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PubMed2004

A novel thermostable membrane protease forming an operon with a stomatin homolog from the hyperthermophilic archaebacterium Pyrococcus horikoshii.

Membrane-bound proteases play several important roles in protein quality control and regulation. In the genome of the hyperthermophilic archaebacterium Pyrococcus horikoshii, the open reading frames PH1510 and PH1511 are homologous to the genes nfed (nodulation formation efficiency D) and stomatin, respectively, and probably form an operon. The nfed proteins are putative membrane proteins, and the N-terminal region shows homology to ClpP-type serine proteases. Stomatin is one of the major integral membrane proteins of human erythrocytes, and its absence is associated with the hemolytic anemia known as hereditary stomatocytosis. Thus, the N-terminal region of PH1510 (1510-N, residues 16-236) was expressed and purified. From activity staining and SDS-PAGE analysis using fluorescein isothiocyanate-casein, 1510-N was identified as a thermostable endo-type protease. From site-directed mutagenesis, the conserved Ser-97 and Lys-138 are involved in proteolysis and, therefore, PH1510 is probably a serine protease with a catalytic Ser-Lys dyad. The sites of cleavage by 1510-N are rich in hydrophobic residues. The site P1 (position -1 relative to the cleavage site) is mainly leucine. P4 and P4' are mainly hydrophobic residues. Interestingly, the 1510-N protease cleaves the C-terminal hydrophobic region of PH1511. From this result and the probability of an operon, PH1510 probably functions in cooperation with PH1511. It is hypothesized that the cleavage of the stomatin-homolog PH1511 by the PH1510 protease causes an ion channel to open.

Binding Sites↗

PubMed2005

Crystal structure of a novel FAD-, FMN-, and ATP-containing L-proline dehydrogenase complex from Pyrococcus horikoshii.

Two novel types of dye-linked L-proline dehydrogenase complex (PDH1 and PDH2) were found in a hyperthermophilic archaeon, Pyrococcus horikoshii OT3. Here we report the first crystal structure of PDH1, which is a heterooctameric complex (alphabeta)4 containing three different cofactors: FAD, FMN, and ATP. The structure was determined by x-ray crystallography to a resolution of 2.86 angstroms. The structure of the beta subunit, which is an L-proline dehydrogenase catalytic component containing FAD as a cofactor, was similar to that of monomeric sarcosine oxidase. On the other hand, the alpha subunit possessed a unique structure composed of a classical dinucleotide fold domain with ATP, a central domain, an N-terminal domain, and a Cys-clustered domain. Serving as a third cofactor, FMN was located at the interface between the alpha and beta subunits in a novel configuration. The observed structure suggests that FAD and FMN are incorporated into an electron transfer system, with electrons passing from the former to the latter. The function of ATP is unknown, but it may play a regulatory role. Although the structure of the alpha subunit differs from that of the beta subunit, except for the presence of an analogous dinucleotide domain with a different cofactor, the structural characteristics of PDH1 suggest that each represents a divergent enzyme that arose from a common ancestral flavoenzyme and that they eventually formed a complex to gain a new function. The structural characteristics described here reveal the PDH1 complex to be a unique diflavin dehydrogenase containing a novel electron transfer system.

Adenosine Triphosphate↗

PubMed2006

Functional similarities of a thermostable protein-disulfide oxidoreductase identified in the archaeon Pyrococcus horikoshii to bacterial DsbA enzymes.

We have isolated and characterized a gene for a putative protein-disulfide oxidoreductase (phdsb) in the archaeon Pyrococcus horikoshii. The open reading frame of phdsb encodes a protein of 170 amino acids with an NH(2)-terminal extension similar to the bacterial signal peptides. The putative mature region of PhDsb includes a sequence motif, Cys-Pro-His-Cys (CPHC), that is conserved in members of the bacterial DsbA family, but otherwise the archaeal and bacterial sequences do not show substantial similarity. A recombinant protein corresponding to the predicted mature form of PhDsb behaved as a monomer and manifested oxidoreductase activities in vitro similar to those of DsbA of Escherichia coli. The catalytic activity of PhDsb was thermostable and was shown by mutation analysis to depend on the NH(2)-terminal cysteine residue of the CPHC motif. Thus, in spite of their low overall sequence similarities, DsbA-like proteins of archaea and bacteria appear to be highly similar in terms of function.

Amino Acid Motifs↗

PubMed2005

L-Threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3: gene cloning and enzymatic characterization.

A gene encoding the L-threonine dehydrogenase homologue has been identified in a hyperthermophlic archaeon Pyrococcus horikoshii OT3 via genome sequencing. The gene was cloned and expressed in Escherichia coli. The purified enzyme from the recombinant E. coli was extremely thermostable; the activity was not lost after incubation at 100 degrees C for 20 min. The enzyme (molecular mass: 192 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme is specific for NAD and utilizes L-threonine, L-serine and DL-threo-3-phenylserine as the substrate. The enzyme required divalent cations such as Zn(2+), Mn(2+) and Co(2+) for the activity, and contained one zinc ion/subunit. The K(m) values for L-threonine and NAD at 50 degrees C were 0.20 mM and 0.024 mM, respectively. Kinetic analyses indicated that the L-threonine oxidation reaction proceeds via a random mechanism with regard to the binding of L-threonine and NAD. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NADH. This is the first description of the characteristics of an L-threonine dehydrogenase from the archaea domain.

Alcohol Oxidoreductases↗

PubMed2006

Purification, crystallization and preliminary X-ray crystallographic analysis of the archaeal phosphoglycerate mutase PH0037 from Pyrococcus horikoshii OT3.

Phosphoglycerate mutases catalyze the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in glycolysis and gluconeogenesis pathways. The archaeal phosphoglycerate mutase PH0037 from Pyrococcus horikoshii OT3 has been overexpressed in Escherichia coli and purified. Crystals were obtained using the oil-microbatch method at 291 K. A native data set extending to a resolution of 2.2 angstroms has been collected and processed in space group R32. Assuming the presence of a dimer in the asymmetric unit, the V(M) value is calculated to be 3.0 angstroms3 Da(-1), consistent with the dynamic light-scattering experiment result, which shows a dimeric state of the protein in solution. Molecular-replacement trials using the crystal structure of Bacilllus stearothermophilus phosphoglycerate mutase as a search model did not provide a satisfactory solution, indicating substantially different structures of these two phophoglycerate mutases.

Archaeal Proteins↗

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↗

PubMed2005

A second novel dye-linked L-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3.

Two distinguishable activity bands for dye-linked l-proline dehydrogenase (PDH1 and PDH2) were detected when crude extract of the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was run on a polyacrylamide gel. After purification, PDH1 was found to be composed of two different subunits with molecular masses of 56 and 43 kDa, whereas PDH2 was composed of four different subunits with molecular masses of 52, 46, 20 and 8 kDa. The native molecular masses of PDH1 and PDH2 were 440 and 101 kDa, respectively, indicating that PDH1 has an alpha4beta4 structure, while PDH2 has an alphabetagammadelta structure. PDH2 was found to be similar to the dye-linked l-proline dehydrogenase complex from Thermococcus profundus, but PDH1 is a different type of enzyme. After production of the enzyme in Escherichia coli, high-performance liquid chromatography showed the PDH1 complex to contain the flavins FMN and FAD as well as ATP. Gene expression and biochemical analyses of each subunit revealed that the beta subunit bound FAD and exhibited proline dehydrogenase activity, while the alpha subunit bound ATP, but unlike the corresponding subunit in the T. profundus enzyme, it exhibited neither proline dehydrogenase nor NADH dehydrogenase activity. FMN was not bound to either subunit, suggesting it is situated at the interface between the alpha and beta subunits. A comparison of the amino-acid sequences showed that the ADP-binding motif in the alpha subunit of PDH1 clearly differs from that in the alpha subunit of PDH2. It thus appears that a second novel dye-linked l-proline dehydrogenase complex is produced in P. horikoshii.

Amino Acid Sequence↗

PubMed2003

Subunit interaction and regulation of activity through terminal domains of the family D DNA polymerase from Pyrococcus horikoshii.

Functions of the terminal domains of the family D DNA polymerase from Pyrococcus horikoshii (PolDPho) were analyzed by making and characterizing various truncated proteins. Based on a co-expression vector developed previously (Shen, Y., Musti, K., Hiramoto, M., Kikuchi, H., Kawabayashi, Y., and Matsui, I. (2001) J. Biol. Chem. 276, 27376-27383), 25 vectors for terminal truncated proteins were constructed. The expressed proteins were characterized in terms of thermostability, subunit interaction, and polymerization and 3'-5' exonuclease activities. The carboxyl-terminal (1255-1332) of the large subunit (DP2Pho) and two regions, the 201-260 and 599-622, of the small subunit (DP1Pho) were found to be critical for the complex formation, and probable subunit interaction of PolDPho. The amino-terminal (1-300) of DP2Pho is essential for the folding of PolDPho and is likely the oligomerization domain of PolDPho. A short region at the extreme C-terminal of DP2Pho (from 1385 to 1434) and the N-terminal of DP1Pho(1-200), which forms a stable protein, are not absolutely necessary for either polymerization or the 3'-5' exonuclease activity. We identified a possible regulatory role of DP1Pho(1-200) for the 3'-5' exonuclease. Deletion of DP1Pho(1-200) increased the exonuclease and DNA binding activities of PolDPho. Adding DP1Pho(1-200) to the truncated protein suppressed the elevated exonuclease activity. We also constructed and analyzed three internal deletion mutants and two site-directed mutants in the region of the putative zinc finger motif (cysteine cluster II) of DP2Pho at the COOH-terminal. We found that the internal region of the zinc finger motif is critical for the 3'-5' exonuclease, but is dispensable for the DNA polymerization.

Amino Acid Sequence↗

PubMed2004

Cloning, purification, crystallization and preliminary crystallographic analysis of acylphosphatase from Pyrococcus horikoshii OT3.

Acylphosphatase is one of the smallest enzymes and catalyzes the hydrolysis of the carboxy-phosphate bond. An extremely thermostable acylphosphatase from a hyperthermophilic archaea, Pyrococcus horikoshii OT3, has been cloned, expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with potassium/sodium tartrate as the precipitant at pH 5.5. X-ray diffraction data have been collected to a highest resolution of 1.72 angstroms on a synchrotron-radiation source. The crystals belong to space group P3(2)21, with approximate unit-cell parameters a = b = 86.6, c = 75.4 angstroms and two monomers in the asymmetric unit.

Acid Anhydride Hydrolases↗

PubMed2004

Structural implications for heavy metal-induced reversible assembly and aggregation of a protein: the case of Pyrococcus horikoshii CutA.

CutA is a small protein that appears to be involved in the mechanism of divalent metal cation tolerance in microorganisms. Here we report the crystal structure of Pyrococcus horikoshii CutA (PhoCutA), with and without Cu(2+), and its metal-binding properties. Crystallographic analyses revealed that PhoCutA forms a stable trimeric structure with intertwined antiparallel beta-strands. The crystal structure of the Cu(2+)-PhoCutA complex shows that the Cu(2+) is located at a trimer-trimer interface and is recognized by the side chains of one Asp(48) from each trimer. In an in vitro experiment, PhoCutA bound to several heavy metals, most of which led to reversible aggregation of the protein; i.e. the aggregates could be completely solubilized by addition of ethylenediamine tetraacetic acid (EDTA) or dialysis against metal free buffer. Substitution of Asp(48) with Ala led to a decrease in the amount of aggregates, suggesting the significant contribution of Asp(48) to the reversible aggregation. To the best of our knowledge, this is the first report which provides the structural evidence for heavy metal-induced multimerization of a protein.

Amino Acid Sequence↗

PubMed2003

Distinct domain functions regulating de novo DNA synthesis of thermostable DNA primase from hyperthermophile Pyrococcus horikoshii.

DNA primases are essential components of the DNA replication apparatus in every organism. Reported here are the biochemical characteristics of a thermostable DNA primase from the thermophilic archaeon Pyrococcus horikoshii, which formed the oligomeric unit L(1)S(1) and synthesized long DNA primers 10 times more effectively than RNA primers. The N-terminal (25KL) and C-terminal halves (20KL) of the large subunit (L) play distinct roles in regulating de novo DNA synthesis of the small catalytic subunit (S). The 25KL domain has a dual function. One function is to depress the large affinity of the intrasubunit domain 20KL for the template DNA until complex (L(1)S(1) unit) formation. The other function is to tether the L subunit tightly to the S subunit, probably to promote effective interaction between the intrasubunit domain 20KL and the active center of the S subunit. The 20KL domain is a central factor to enhance the de novo DNA synthesis activity of the catalytic S subunit since the total affinity of the L(1)S(1) unit is mainly derived from the affinity of 20KL, which is elevated more than 10 times by the heterodimer formation, presumably due to the cancellation of the inhibitory activity of 25KL through tight binding to the S subunit.

Amino Acid Sequence↗

PubMed2004

Crystal structure of the Pyrococcus horikoshii isopropylmalate isomerase small subunit provides insight into the dual substrate specificity of the enzyme.

Recent studies have implied that the isopropylmalate isomerase small subunit of the hyperthermophilic archaea Pyrococcus horikoshii (PhIPMI-s) functions as isopropylmalate isomerase in the leucine biosynthesis pathway, and as homoaconitase (HACN) in the lysine biosynthesis pathway via alpha-aminoadipic acid. PhIPMI is thus considered a key to understanding the fundamental metabolism of the earliest organisms. We describe for the first time the crystal structure of PhIPMI-s, which displays dual substrate specificity. The crystal structure unexpectedly shows that four molecules create an interlocked assembly with intermolecular disulfide linkages having a skewed 222 point-group symmetry. Although the overall fold of the PhIPMI-s monomer is related closely to domain 4 of the aconitase (ACN), one alpha-helix in the ACN structure is replaced by a short loop with relatively high temperature factor values. Because this region is essential for discriminating the structurally similar substrate based on interactions with its diversified gamma-moiety, the loop structure in the PhIPMI-s must be dependent on the presence of a substrate. The flexibility of the loop region might be a structural basis for recognizing both hydrophobic and hydrophilic gamma-moieties of two distinct substrates, isopropylmalate and homocitrate.

Amino Acid Sequence↗

PubMed2004

Crystal structure of the ribonuclease P protein Ph1877p from hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of pre-tRNA. Protein Ph1877p is one of essential components of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 RNase P [Biochem. Biophys. Res. Commun. 306 (2003) 666]. The crystal structure of Ph1877p was determined at 1.8A by X-ray crystallography and refined to a crystallographic R factor of 22.96% (Rfree of 26.77%). Ph1877p forms a TIM barrel structure, consisting of ten alpha-helices and seven beta-strands, and has the closest similarity to the TIM barrel domain of Escherichia coli cytosine deaminase with a root-mean square deviation of 3.0A. The protein Ph1877p forms an oblate ellipsoid, approximate dimensions being 45Ax43Ax39A, and the electrostatic representation indicated the presence of several clusters of positively charged amino acids present on the molecular surface. We made use of site-directed mutagenesis to assess the role of twelve charged amino acids, Lys42, Arg68, Arg87, Arg90, Asp98, Arg107, His114, Lys123, Lys158, Arg176, Asp180, and Lys196 related to the RNase P activity. Individual mutations of Arg90, Arg107, Lys123, Arg176, and Lys196 by Ala resulted in reconstituted particles with reduced enzymatic activities (32-48%) as compared with that reconstituted RNase P by wild-type Ph1877p. The results presented here provide an initial step for definite understanding of how archaeal and eukaryotic RNase Ps mediate substrate recognition and process 5'-leader sequence of pre-tRNA.

Amino Acid Sequence↗

PubMed2005

Protein-protein interactions in the subunits of ribonuclease P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of RNA and five protein subunits (Ph1481p, Ph1496p, Ph1601p, Ph1771p, and Ph1877p). In vivo interactions among five protein subunits of RNase P in P. horikoshii OT3 were examined using a yeast two-hybrid system. The analysis indicates that proteins Ph1481p and Ph1601p interact strongly with Ph1877p and Ph1771p respectively, whereas Ph1481p interacts moderately with Ph1601p. In contrast, no interaction was detected between Ph1496p and the other four proteins. Co-immunoprecipitation analysis confirmed the interactions obtained by yeast two-hybrid assay.

Archaeal Proteins↗

PubMed2006

Purification, crystallization and preliminary crystallographic analysis of RecA superfamily ATPase PH0284 from Pyrococcus horikoshii OT3.

Circadian (daily) protein clocks are found in cyanobacteria, where a complex of the KaiA, KaiB and KaiC proteins generates circadian rhythms. The 28.09 kDa KaiC homologue PH0284 protein from Pyrococcus horikoshii OT3 was cloned and expressed and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data from the crystal were collected to 2.0 angstroms resolution using synchrotron radiation at 100 K. The crystal belongs to the trigonal space group P3(2)21, with unit-cell parameters a = b = 96.06, c = 298.90 angstroms. Assuming the presence of one hexamer in the asymmetric unit gives a V(M) value of 2.36 angstroms3 Da(-1) and a solvent content of 47.9%. A cocrystal with ATP was prepared and a diffraction data set was collected at 2.3 angstroms resolution.

Adenosine Triphosphatases↗

PubMed1998

Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3.

The complete sequence of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3, has been determined by assembling the sequences of the physical map-based contigs of fosmid clones and of long polymerase chain reaction (PCR) products which were used for gap-filling. The entire length of the genome was 1,738,505 bp. The authenticity of the entire genome sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2061 open reading frames (ORFs) were assigned, and by similarity search against public databases, 406 (19.7%) were related to genes with putative function and 453 (22.0%) to the sequences registered but with unknown function. The remaining 1202 ORFs (58.3%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs in the genome provided evidence that a considerable number of ORFs were generated by sequence duplication. By similarity search, 11 ORFs were assumed to contain the intein elements. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 46 tRNA genes including two with the intron structure. All the assigned ORFs and RNA coding regions occupied 91.25% of the whole genome. The data presented in this paper are available on the internet at http:@www.nite.go.jp.

Chromosomes, Archaeal↗

PubMed2004

Crystallization and preliminary X-ray diffraction study of the catalytic subunit of archaeal H+ -transporting ATP synthase from Pyrococcus horikoshii OT3.

H+ -transporting ATP synthase (H+ -ATPase) is a multi-subunit complex which acts to produce ATP molecules. The catalytic subunit A of the archaeal-type H+ -ATPase from Pyrococcus horikoshii OT3 was cloned, expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method with MPD as a precipitant. X-ray intensity data were collected to 2.55 A resolution at beamline BL41XU of SPring-8. The crystals belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 128.0, c = 104.7 A, and contain one molecule per asymmetric unit.

Catalytic Domain↗

PubMed2005

Purification, crystallization and preliminary crystallographic analysis of the biotin-protein ligase from Pyrococcus horikoshii OT3.

Biotin-protein ligase is an enzyme that catalyzes the ATP-dependent biotinylation of a specific lysine residue in acetyl-CoA carboxylase. The biotin-protein ligase from Pyrococcus horikoshii OT3 has been cloned, overexpressed and purified. Crystallization was performed by the microbatch method or the vapour-diffusion method using PEG 2000 as a precipitant at 295 K. X-ray diffraction data have been collected to 1.6 A resolution from a native crystal and to 1.55 A resolution from a selenomethionine-derivative crystal for multiple anomalous dispersion phasing using synchrotron radiation at 100 K. The native crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 38.601, b = 78.264, c = 70.147 A, beta = 101.48 degrees. Assuming a homodimer per asymmetric unit gives a VM value of 2.14 A3 Da(-1) and a solvent content of 42.5%. Cocrystals with biotin, ADP and biotinyl-5'-AMP were prepared and diffraction data sets were collected to 1.6, 1.6 and 1.45 A resolution, respectively.

Bacterial Proteins↗