcus woesei (DSM 3773) beta-galactosidase gene amplified by polymerase chain reaction was cloned into KpnI and HindIII binding sites of pET-30LIC expression plasmid. The obtained pGal2 (6785 bp) transcription vector was then transferred to Escherichia coli B121 (DE3) cells. High identity (99.9%) of DNA sequences suggests that beta-galactosidases from P. woesei and Pyrococcus furiosus are closely related. This enzyme from E. coli transformant is a unique thermostable protein in the cells and can be successfully separated by thermal precipitation of other bacterial proteins at 85 degrees C. The crude beta-galactosidase remaining in the solution comprises about 21% of the total amount of proteins extracted from E. coli cells and has maximal activity at pH 5.4 and temperature of 93 degrees C. Isolated enzyme is active at temperatures up to 110 degrees C and the activity loss after 4 h of incubation at 85 and 93 degrees C did not exceed 11 and 15% of the initial value respectively.

Amino Acid Sequence↗
PubMed2000

Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from Pyrococcus furiosus; evidence for a [2Fe-2S] cluster with Asp(Cys)3 ligands.

The consecutive structural genes for the iron-sulfur flavoenzyme sulfide dehydrogenase, sudB and sudA, have been identified in the genome of Pyrococcus furiosus. The translated sequences encode a heterodimeric protein with an alpha-subunit, SudA, of 52598 Da and a beta-subunit, SudB, of 30686 Da. The alpha-subunit carries a FAD, a putative nucleotide binding site for NADPH, and a [2Fe-2S]2+,+ prosthetic group. The latter exhibit EPR g-values, 2.035, 1.908, 1.786, and reduction potential, Em,8 = +80 mV, reminiscent of Rieske-type clusters; however, comparative sequence analysis indicates that this cluster is coordinated by a novel motif of one Asp and three Cys ligands. The motif is not only found in the genome of hyperthermophilic archaea and hyperthermophilic bacteria, but also in that of mesophilic Treponema pallidum. The beta-subunit of sulfide dehydrogenase contains another FAD, another putative binding site for NADPH, a [3Fe-4S]+,0 cluster, and a [4Fe-4S]2+,+ cluster. The 3Fe cluster has an unusually high reduction potential, Em,8 = +230 mV. The reduced 4Fe cluster exhibits a complex EPR signal, presumably resulting from magnetic interaction of its S = 1/2 spin with the S=2 spin of the reduced 3Fe cluster. The 4Fe cluster can be reduced with deazaflavin/EDTA/light but not with sodium dithionite; however, it is readily reduced with NADPH. SudA is highly homologous to KOD1-GO-GAT (or KOD1-GltA), a single-gene encoded protein in Pyrococcus kodakaraensis, which has been putatively identified as hyperthermophilic glutamate synthase. However, P. furiosus sulfide dehydrogenase does not have glutamate synthase activity. SudB is highly homologous to HydG, the gamma-subunit of P. furiosus NiFe hydrogenase. The latter enzyme also has sulfide dehydrogenase activity. The P. furiosus genome contains a second set of consecutive genes, sudY and sudX, with very high homology to the sudB and sudA genes, and possibly encoding a sulfide dehydrogenase isoenzyme. Each subunit of sulfide dehydrogenase is a primary structural paradigm for a different class of iron-sulfur flavoproteins.

Amino Acid Motifs↗