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PubMed2002

Molecular structure and novel DNA binding sites located in loops of flap endonuclease-1 from Pyrococcus horikoshii.

The crystal structure of flap endonuclease-1 from Pyrococcus horikoshii (phFEN-1) was determined to a resolution of 3.1 A. The active cleft of the phFEN-1 molecule is formed with one large loop and four small loops. We examined the function of the conserved residues and positively charged clusters on these loops by kinetic analysis with 45 different mutants. Arg(40) and Arg(42) on small loop 1, a cluster Lys(193)-Lys(195) on small loop 2, and two sites, Arg(94) and Arg(118)-Lys(119), on the large loop were identified as binding sites. Lys(87) on the large loop may play significant roles in catalytic reaction. Furthermore, we successfully elucidated the function of the four DNA binding sites that form productive ES complexes specific for each endo- or exo-type hydrolysis, probably by bending the substrates. For the endo-activity, Arg(94) and Lys(193)-Lys(195) located at the top and bottom of the molecule were key determinants. For the exo-activity, all four sites were needed, but Arg(118)-Lys(119) was dominant. The major binding sites for both the nick substrate and double-stranded DNA might be the same.

Amino Acid Sequence↗

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↗

PubMed2004

Analysis of the function of a hyperthermophilic endoglucanase from Pyrococcus horikoshii that hydrolyzes crystalline cellulose.

A hyperthermophilic beta-1,4 endoglucanase was identified in Pyrococcus horikoshii, a hyperthermophilic archaeon. In order to clarify the function of the protein in detail, structural and catalytic site studies were performed using protein engineering. By removing some of the C-terminal sequence of the ORF of the endoglucanase (PH1171), two types of recombinant proteins were expressed from one ORF, using Escherichia coli. One exhibited endoglucanase activity, and the other did not. An SD-like sequence was identified in the ORF of the endoglucanase. By removing the SD-like sequence without changing the amino acid sequence of the endoglucanase, one recombinant endoglucanase was prepared effectively from E. coli. From the analysis of the N- and C-terminal regions of the ORF, this endoglucanase appears to be a secreted and membrane-binding enzyme of P. horikoshii. A mutation analysis of the endoglucanase, using the synthetic substrate, indicated that Glu342 is a candidate for the active center and plays a critical role in the activity of the enzyme. Additional catalytic amino acid residues were not found. These results indicate that the catalytic residue of the enzyme is different from that of typical family 5 endoglucanase, even though it has a high homology to the endoglucanase from Acidothermus celluloliticus. The activity of the enzyme, using carboxy methylcellulose and crystalline cellulose as the substrates, was increased, but not for a synthetic low-molecular substrate when a carbohydrate-binding module of chitinase from P. furiosus was added to the C-terminal region.

Amino Acid Sequence↗

PubMed2005

Crystal structure and structural stability of acylphosphatase from hyperthermophilic archaeon Pyrococcus horikoshii OT3.

To elucidate the structural basis for the high stability of acylphosphatase (AcP) from Pyrococcus horikoshii OT3, we determined its crystal structure at 1.72 A resolution. P. horikoshii AcP possesses high stability despite its approximately 30% sequence identity with eukaryotic enzymes that have moderate thermostability. The overall fold of P. horikoshii AcP was very similar to the structures of eukaryotic counterparts. The crystal structure of P. horikoshii AcP shows the same fold betaalphabetabetaalphabeta topology and the conserved putative catalytic residues as observed in eukaryotic enzymes. Comparison with the crystal structure of bovine common-type AcP and that of D. melanogaster AcP (AcPDro2) as representative of eukaryotic AcP revealed some significant characteristics in P. horikoshii AcP that likely play important roles in structural stability: (1) shortening of the flexible N-terminal region and long loop; (2) an increased number of ion pairs on the protein surface; (3) stabilization of the loop structure by hydrogen bonds. In P. horikoshii AcP, two ion pair networks were observed one located in the loop structure positioned near the C-terminus, and other on the beta-sheet. The importance of ion pairs for structural stability was confirmed by site-directed mutation and denaturation induced by guanidium chloride.

Acid Anhydride Hydrolases↗

PubMed1999

Thermostable aminopeptidase from Pyrococcus horikoshii.

From the genome sequence data of the thermophilic archaeon Pyrococcus horikoshii, an open reading frame was found which encodes a protein (332 amino acids) homologous with an endoglucanase from Clostridium thermocellum (42% identity), deblocking aminopeptidase from Pyrococcus furiosus (42% identity) and an aminopeptidase from Aeromonas proteolytica (18% identity). This gene was cloned and expressed in Escherichia coli, and the characteristics of the expressed protein were examined. Although endoglucanase activity was not detected, this protein was found to have aminopeptidase activity to cleave the N-terminal amino acid from a variety of substrates including both N-blocked and non-blocked peptides. The enzyme was stable at 90 degrees C, with the optimum temperature over 90 degrees C. The metal ion bound to this enzyme was calcium, but it was not essential for the aminopeptidase activity. Instead, this enzyme required the cobalt ion for activity. This enzyme is expected to be useful for the removal of N(alpha)-acylated residues in short peptide sequence analysis at high temperatures.

Aminopeptidases↗

PubMed2005

Crystallization and preliminary X-ray analysis of PH1566, a putative ribosomal RNA-processing factor from the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

A putative ribosomal RNA-processing factor consisting of two KH domains from Pyrococcus horikoshii OT3 (PH1566; 25 kDa) was crystallized by the sitting-drop vapour-diffusion method using PEG 3000 as the precipitant. The crystals diffracted X-rays to beyond 2.0 A resolution using a synchrotron-radiation source. The space group of the crystals was determined as primitive orthorhombic P2(1)2(1)2(1), with unit-cell parameters a = 45.9, b = 47.4, c = 95.7 A. The crystals contain one molecule in the asymmetric unit (VM = 2.5 A3 Da(-1)) and have a solvent content of 50%.

Archaeal Proteins↗

PubMed2006

Cloning, expression and characterization of a thermostable exo-beta-D-glucosaminidase from the hyperthermophilic archaeon Pyrococcus horikoshii.

An exo-beta-D-glucosaminidase gene (PH0511) was cloned from the hyperthermophilic archaeon, Pyrococcus horikoshii, and expressed in Escherichia coli. The purified protein showed a strong exo-beta-D: -glucosaminidase activity by TLC analysis. DTT (50 mM) had little effect on its homodimeric structure during SDS-PAGE. The enzyme was optimally active at 90 degrees C (over 20 min) and pH 6. It had a half-life of 9 h at 90 degrees C and is the most thermostable glucosaminidase described up to now. The activity was not inhibited by ethanol, 2-propanol, DMSO, PEG-400, denaturing agents SDS (5%, w/v), urea, guanidine hydrochloride (5 M) and Mg(2+), Mn(2+), Co(2+), Ca(2+), Sr(2+), Ni(2+) (at up to 10 mM).

Cations, Divalent↗

PubMed2005

Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii. Implications for this disulfide metabolism of anaerobic hyperthermophiles.

We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P. furiosus, and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia coli. Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H+-->2CoA-SH + NAD(P)+). The P. horikoshii enzyme shows a kcat app of 7.2 s(-1) with NADPH at 75 degrees C. While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equal kcats, while the Km for NADPH is roughly eightfold lower than that for NADH. The enzyme is specific for the CoA disulfide, and does not show significant reductase activity with other disulfides, including dephospho-CoA. Anaerobic reductive titration of the enzyme with NAD(P)H proceeds in two stages, with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH2 form of the enzyme. Addition of a second of NADPH results in the formation of an apparent FAD-NAD(P)H complex. The behavior of this enzyme is quite different from the mesophilic staphylococcal version of the enzyme. This is only the second enzyme with this activity discovered, and the first from a strict anaerobe, an Archaea, or hyperthermophilic source. P. furiosus cells were assayed for small molecular mass thiols and found to contain 0.64 micromol CoA.g dry weight(-1) (corresponding to 210 microM CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents.

Amino Acid Sequence↗

PubMed2005

First archaeal inorganic polyphosphate/ATP-dependent NAD kinase, from hyperthermophilic archaeon Pyrococcus horikoshii: cloning, expression, and characterization.

The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon Pyrococcus horikoshii was identified in the genome database, cloned, and functionally expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment at 90 degrees C for 20 min and one successive HiTrap affinity chromatography step. The purified enzyme was easily precipitated by dialysis against phosphate buffer without NaCl and imidazole and was usually stored in buffer containing 0.5 M NaCl and 0.5 M imidazole to avoid precipitation. The molecular mass of the active enzyme was determined to be 145 kDa by a gel filtration method, and the enzyme was composed of a tetramer of 37-kDa subunits. The archaeal enzyme utilized several nucleoside triphosphates, such as GTP, CTP, UTP, and ITP, as well as ATP and inorganic polyphosphates [poly(P)] as phosphoryl donors for NAD phosphorylation. The enzyme utilized poly(P)27 (the average length of the phosphoryl chain was 27) as the most active inorganic polyphosphate for NAD phosphorylation. Thus, this enzyme is categorized as an inorganic polyphosphate/ATP-dependent NAD kinase. The enzyme was the most thermostable NAD kinase found to date: its activity was not lost by incubation at 95 degrees C for 10 min. The enzyme showed classical Michaelis-Menten-type kinetics for NAD and ATP, but not for poly(P)27. The Km values for NAD were determined to be 0.30 and 0.40 mM when poly(P)27 and ATP, respectively, were used as the phosphoryl donors. The Km value for ATP was 0.29 mM, and the concentration of poly(P)27 which gave half of the maximum enzyme activity was 0.59 mM. The enzyme required several metal cations, such as Mg2+, Mn2+, or Ni2+, for its activity. The deduced amino acid sequence showed a low level of identity to those of E. coli ATP-dependent NAD kinase (31%) and the inorganic polyphosphate/ATP-dependent NAD kinase of Mycobacterium tuberculosis (29%). This is the first description of the characteristics of a poly(P)/ATP-dependent NAD kinase from a hyperthermophilic archaeon.

Adenosine Triphosphate↗

PubMed2004

Structure analysis of PH1161 protein, a transcriptional activator TenA homologue from the hyperthermophilic archaeon Pyrococcus horikoshii.

The crystal structure of the Bacillus subtilis TenA-homologue protein PH1161 from the hyperthermophilic archaebacterium Pyrococcus horikoshii was determined. TenA is known to belong to a new family of activators that stimulate the production of extracellular proteases in B. subtilis. A sequence-similarity search revealed that TenA-homologue proteins are widespread in bacteria and archaea, suggesting that this family of proteins plays an essential role in these organisms. In the present study, the first three-dimensional structure of a member of the TenA family of proteins was determined, unexpectedly revealing that the protein has a fold identical to that of haem oxygenase-1. Analysis has also shown that the protein has a unique ligand-binding pocket. Electron density of a bound ligand molecule was observed in this pocket. These results provide a valuable insight into the functional understanding of the TenA family of proteins.

Amino Acid Sequence↗

PubMed2005

A novel trehalose-synthesizing glycosyltransferase from Pyrococcus horikoshii: molecular cloning and characterization.

A gene (ORF PH1035), annotated to encode an uncharacterized hypothetical protein in Pyrococcus horikoshii, was first cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by Ni-NTA affinity chromatography and its molecular mass was determined to be 49,871Da by MALDI-TOF mass spectrometry. When the purified enzyme was reacted with nucleoside diphosphate-glucoses including UDP-glucose as a donor and glucose, rather than glucose-6-phosphate, as an acceptor, it specifically created a free trehalose. The enzyme was also able to partly hydrolyze the trehalose to glucose. The optimum pH was 5.5 and the enzyme was highly stable from pH 6 to 8. The deduced amino acid sequence showed a high homology with that of the glycosyl transferase group 1 (Pfam00534) in the BLAST search. The results suggest that the enzyme is a novel glycosyltransferase catalyzing the synthesis of the trehalose in the archaeon.

Amino Acid Sequence↗

PubMed2005

Structure of the type I L-asparaginase from the hyperthermophilic archaeon Pyrococcus horikoshii at 2.16 angstroms resolution.

The crystal structure of the L-asparaginase from the hyperthermophilic archaeon Pyrococcus horikoshii (PhA) was determined by the multiwavelength anomalous diffraction (MAD) method and was refined to a resolution of 2.16 angstroms with a crystallographic R factor and free R factor of 21.1 and 25.3%, respectively. This is the first report of the three-dimensional structure of a type I L-asparaginase. These enyzmes are known as cytosolic L-asparaginases with lower affinities for substrate than the type II L-asparaginases. Although the overall fold of PhA was closely related to the structure of the well characterized type II L-asparaginase, structural differences were also detected. PhA forms a homodimer that corresponds to half the homotetramer of type II L-asparaginases. Structure comparison at the active site reveals that most catalytic residues are conserved except for two residues that recognize the amino group of the substrate. Additionally, a remarkable structural difference is found in the so-called 'active-site flexible loop'. In PhA this loop is stabilized by beta-hairpin formation and by elaborate interactions with the type-I-specific alpha-helical region derived from the other subunit forming the PhA dimer. The flexible loop of the type II enzyme is considered to serve as a mobile gate to the active site. Therefore, the loop stabilization observed in the PhA structure may cause limitation of the access of the substrate to the active site.

Amino Acid Sequence↗

PubMed2006

Crystal structure of protein Ph1481p in complex with protein Ph1877p of archaeal RNase P from Pyrococcus horikoshii OT3: implication of dimer formation of the holoenzyme.

Ribonuclease P (RNase P) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of a catalytic RNA and five protein subunits. We previously determined crystal structures of four protein subunits. Ph1481p, an archaeal homologue for human hPop5, is the protein component of the P.horikoshii RNase P for which no structural information is available. Here we report the crystal structure of Ph1481p in complex with another protein subunit, Ph1877p, determined at 2.0 A resolution. Ph1481p consists of a five-stranded antiparallel beta-sheet and five helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. Ph1481p is, however, distinct from the typical RNP domain in that it has additional helices at the C terminus, which pack against one face of the beta-sheet. The presence of two complexes in the asymmetric unit, together with gel filtration chromatography indicates that the heterotetramer is stable in solution and represents a fundamental building block in the crystals. In the heterotetrameric structure (Ph1877p-(Ph1481p)(2)-Ph1877p), a homodimer of Ph1481p sits between two Ph1877p monomers. Ph1481p dimerizes through hydrogen bonding interaction from the loop between alpha1 and alpha2 helices, and each Ph1481p interacts with two Ph1877p molecules, where alpha2 and alpha3 in Ph1481p interact with alpha7 in one Ph1877p and alpha8 in the other Ph1877p molecule, respectively. Deletion of the alpha1-alpha2 loop in Ph1481p caused heterodimerization with Ph1877p, and abolished ability to homodimerize itself and heterotetramerize with Ph1877p. Furthermore, the reconstituted particle containing the deletion mutant Ph1481p (mPh1481p) exhibited significantly reduced nuclease activity. These results suggest the presence of the heterotetramer of Ph1481p and Ph1877p in P.horikoshii RNase P.

Amino Acid Sequence↗

PubMed2005

Characterization of the Family I inorganic pyrophosphatase from Pyrococcus horikoshii OT3.

A gene encoding for a putative Family I inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) from P. horikoshii showed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase from P. horikoshii (PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 degrees C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 degrees C. The heat stability of PhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+ being most effective; Zn2+, Co2+ and Mn2+ efficiently supported hydrolytic activity in a narrow range of concentrations (0.05-0.5 mM). The K(m) for pyrophosphate and Mg2+ were 113 and 303 microM, respectively; and maximum velocity, V(max), was estimated at 930 U mg(-1).

Diphosphates↗

PubMed2003

Solution NMR structure of the 30S ribosomal protein S28E from Pyrococcus horikoshii.

We report NMR assignments and solution structure of the 71-residue 30S ribosomal protein S28E from the archaean Pyrococcus horikoshii, target JR19 of the Northeast Structural Genomics Consortium. The structure, determined rapidly with the aid of automated backbone resonance assignment (AutoAssign) and automated structure determination (AutoStructure) software, is characterized by a four-stranded beta-sheet with a classic Greek-key topology and an oligonucleotide/oligosaccharide beta-barrel (OB) fold. The electrostatic surface of S28E exhibits positive and negative patches on opposite sides, the former constituting a putative binding site for RNA. The 13 C-terminal residues of the protein contain a consensus sequence motif constituting the signature of the S28E protein family. Surprisingly, this C-terminal segment is unstructured in solution.

Amino Acid Sequence↗

PubMed2001

Crystallization and preliminary X-ray analysis of the archaeosine tRNA-guanine transglycosylase from Pyrococcus horikoshii.

The archaeosine tRNA-guanine transglycosylase from the hyperthermophilic archaeon Pyrococcus horikoshii was crystallized and preliminary X-ray characterization was performed. Single crystals were grown by the hanging-drop vapour-diffusion method, using sodium/potassium phosphate and sodium acetate as precipitants. The space group is P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 99.28 (14), c = 363.74 (56) A. The cryocooled crystals diffracted X-rays beyond 2.2 A resolution using synchrotron radiation from station BL44XU at SPring-8 (Harima). Selenomethionine-substituted protein crystals were prepared in order to solve the structure by the MAD phasing method.

Crystallization↗

PubMed2004

Crystal structure of the hyperthermophilic inorganic pyrophosphatase from the archaeon Pyrococcus horikoshii.

A homolog to the eubacteria inorganic pyrophosphatase (PPase, EC 3.6.1.1) was found in the genome of the hyperthermophilic archaeon Pyrococcus horikoshii. This inorganic pyrophosphatase (Pho-PPase) grows optimally at 88 degrees C. To understand the structural basis for the thermostability of Pho-PPase, we have determined the crystal structure to 2.66 A resolution. The crystallographic asymmetric unit contains three monomers related by approximate threefold symmetry, and a hexamer is built up by twofold crystallographic symmetry. The main-chain fold of Pho-PPase is almost identical to that of the known crystal structure of the model from Sulfolobus acidocaldarius. A detailed comparison of the crystal structure of Pho-PPase with related structures from S. acidocaldarius, Thermus thermophilus, and Escherichia coli shows significant differences that may account for the difference in their thermostabilities. A reduction in thermolabile residues, additional aromatic residues, and more intimate association between subunits all contribute to the larger thermophilicity of Pho-PPase. In particular, deletions in two loops surrounding the active site help to stabilize its conformation, while ion-pair networks unique to Pho-PPase are located in the active site and near the C-terminus. The identification of structural features that make PPases more adaptable to extreme temperature should prove helpful for future biotechnology applications.

Amino Acid Sequence↗

PubMed2005

Purification, crystallization and initial X-ray crystallographic analysis of the putative GTPase PH0525 from Pyrococcus horikoshii OT3.

GTPases are involved in diverse cellular functions including cell proliferation, cytoskeleton organization and intracellular traffic. The putative GTPase PH0525 from Pyrococcus horikoshii OT3 has been overexpressed in Escherichia coli and purified. Two distinct crystal forms were grown by the microbatch method at 291 K using a very high protein concentration (80 mg ml(-1)). Native data sets extending to resolutions of 2.3 and 2.4 A have been collected and processed in space groups P2(1) and C222(1), respectively. Assuming the presence of one monomer per asymmetric unit gives VM values of 2.6 and 2.4 A3 Da(-1) for the P2(1) and C222(1) forms, respectively, which is consistent with dynamic light-scattering experiments, which show a monomeric state of the protein in solution.

Amino Acid Motifs↗