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At least 775 records · Page 43Linked to original sources
PubMed2003

Reconstitution of archaeal ribonuclease P from RNA and four protein components.

Ribonuclease P (RNase P) is an endonuclease responsible for generating the 5(') end of matured tRNA molecules. A homology search of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 genome database revealed that the four genes, PH1481, PH1601, PH1771, and PH1877, have a significant homology to those encoding RNase P protein subunits, hpop5, Rpp21, Rpp29, and Rpp30, of human, respectively. These genes were expressed in Escherichia coli cells, and the resulting proteins Ph1481p, Ph1601p, Ph1771p, and Ph1877p were purified to apparent homogeneity in a set of column chromatographies. The four proteins were characterized in terms of their capability to bind the cognate RNase P RNA from P. horikoshii. All four proteins exhibited the binding activity to the RNase P RNA. In vitro reconstitution of four putative RNase P proteins with the in vitro transcripted P. horikoshii RNase P RNA revealed that three proteins Ph1481p, Ph1601p, and Ph1771p, and RNase P RNA are minimal components for the RNase P activity. However, addition of the fourth protein Ph1877p strongly stimulated enzymatic activity, indicating that all four proteins and RNase P RNA are essential for optimal RNase P activity. The present data will pave the way for the elucidation of the reaction mechanism for archaeal as well as eukaryotic RNase P.

Animals↗
PubMed2001

Multiple Holliday junction resolving enzyme activities in the Crenarchaeota and Euryarchaeota.

Holliday junction resolving enzymes are required by all life forms that catalyse homologous recombination, including all cellular organisms and many bacterial and eukaryotic viruses. Here we report the identification of three distinct Holliday junction resolving enzyme activities present in two highly divergent archaeal species. Both Sulfolobus and Pyrococcus share the Hjc activity, and in addition possess unique secondary activities (Hje and Hjr). We propose by analogy with the two other domains of life that the latter enzymes are viral in origin, suggesting the widespread existence of archaeal viruses that rely on homologous recombination as part of their life cycle.

Archaea↗
PubMed2001

An archaeal protein homologous to mammalian SRP54 and bacterial Ffh recognizes a highly conserved region of SRP RNA.

The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf-SRP54) and the N-terminal G-domain and C-terminal M-domain (pf-SRP54M) of pf-SRP54 with an amino-terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non-conserved 213-nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107-nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine-tagged pf-SRP54M bound specifically to the conserved 107-nucleotide RNA in the absence of pf-SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.

Amino Acid Sequence↗
PubMed2002

Structural insight into gene duplication, gene fusion and domain swapping in the evolution of PLP-independent amino acid racemases.

The X-ray crystal structure has revealed two similar alpha/beta domains of aspartate racemase (AspR) from Pyrococcus horikoshii OT3, and identified a pseudo mirror-symmetric distribution of the residues around its active site [Liu et al. (2002) J. Mol. Biol. 319, 479-489]. Structural homology and functional similarity between the two domains suggested that this enzyme evolved from an ancestral domain by gene duplication and gene fusion. We have expressed solely the C-terminal domain of this AspR and determined its three-dimensional structure by X-ray crystallography. The high structural stability of this domain supports the existence of the ancestral domain. In comparison with other amino acid racemases (AARs), we suggest that gene duplication and gene fusion are conventional ways in the evolution of pyridoxal 5'-phosphate-independent AARs.

Amino Acid Isomerases↗
PubMed2002

Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM).

The genome of the hyperthermophilic bacterium Thermotoga maritima (Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously. The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino-terminus. The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide in Escherichia coli and found to be optimally active at pH 6, 90 degrees C, with a melting temperature of approximately 105 degrees C. The cel74 gene was previously found to be induced during T. maritima growth on a variety of polysaccharides, including barley glucan, carboxymethyl cellulose (CMC), glucomannan, galactomannan and starch. However, while Tm Cel74 was most active towards barley glucan and to a lesser extent CMC, glucomannan and tamarind (xyloglucan), no activity was detected on other glycans, including galactomannan, laminarin and starch. Also, Tm Cel74 did not contain a carbohydrate binding module (CBM), versions of which have been identified in the amino acid sequences of other family 74 enzymes. As such, a CBM associated with a chitinase in another hyperthermophile, Pyrococcus furiosus, was used to create a fusion protein that was active on crystalline cellulose; Tm Cel74 lacked activity on this substrate. Based on the cleavage pattern determined for Tm Cel74 on glucan-based substrates, this enzyme likely initiates recruitment of carbohydrate carbon and energy sources by creating oligosaccharides that are transported into the cell for further processing.

Amino Acid Sequence↗
PubMed2004

Structural characteristics and refolding of in vivo aggregated hyperthermophilic archaeon proteins.

Several recombinant proteins in inclusion bodies expressed in Escherichia coli have been measured by Fourier transform infrared and solid-state nuclear magnetic resonance spectra to provide the secondary structural characteristics of the proteins from hyperthermophilic archaeon Pyrococcus horikoshii OT3 (hyperthermophilic proteins) in inclusion bodies. The beta-strand-rich single chain Fv fragment (scFv) and alpha-helix-rich interleukin (IL)-4 lost part of the native-like secondary structure in inclusion bodies, while the inclusion bodies composed of the hyperthermophilic proteins of which the native form is alpha-helix rich, are predominated by alpha-helix structure. Further, the secondary structure of the recombinant proteins solubilized from inclusion bodies by detergent or denaturant was observed by circular dichroism (CD) spectra. The solubilization induced the denaturation of the secondary structure for scFv and IL-4, whereas the solubilized hyperthermophilic proteins have retained the alpha-helix structure with the CD properties resembling those of their native forms. This indicates that the hyperthermophilic proteins form native-like secondary structure in inclusion bodies. Refolding of several hyperthermophilic proteins from in vivo aggregated form without complete denaturation could be accomplished by solubilization with lower concentration (e.g. 2 M) of guanidine hydrochloride and removal of the denaturant via stepwise dialysis. This supports the existence of proteins with native-like structure in inclusion bodies and suggests that non-native association between the secondary structure elements leads to in vivo aggregation. We propose a refolding procedure on the basis of the structural properties of the aggregated archaeon proteins.

Archaeal Proteins↗
PubMed1998

A transcription frame-based analysis of the genomic DNA sequence of a hyper-thermophilic archaeon for the identification of genes, pseudo-genes and operon structures.

An algorithm for identifying transcription units, independently regulated genes and operons, and pseudo-genes that are not expected to be expressed, has been developed by combining a system for predicting transcription and translation signals, and a system for scoring the triplet periodicity in ORF candidates. By using the algorithm, the 1.09 Mb sequence that covers approximately 60% of the genome of Pyrococcus sp. OT3 has been analyzed. The identified ORFs show the expected biological and physical characteristics, while the rejected ORF candidates do not. Frequent use of operon structures for transcription, and gene duplication followed by mutation or termination of the duplicated genes, are discussed.

Base Composition↗
PubMed1998

In vitro heat effect on heterooligomeric subunit assembly of thermostable indolepyruvate ferredoxin oxidoreductase.

Indolepyruvate ferredoxin oxidoreductase (IOR) from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 catalyzes the oxidative decarboxylation of arylpyruvates by forming a heterooligomeric complex (alpha2beta2). The genes iorA and iorB which encode respective alpha and beta subunits, were coexpressed heterologously in Escherichia coli cells under anaerobic conditions. IOR activity was detected from the cell extract containing both subunits and its activity was enhanced by in vitro heat treatment prior to the assay. The iorA and iorB were expressed individually and each subunit was examined for enzymatic activity with and without heat treatment. IOR activity was detected neither from the extract of alpha subunit nor beta subunit. The alpha and beta subunits were mixed and then IOR activity was examined. Weak IOR activity was detected without heat treatment, however, upon heat treatment its activity was enhanced. The mixture of individually heat treated alpha and beta subunits did not possess any IOR activity even though the mixed sample was heat treated again. IOR alpha and beta subunits were individually purified to homogeneity, mixed with or without heat treatment and subunit assembly was examined by determining molecular mass. Upon heat treatment, inactive alpha and beta were converted to an active high molecular weight complex (195 kDa) which corresponds to the alpha2beta2 structure. However, the active complex was not formed without heat treatment, suggesting that high temperature environments are important for the heterooligomerization of IOR subunits.

Base Sequence↗
PubMed1998

Hyperthermophilic redox chemistry: a re-evaluation.

The redox chemistry of Pyrococcus furiosus rubredoxin and ferredoxin has been studied as a function of temperature in direct voltammetry and in EPR monitored bulk titrations. The Ems of both proteins, measured with direct voltammetry, have a normal (linear) temperature dependence and show no pH dependence. EPR monitoring is not a reliable method to determine the temperature dependence of the Em: upon rapid freezing the proteins take their conformation corresponding to the freezing point of the solution.

Electron Spin Resonance Spectroscopy↗
PubMed1999

A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon.

A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein.

Adenosine Triphosphatases↗
PubMed1999

Isolation of TBP-interacting protein (TIP) from a hyperthermophilic archaeon that inhibits the binding of TBP to TATA-DNA.

We have isolated TBP (TATA-binding protein)-interacting protein (TIP) from cell lysates of a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1, by affinity chromatography with TBP-agarose. Based on the internal amino acid sequence information, PCR primers were synthesized and used to amplify the gene encoding this protein (Pk-TIP). Determination of the nucleotide sequence and characterization of the recombinant protein revealed that Pk-TIP is composed of 224 amino acid residues (molecular weight of 25,558) and exists in a dimeric form. BIAcore analyses for the interaction between recombinant Pk-TIP and recombinant Pk-TBP indicated that they interact with each other with an equilibrium dissociation constant, KD, of 1.24-1.46 microM. A gel mobility shift assay indicated that Pk-TIP inhibited the interaction between Pk-TBP and a TATA-DNA. Pk-TIP may be one of the archaeal factors which negatively regulate transcription.

Amino Acid Sequence↗
PubMed1999

Cloning of an alpha-glucosidase gene from Thermococcus hydrothermalis by functional complementation of a Saccharomyces cerevisiae mal11 mutant strain.

alpha-Glucosidase is found in methanogenic and thermophilic archaea and also in eukaryotes and bacteria. The gene encoding the enzyme was cloned from Thermococcus hydrothermalis by complementation of a Saccharomyces cerevisiae deficiency maltase mutant strain. The gDNA clone isolated encodes an open reading frame corresponding to a protein of 242 amino acids. The protein shows 42% identity to a Pyrococcus horikoshii unknown ORF but no similarities were obtained with polysaccharidase sequences.

Amino Acid Sequence↗
PubMed2002

Crystal structure of archaeosine tRNA-guanine transglycosylase.

Archaeosine tRNA-guanine transglycosylase (ArcTGT) catalyzes the exchange of guanine at position 15 in the D-loop of archaeal tRNAs with a free 7-cyano-7-deazaguanine (preQ(0)) base, as the first step in the biosynthesis of an archaea-specific modified base, archaeosine (7-formamidino-7-deazaguanosine). We determined the crystal structures of ArcTGT from Pyrococcus horikoshii at 2.2 A resolution and its complexes with guanine and preQ(0), at 2.3 and 2.5 A resolutions, respectively. The N-terminal catalytic domain folds into an (alpha/beta)(8) barrel with a characteristic zinc-binding site, showing structural similarity with that of the bacterial queuosine TGT (QueTGT), which is involved in queuosine (7-[[(4,5-cis-dihydroxy-2-cyclopenten-1-yl)-amino]methyl]-7-deazaguanosine) biosynthesis and targets the tRNA anticodon. ArcTGT forms a dimer, involving the zinc-binding site and the ArcTGT-specific C-terminal domain. The C-terminal domains have novel folds, including an OB fold-like "PUA domain", whose sequence is widely conserved in eukaryotic and archaeal RNA modification enzymes. Therefore, the C-terminal domains may be involved in tRNA recognition. In the free-form structure of ArcTGT, an alpha-helix located at the rim of the (alpha/beta)(8) barrel structure is completely disordered, while it is ordered in the guanine-bound and preQ(0)-bound forms. Structural comparison of the ArcTGT.preQ(0), ArcTGT.guanine, and QueTGT.preQ(1) complexes provides novel insights into the substrate recognition mechanisms of ArcTGT.

Amino Acid Sequence↗
PubMed2003

Modeling of flap endonuclease interactions with DNA substrate.

Structure-specific 5' nucleases play an important role in DNA replication and repair uniquely recognizing an overlap flap DNA substrate and processing it into a DNA nick. However, in the absence of a high-resolution structure of the enzyme/DNA complex, the mechanism underlying this recognition and substrate specificity, which is key to the enzyme's function, remains unclear. Here, we propose a three-dimensional model of the structure-specific 5' flap endonuclease from Pyrococcus furiosus in its complex with DNA. The model is based on the known X-ray structure of the enzyme and a variety of biochemical and molecular dynamics (MD) data utilized in the form of distance restraints between the enzyme and the DNA. Contacts between the 5' flap endonuclease and the sugar-phosphate backbone of the overlap flap substrate were identified using enzyme activity assays on substrates with methylphosphonate or 2'-O-methyl substitutions. The enzyme footprint extends two to four base-pairs upstream and eight to nine base-pairs downstream of the cleavage site, thus covering 10-13 base-pairs of duplex DNA. The footprint data are consistent with a model in which the substrate is bound in the DNA-binding groove such that the downstream duplex interacts with the helix-hairpin-helix motif of the enzyme. MD simulations to identify the substrate orientation in this model are consistent with the results of the enzyme activity assays on the methylphosphonate and 2'-O-methyl-modified substrates. To further refine the model, 5' flap endonuclease variants with alanine point substitutions at amino acid residues expected to contact phosphates in the substrate and one deletion mutant were tested in enzyme activity assays on the methylphosphonate-modified substrates. Changes in the enzyme footprint observed for two point mutants, R64A and R94A, and for the deletion mutant in the enzyme's beta(A)/beta(B) region, were interpreted as being the result of specific interactions in the enzyme/DNA complex and were used as distance restraints in MD simulations. The final structure suggests that the substrate's 5' flap interacts with the enzyme's helical arch and that the helix-hairpin-helix motif interacts with the template strand in the downstream duplex eight base-pairs from the cleavage site. This model suggests specific interactions between the 3' end of the upstream oligonucleotide and the enzyme. The proposed structure presents the first detailed description of substrate recognition by structure-specific 5' nucleases.

Base Sequence↗
PubMed2000

Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.

To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins.

Adenosine Triphosphatases↗
PubMed1998

Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity.

Flap endonuclease (FEN-1) removes 5' overhanging flaps in DNA repair and processes the 5' ends of Okazaki fragments in lagging strand DNA synthesis. The crystal structure of Pyrococcus furiosus FEN-1, active-site metal ions, and mutational information indicate interactions for the single- and double-stranded portions of the flap DNA substrate and identify an unusual DNA-binding motif. The enzyme's active-site structure suggests that DNA binding induces FEN-1 to clamp onto the cleavage junction to form the productive complex. The conserved FEN-1 C terminus binds proliferating cell nuclear antigen (PCNA) and positions FEN-1 to act primarily as an exonuclease in DNA replication, in contrast to its endonuclease activity in DNA repair. FEN-1 mutations altering PCNA binding should reduce activity during replication, likely causing DNA repeat expansions as seen in some cancers and genetic diseases.

Amino Acid Sequence↗
PubMed2001

Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase.

To clarify functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair, we report Pyrococcus furiosus Mre11 crystal structures, revealing a protein phosphatase-like, dimanganese binding domain capped by a unique domain controlling active site access. These structures unify Mre11's multiple nuclease activities in a single endo/exonuclease mechanism and reveal eukaryotic macromolecular interaction sites by mapping human and yeast Mre11 mutations. Furthermore, the structure of the P. furiosus Rad50 ABC-ATPase with its adjacent coiled-coil defines a compact Mre11/Rad50-ATPase complex and suggests that Rad50-ATP-driven conformational switching directly controls the Mre11 exonuclease. Electron microscopy, small angle X-ray scattering, and ultracentrifugation data of human and P. furiosus MR reveal a dual functional complex consisting of a (Mre11)2/(Rad50)2 heterotetrameric DNA processing head and a double coiled-coil linker.

Amino Acid Sequence↗
PubMed2000

Single-step purification of a recombinant thermostable alpha-amylase after solubilization of the enzyme from insoluble aggregates.

The expression of the gene encoding a thermostable alpha-amylase (EC 3.2.1.1) (optimal activity at 100 degrees C) from the hyperthermophilic archaeon Pyrococcus woesei in the mesophilic hosts Escherichia coli and Halomonas elongata resulted in the formation of insoluble aggregates. More than 85% of the recombinant enzyme was present within the cells as insoluble but catalytically active aggregates. The recombinant alpha-amylase was purified to homogeneity in a single step by hydrophobic interaction chromatography on a phenyl superose column after solubilization of the enzyme under nondenaturing conditions. The enzyme was purified 258-fold with a final yield of 54%.

Chromatography, Liquid↗