Crystal structure of the ferritin from the hyperthermophilic archaeal anaerobe <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>

The crystal structure of the ferritin from the archaeon, hyperthermophile and anaerobe Pyrococcus furiosus (PfFtn) is presented. While many ferritin structures from bacteria to mammals have been reported, until now only one was available from archaea, the ferritin from Archaeoglobus fulgidus (AfFtn). The PfFtn 24-mer exhibits the 432 point-group symmetry that is characteristic of most ferritins, which suggests that the 23 symmetry found in the previously reported AfFtn is not a common feature of archaeal ferritins. Consequently, the four large pores that were found in AfFtn are not present in PfFtn. The structure has been solved by molecular replacement and refined at 2.75-Å resolution to R = 0.195 and R _free = 0.247. The ferroxidase center of the aerobically crystallized ferritin contains one iron at site A and shows sites B and C only upon iron or zinc soaking. Electron paramagnetic resonance studies suggest this iron depletion of the native ferroxidase center to be a result of a complexation of iron by the crystallization salt. The extreme thermostability of PfFtn is compared with that of eight structurally similar ferritins and is proposed to originate mostly from the observed high number of intrasubunit hydrogen bonds. A preservation of the monomer fold, rather than the 24-mer assembly, appears to be the most important factor that protects the ferritin from inactivation by heat.     

Characterization of a thermostable cyclodextrin glucanotransferase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> DSM3638

A gene that encodes the enzyme Pyrococcus furiosus cyclodextrin glucanotransferase (PFCGT) was cloned in Escherichia coli. PFCGT was highly expressed in recombinant E. coli after compensation for codon usage bias using the pRARE plasmid. Purified PFCGT was extremely thermostable with an optimal temperature and pH of 95°C and 5.0, respectively, retaining 97% of its activity at 100°C. Incubation at 60°C for 20 min during the purification process led to a 1.5-fold increase in enzymatic activity. A time course assay of the PFCGT reaction with starch indicated that cyclic α-1,4-glucans with DPs greater than 20 were produced at the beginning of the incubation followed by an increase in β-CD. The major final product of PFCGT cyclization was β-CD, and thus the enzyme is a β-CGTase.     

Characterization of malate dehydrogenase from the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrobaculum islandicum</Emphasis>

Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins are the same. The apparent K _m values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80°C and pH 8.0) were 15 and 86 μM, respectively, with specific activity as high as 470 U mg⁻¹. Activity decreased more than 90% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific activity of the P. islandicum MDH back-reaction also decreased more than 90% when malate and NAD⁺ were used as substrates and was not detected with NADP⁺. A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines suggesting minimal lateral mdh transfer between Archaea.     

Re-Annotation of Two Hyperthermophilic Archaea <Emphasis Type="Italic">Pyrococcus abyssi</Emphasis> GE5 and <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> DSM 3638

Pyrococcus abyssi GE5 (P. aby) and Pyrococcus furiosus DSM 3638 (P. fur) are two model hyperthermophilic archaea. However, their annotations in public databases are unsatisfactory. In this article, the two genomes were re-annotated according to the following steps. (i) All “hypothetical genes” in the original annotation were re-identified based on the Z-curve method, and some of them were recognized as non-coding open reading frames (ORFs). Evidence showed that the recognized non-coding ORFs were highly unlikely to encode proteins. (ii) The translation initiation sites (TISs) of all the annotated genes were re-located, and more than 10% of the TISs were shifted to 5′-upstream or 3′-downstream regions. (iii) The functions of the refined “hypothetical genes” were predicted using sequence alignment tools, more than 200 originally annotated “hypothetical genes” in either of the two hyperthermophiles were assigned functions. A large number of these functions have reference support or experimentally characterized homologues. All the refined information will serve as a valuable resource for research on P. aby and P. fur, which may be helpful in the exploration of thermal adaptation mechanisms. The complete re-annotation files of P. aby and P. fur are available at .     

An archaeal bi-species biofilm formed by <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> and <Emphasis Type="Italic">Methanopyrus kandleri</Emphasis>

Recently it was shown that Pyrococcus furiosus uses its flagella not only for swimming, but also for establishment of cell-cell connections, and for adhesion to abiotic surfaces. Therefore, it was asked here if P. furiosus might be able to adhere also to biotic surfaces. Since Methanopyrus kandleri can be found in habitats similar to those of P. furiosus (seawater close to the boiling point and anaerobic conditions) it was tested if interactions between both archaea occur. Using a standard medium and a gas phase reduced in H(2) (compared with the optimal gas phase for M. kandleri) we were able to grow both species in a stable coculture. Very interestingly, M. kandleri could adhere to glass under such conditions, but not P. furiosus. This latter archaeum, however, was able to adhere onto M. kandleri cells and onto itself, resulting in structured biofilms on glass. These very often appeared as a bottom layer of M. kandleri cells covered by a multitude of P. furiosus cells. Interactions between P. furiosus and M. kandleri were mediated not only by flagella, but also by direct cell-cell contact.     

Improvement of the enzymatic activity of the hyperthermophilic cellulase from <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis>

A hyperthermophilic β-1,4 endoglucanase (EGPh) from the hyperthermophilic archaeon Pyrococcus horikoshii exhibits a strong hydrolyzing activity toward crystalline cellulose. The characteristic features of EGPh are: (1) it appears to have disulfide bonds, which is rare among anaerobic hyperthermophilic archaeon proteins, and (2) it lacks a carbohydrate-binding domain, which is necessary for effective hydrolysis of cellulose. We first examined the relationship between the disulfide bonds and the catalytic activity by analyzing various cysteine mutations. The activities of the mutated enzymes toward carboxy methyl cellulose (CMC) increased without any loss in thermostability. Second, we prepared a fusion enzyme so that the thermostable chitin-binding domain of chitinase from P. furiosus was joined to the C-terminus of EGPh and its variants. These fusion enzymes showed stronger activities than did the wild-type EGPh toward both CMC and crystalline cellulose (Avicel).     

DNA protection mechanisms are not involved in the radioresistance of the hyperthermophilic archaea<Emphasis Type="Italic"> Pyrococcus abyssi</Emphasis> and<Emphasis Type="Italic"> P. furiosus</Emphasis>

Hyperthermophilic archaea of the genus Pyrococcus are resistant to gamma radiation, suggesting that efficient mechanisms for DNA repair exist in these organisms. To determine whether protective mechanisms might also be implicated in this radioresistance, we have estimated the linear density of DNA double-stranded breaks caused by gamma irradiation in the genomic DNA of two Pyrococcus species, using Escherichia coli and the radioresistant bacterium Deinococcus radiodurans as controls. The linear density of double-stranded breaks was essentially the same in all four microorganisms when irradiation was carried under similar anaerobic conditions, indicating that no specific DNA protection mechanisms exist in Pyrococcus species. Using one- and two-dimensional gel electrophoresis we compared the protein patterns from Pyrococcus abyssi and P. furiosus cells that had or had not been exposed to gamma rays. We did not detect any significant protein induction following DNA damage in either species.     

New DNA polymerase from the hyperthermophilic marine archaeon <Emphasis Type="Italic">Thermococcus thioreducens</Emphasis>

The family B DNA polymerase gene of Thermococcus thioreducens, an archaeon recently isolated from the Rainbow hydrothermal vent field, was cloned and its protein product expressed, purified and characterized. The gene was found to encode a 1,311 amino acid chain including an intein sequence of 537 residues. Phylogenetic analysis revealed a predominantly vertical type of inheritance of the intein in the Thermococcales order. Primary sequence analysis of the mature protein (TthiPolB) showed significant sequence conservation among DNA polymerases in this family. The structural fold of TthiPolB was predicted against the known crystallographic structure of a family B DNA polymerase from Thermococcus gorgonarius, allowing regional domain assignments within the TthiPolB sequence. The recombinant TthiPolB was overexpressed in Escherichia coli and purified for biochemical characterization. Compared with other DNA polymerases from the Thermococcales order, TthiPolB was found to have moderate thermal stability and fidelity, and a high extension rate, consistent with an extremely low K _m corresponding to the dNTP substrate. TthiPolB performed remarkably well in a wide range of PCR conditions, being faster, more stable and more accurate than many commonly used enzymes.     

Characterization of a novel dye-linked <Emphasis Type="SmallCaps">l</Emphasis>-proline dehydrogenase from an aerobic hyperthermophilic archaeon, <Emphasis Type="Italic">Pyrobaculum calidifontis</Emphasis>

The activity of a dye-linked l-proline dehydrogenase (dye-l-proDH) was found in the crude extract of an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis JCM 11548, and was purified 163-fold through four sequential chromatography steps. The enzyme has a molecular mass of about 108 kDa and is a homodimer with a subunit molecular mass of about 46 kDa. The enzyme retained more than 90% of its activity after incubation at 100 °C for 120 min (pH 7.5) or after incubation at pHs 4.5–9.0 for 30 min at 50 °C. The enzyme catalyzed l-proline dehydrogenation to Δ¹-pyroline-5-carboxylate using 2,6-dichloroindophenol (DCIP) as the electron acceptor and the Michaelis constants for l-proline and DCIP were 1.67 and 0.026 mM, respectively. The prosthetic group on the enzyme was identified as flavin adenine dinucleotide by high-performance liquid chromatography. The subunit N-terminal amino acid sequence was MYDYVVVGAG. Using that sequence and previously reported genome information, the gene encoding the enzyme (Pcal_1655) was identified. The gene was then cloned and expressed in Escherichia coli and found to encode a polypeptide of 415 amino acids with a calculated molecular weight of 46,259. The dye-l-proDH gene cluster in P. calidifontis inherently differs from those in the other hyperthermophiles reported so far.     

A cryo-crystallographic time course for peroxide reduction by rubrerythrin from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>

High-resolution crystal structures of Pyrococcus furiosus rubrerythrin (PfRbr) in the resting (all-ferrous) state and at time points following exposure of the crystals to hydrogen peroxide are reported. This approach was possible because of the relativity slow turnover of PfRbr at room temperature. To this end, we were able to perform time-dependent peroxide treatment of the fully reduced enzyme, under strictly anaerobic conditions, in the crystalline state. In this work we demonstrate, for the first time, that turnover of a thermophilic rubrerythrin results in approximately 2-Å movement of one iron atom in the diiron site from a histidine to a carboxylate ligand. These results confirm that, despite the domain-swapped architecture, the hyperthermophilic rubrerythrins also utilize the classic combination of iron sites together with redox-dependent iron toggling to selectively reduce hydrogen peroxide over dioxygen. In addition, we have identified previously unobserved intermediates in the reaction cycle and observed structural changes that may explain the enzyme precipitation observed for the all-iron form of PfRbr upon oxidation to the all-ferric state.     

Formate hydrogenlyase in the hyperthermophilic archaeon, <Emphasis Type="Italic">Thermococcus litoralis</Emphasis>

Background  

Thermococcus litoralis is a heterotrophic facultative sulfur dependent hyperthermophilic Archaeon, which was isolated from a shallow submarine thermal spring. It has been successfully used in a two-stage fermentation system, where various keratinaceous wastes of animal origin were converted to biohydrogen. In this system T. litoralis performed better than its close relative, P. furiosus. Therefore, new alternative enzymes involved in peptide and hydrogen metabolism were assumed in T. litoralis.     

Purification and biochemical properties of a cytochrome <Emphasis Type="Italic">bc</Emphasis> complex from the aerobic hyperthermophilic archaeon <Emphasis Type="Italic">Aeropyrum pernix</Emphasis>

Background  

The bioenergetics of Archaea with respect to the evolution of electron transfer systems is very interesting. In contrast to terminal oxidases, a canonical bc ₁ complex has not yet been isolated from Archaea. In particular, c -type cytochromes have been reported only for a limited number of species.     

Contribution of inter-subunit interactions to the thermostability of <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> citrate synthase

Using citrate synthase from the hyperthermophile Pyrococcus furiosus (PfCS) as our test molecule, we show through guanidine hydrochloride-induced unfolding that the dimer separates into folded, but inactive, monomers before individual subunit unfolding takes place. Given that forces across the dimer interface are vital for thermostability, a robust computational method was derived that uses the University of Houston Brownian Dynamics (UHBD) program to calculate both the hydrophobic and electrostatic contribution to the dimerisation energy at 100°C. The results from computational and experimental determination of the lowered stability of interface mutants were correlated, being both of the same order of magnitude and placing the mutant proteins in the same order of stability. This computational method, optimised for hyperthermophilic molecules and tested in the laboratory, after further testing on other examples, could be of widespread use in the prediction of thermostabilising mutations in other oligomeric proteins for which dissociation is the first step in unfolding.     

Substrate specificity of a glucose-6-phosphate isomerase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> for monosaccharides

We purified recombinant glucose-6-phosphate isomerase from Pyrococcus furiosus using heat treatment and Hi-Trap anion-exchange chromatography with a final specific activity of 0.39 U mg⁻¹. The activity of the glucose-6-phosphate isomerase for l-talose isomerization was optimal at pH 7.0, 95°C, and 1.5 mM Co²⁺. The half-lives of the enzyme at 65°C, 75°C, 85°C, and 95°C were 170, 41, 19, and 7.9 h, respectively. Glucose-6-phosphate isomerase catalyzed the interconversion between two different aldoses and ketose for all pentoses and hexoses via two isomerization reactions. This enzyme has a unique activity order as follows: aldose substrates with hydroxyl groups oriented in the same direction at C2, C3, and C4 > C2 and C4 > C2 and C3 > C3 and C4. l-Talose and d-ribulose exhibited the most preferred substrates among the aldoses and ketoses, respectively. l-Talose was converted to l-tagatose and l-galactose by glucose-6-phosphate isomerase with 80% and 5% conversion yields after about 420 min, respectively, whereas d-ribulose was converted to d-ribose and d-arabinose with 53% and 8% conversion yields after about 240 min, respectively.     

Bifunctional phosphoglucose/phosphomannose isomerase from the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrobaculum aerophilum</Emphasis>

ORF PAE1610 from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum was first annotated as the conjectural pgi gene coding for hypothetical phosphoglucose isomerase (PGI). However, we have recently identified this ORF as the putative pgi/pmi gene coding for hypothetical bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI). To prove its coding function, ORF PAE1610 was overexpressed in Escherichia coli, and the recombinant enzyme was characterized. The 65-kDa homodimeric protein catalyzed the isomerization of both glucose-6-phosphate and mannose-6-phosphate to fructose-6-phosphate at similar catalytic rates, thus characterizing the enzyme as bifunctional PGI/PMI. The enzyme was extremely thermoactive; it had a temperature optimum for catalytic activity of about 100°C and a melting temperature for thermal unfolding above 100°C.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

A thermostable dolichol phosphoryl mannose synthase responsible for glycoconjugate synthesis of the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis>

Dolichol phosphoryl mannose synthase (DPM synthase) is an essential enzyme in the synthesis of N- and O-linked glycoproteins and the glycosylphosphatidyl-inositol anchor. An open reading frame, PH0051, from the hyperthermophilic archaeon Pyrococcus horikoshii encodes a DPM synthase ortholog, PH0051p. A full-length version of PH0051p was produced using an E. coli in vitro translation system and its thermostable activity was confirmed with a DPM synthesis assay, although the in vitro productivity was not sufficient for further characterization. Then, a yeast expression vector coding for the N-terminal catalytic domain of PH0051p was constructed. The N-terminal domain, named DPM(1-237), was successfully expressed, and turned out to be a membrane-bound form in Saccharomyces cerevisiae cells, even without its hydrophobic C-terminal domain. The membrane-bound DPM(1-237) was solubilized with a detergent and purified to homogeneity. The purified DPM(1-237) showed thermostability at up to 75 degrees C and an optimum temperature of 60 degrees C. The truncated mutant DPM(1-237) required Mg(2+) and Mn(2+) ions as cofactors the same as eukaryotic DPM synthases. By site-directed mutagenesis, Asp(89) and Asp(91) located at the most conserved motif, DXD, were confirmed as the catalytic residues, the latter probably bound to a cofactor, Mg(2+). DPM(1-237) was able to utilize both acceptor lipids, dolichol phosphate and the prokaryotic carrier lipid C(55)-undecaprenyl phosphate, with Km values of 1.17 and 0.59 muM, respectively. The DPM synthase PH0051p seems to be a key component of the pathway supplying various lipid-linked phosphate sugars, since P. horikoshii could synthesize glycoproteins as well as the membrane-associated PH0051p in vivo.