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1.  Crystallization and preliminary X-ray diffraction study of glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis  
Glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis was crystallized and preliminary crystallographic studies of the crystals were performed.
Glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis was crystallized and preliminary crystallographic studies of the crystals were performed. Crystals were grown at 293 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 2.4 Å resolution using synchrotron radiation at station BL44XU of SPring-8. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 217.48, c = 66.48 Å. Assuming the presence of two molecules in the asymmetric unit, the V M value was 2.7 Å3 Da−1 and the solvent content was 54.1%. The protein was also cocrystallized with substrates and diffraction data were collected to 2.7 Å resolution.
doi:10.1107/S1744309107001388
PMCID: PMC2330122  PMID: 17277457
glycerol kinase; Thermococcus kodakaraensis; thermostability
2.  Crystallization and preliminary crystallographic analysis of type 1 RNase H from the hyperthermophilic archaeon Sulfolobus tokodaii 7 
Type 1 RNase H from the hyperthermophilic archaeon S. tokodaii 7 was overproduced in E. coli, purified, and crystallized. Preliminary crystallographic studies indicated that the crystal belongs to space group P43, with unit-cell parameters a = b = 39.21, c = 91.15 Å.
Crystallization and preliminary crystallographic studies of type 1 RNase H from the hyperthermophilic archaeon Sulfolobus tokodaii 7 were performed. A crystal was grown at 277 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 1.5 Å resolution using synchrotron radiation from station BL41XU at SPring-8. The crystal belongs to space group P43, with unit-cell parameters a = b = 39.21, c = 91.15 Å. Assuming the presence of one molecule in the asymmetric unit, the Matthews coefficient V M was calculated to be 2.1 Å3 Da−1 and the solvent content was 40.5%. The structure of a selenomethionine Sto-RNase HI mutant obtained using a MAD data set is currently being analysed.
doi:10.1107/S1744309106024420
PMCID: PMC2242919  PMID: 16880556
type 1 RNase H; Sulfolobus tokodaii 7
3.  Ca2+-Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis: the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding 
Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca2+ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca2+ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca2+ but was activated upon incubation with Ca2+ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ∼5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a Ki of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.
doi:10.1128/AEM.02696-05
PMCID: PMC1489632  PMID: 16751527
4.  Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence 
The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly−82 to Gly316), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca2+ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.
doi:10.1128/AEM.67.6.2445-2452.2001
PMCID: PMC92893  PMID: 11375149
5.  Crystallization and preliminary X-ray analysis of hyperthermophilic l-threonine dehydrogenase from the archaeon Pyrococcus horikoshii  
l-Threonine dehydrogenase from the hyperthermophilic archaeon P. horikoshii was crystallized and preliminary X-ray crystallographic analysis was carried out.
Recombinant l-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii was prepared using an Escherichia coli expression system. The hyperthermostable l-threonine dehydrogenase consists of 348 amino acids with a molecular weight of 37.7 kDa. The enzyme was crystallized by the hanging-drop vapour-diffusion method at 277 K and preliminary X-ray crystallographic analysis was carried out. Diffraction data were collected to 2.20 Å resolution under cryogenic conditions. P. horikoshii l-threonine dehydrogenase crystals belong to space group I4122, with unit-cell parameters a = b = 143.84, c = 304.13 Å. The presence of three subunits of the enzyme per asymmetric unit was estimsted to give a Matthews coefficient (V M) of 3.5 Å3 Da−1 and a solvent content of 64.7%(v/v).
doi:10.1107/S174430910500881X
PMCID: PMC1952418  PMID: 16511061
archaea; Pyrococcus horikoshii; hyperthermostability; threonine dehydrogenase
6.  Crystallization and preliminary X-ray diffraction analysis of l-threonine dehydrogenase (TDH) from the hyperthermophilic archaeon Thermococcus kodakaraensis  
The l-threonine dehydrogenase from T. kodakaraensis KOD1 has been crystallized in the tetragonal space group P43212, with unit-cell parameters a = b = 124.5, c = 271.1 Å. Diffraction data were collected to 2.6 Å resolution and preliminary analysis indicates that there are four molecules in the crystallographic asymmetric unit.
The enzyme l-threonine dehydrogenase catalyses the NAD+-dependent conversion of l-threonine to 2-amino-3-ketobutyrate, which is the first reaction of a two-step biochemical pathway involved in the metabolism of threonine to glycine. Here, the crystallization and preliminary crystallographic analysis of l-­threonine dehydrogenase (Tk-TDH) from the hyperthermophilic organism Thermococcus kodakaraensis KOD1 is reported. This threonine dehydrogenase consists of 350 amino acids, with a molecular weight of 38 kDa, and was prepared using an Escherichia coli expression system. The purified native protein was crystallized using the hanging-drop vapour-diffusion method and crystals grew in the tetragonal space group P43212, with unit-cell parameters a = b = 124.5, c = 271.1 Å. Diffraction data were collected to 2.6 Å resolution and preliminary analysis indicates that there are four molecules in the asymmetric unit of the crystal.
doi:10.1107/S1744309108025384
PMCID: PMC2531275  PMID: 18765916
l-threonine dehydrogenase; thermophiles
7.  Superoxide reductase from Nanoarchaeum equitans: expression, purification, crystallization and preliminary X-ray crystallographic analysis 
Diffraction-quality crystals of N. equitans neelaredoxin have been produced. The expression, purification and crystallization of the protein and preliminary X-ray crystallographic analysis of the crystals are reported.
Superoxide reductases (SORs) are the most recent oxygen-detoxification system to be identified in anaerobic and microaerobic bacteria and archaea. SORs are metalloproteins that are characterized by their possession of a catalytic nonhaem iron centre in the ferrous form coordinated by four histidine ligands and one cysteine ligand. Ignicoccus hospitalis, a hyperthermophilic crenarchaeon, is the only organism known to date to serve as a host for Nanoarchaeum equitans, a nanosized hyperthermophilic archaeon isolated from a submarine hot vent which completely depends on the presence of and contact with I. hospitalis cells for growth to occur. Similarly to I. hospitalis, N. equitans has a neelaredoxin (a 1Fe-type SOR) that keeps toxic oxygen species under control, catalysing the one-electron reduction of superoxide to hydrogen peroxide. Blue crystals of recombinant N. equitans SOR in the oxidized form (12.7 kDa, 109 residues) were obtained using polyethylene glycol (PEG 2000 MME) as precipitant. These crystals diffracted to 1.9 Å resolution at 100 K and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 51.88, b = 82.01, c = 91.30 Å. Cell-content analysis suggested the presence of four monomers in the asymmetric unit. The Matthews coefficient (V M) was determined to be 1.9 Å3 Da−1, corresponding to an estimated solvent content of 36%. Self-rotation function and native Patterson calculations suggested a tetramer with 222 point-group symmetry, similar to other 1Fe-SORs. The three-dimensional structure will be determined by the molecular-replacement method.
doi:10.1107/S1744309111009432
PMCID: PMC3087648  PMID: 21543869
superoxide reductases; Nanoarchaeum equitans; oxidative stress; neelaredoxins
8.  Crystallization and preliminary X-ray crystallographic study of [NiFe]-hydrogenase maturation factor HypE from Thermococcus kodakaraensis KOD1 
The [NiFe]-hydrogenase maturation protein HypE was purified and crystallized. Crystals of HypE suitable for data collection diffracted to 1.55 Å resolution.
The hydrogenase maturation protein HypE is involved in the biosynthesis of the CN ligands of the active-site iron of [NiFe] hydrogenases using carbamoylphosphate as a substrate. Here, the crystallization and preliminary crystallographic analysis of HypE from Thermococcus kodakaraensis KOD1 are reported. Crystals of HypE (338 amino acids, 35.9 kDa) have been obtained by the sitting-drop vapour-diffusion method using 2-methyl-2,4-pentanediol (MPD) as a precipitant. The crystals belong to space group P21212, with unit-cell parameters a = 88.3, b = 45.8, c = 75.1 Å. There is one HypE molecule in the asymmetric unit. A complete native X-ray diffraction data set was collected to a maximum resolution of 1.55 Å at 100 K.
doi:10.1107/S1744309107038833
PMCID: PMC2376326  PMID: 17768349
[NiFe] hydrogenase; maturation; CN-ligand synthesis
9.  Expression, purification and preliminary X-ray analysis of proliferating cell nuclear antigen from the archaeon Thermococcus thioreducens  
The proliferating cell nuclear antigen (PCNA) from a novel hyperthermophilic archaeon Thermococcus thioreducens has been crystallized, and diffraction data have been collected to 1.86 Å.
Proliferating cell nuclear antigen (PCNA) is a DNA sliding clamp which confers processivity on replicative DNA polymerases. PCNA also acts as a sliding platform that enables the association of many DNA-processing proteins with DNA in a non-sequence-specific manner. In this investigation, the PCNA from the hyperthermophilic archaeon Thermococcus thioreducens (TtPCNA) was cloned, overexpressed in Escherichia coli and purified to greater than 90% homogeneity. TtPCNA crystals were obtained by sitting-drop vapor-diffusion methods and the best ordered crystal diffracted to 1.86 Å resolution using synchrotron radiation. The crystals belonged to the hexagonal space group P63, with unit-cell parameters a = b = 89.0, c = 62.8 Å. Crystals of TtPCNA proved to be amenable to complete X-ray analysis and future structure determination.
doi:10.1107/S174430910903036X
PMCID: PMC2795597  PMID: 19724129
proliferating cell nuclear antigen; Thermococcus thioreducens
10.  Crystallization and preliminary X-ray diffraction analysis of a flavoenzyme amine dehydrogenase/oxidase from Pyrococcus furiosus DSM 3638 
This report describes the crystallization of a recombinant flavoprotein amine dehydrogenase/oxidase with specificity for l-proline from the hyperthermophile P. furiosus DSM 3638 and X-ray diffraction data collection. Crystals belonged to space group P1 and diffracted to a resolution of 3.3 Å.
A flavoprotein amine dehydrogenase/oxidase with subunit molecular weights of 54.8 kDa (α-subunit) and 42.4 kDa (β-subunit) and specificity for l-proline was cloned from the genomic DNA of the hyperthermophilic marine archaeon Pyrococcus furiosus DSM 3638. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The enzyme was crystallized using the sitting-drop vapour-diffusion technique. Diffraction data from two different crystal forms were collected to 3.3 and 3.6 Å, respectively, using synchrotron radiation. Both crystals belonged to space group P1, with unit-cell parameters a = 91.3, b = 136.3, c = 203.8 Å, α = 94.5, β = 99.4, γ = 102.7° and a = 93.7, b = 116.3, c = 126.9 Å, α = 97.3, β = 99.9, γ = 104.6°.
doi:10.1107/S1744309105020737
PMCID: PMC1952344  PMID: 16511149
Pyrococcus furiosus DSM 3638; flavoprotein amine dehydrogenase; flavin adenine dinucleotide
11.  Expression, purification, crystallization and preliminary crystallographic analysis of a thermostable DNA ligase from the archaeon Thermococcus sibiricus  
A recombinant ATP-dependent DNA ligase from the hyperthermophilic anaerobic archaeon Thermococcus sibiricus was expressed in Escherichia coli and purified. Crystals were grown by vapour diffusion using the hanging-drop method.
DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5′-­phosphate and 3′-­hydroxyl termini. Their function is essential to maintain the integrity of the genome in DNA replication, recombination and repair. A recombinant ATP-dependent DNA ligase from the hyperthermophilic anaerobic archaeon Thermococcus sibiricus was expressed in Escherichia coli and purified. Crystals were grown by vapour diffusion using the hanging-drop method with 17%(w/v) PEG 4000 and 8.5%(v/v) 2-propanol as precipitants. A diffraction experiment was performed with a single crystal, which diffracted X-­rays to 3.0 Å resolution. The crystal belonged to space group P212121, with unit-cell parameters a = 58.590, b = 87.540, c = 126.300 Å.
doi:10.1107/S1744309111050913
PMCID: PMC3274393  PMID: 22297989
DNA ligases; archaea
12.  Crystallization and preliminary X-ray diffraction study of thermostable RNase HIII from Bacillus stearothermophilus  
A thermostable ribonuclease HIII from B. stearothermophilus (Bst RNase HIII) was crystallized and preliminary crystallographic studies were performed. Plate-like overlapping polycrystals were grown by the sitting-drop vapour-diffusion method at 283 K.
A thermostable ribonuclease HIII from Bacillus stearothermophilus (Bst RNase HIII) was crystallized and preliminary crystallographic studies were performed. Plate-like overlapping polycrystals were grown by the sitting-drop vapour-diffusion method at 283 K. Native X-ray diffraction data were collected to 2.8 Å resolution using synchrotron radiation from station BL44XU at SPring-8. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 66.73, b = 108.62, c = 48.29 Å. Assuming one molecule per asymmetric unit, the V M value was 2.59 Å3 Da−1 and the solvent content was 52.2%.
doi:10.1107/S1744309105003659
PMCID: PMC1952286  PMID: 16511022
ribonuclease HIII
13.  Inorganic pyrophosphatase crystals from Thermococcus thioreducens for X-ray and neutron diffraction 
Inorganic pyrophosphatase from T. thioreducans has been crystallized and the crystals were deemed to be suitable for both X-ray and neutron diffraction at room temperature.
Inorganic pyrophosphatase (IPPase) from the archaeon Thermococcus thioreducens was cloned, overexpressed in Escherichia coli, purified and crystallized in restricted geometry, resulting in large crystal volumes exceeding 5 mm3. IPPase is thermally stable and is able to resist denaturation at temperatures above 348 K. Owing to the high temperature tolerance of the enzyme, the protein was amenable to room-temperature manipulation at the level of protein preparation, crystallization and X-ray and neutron diffraction analyses. A complete synchrotron X-ray diffraction data set to 1.85 Å resolution was collected at room temperature from a single crystal of IPPase (monoclinic space group C2, unit-cell parameters a = 106.11, b = 95.46, c = 113.68 Å, α = γ = 90.0, β = 98.12°). As large-volume crystals of IPPase can be obtained, preliminary neutron diffraction tests were undertaken. Consequently, Laue diffraction images were obtained, with reflections observed to 2.1 Å resolution with I/σ(I) greater than 2.5. The preliminary crystallographic results reported here set in place future structure–function and mechanism studies of IPPase.
doi:10.1107/S1744309112032447
PMCID: PMC3509969  PMID: 23192028
inorganic pyrophosphatase; Thermococcus thioreducens; neutron diffraction
14.  Expression, purification, crystallization and preliminary X-ray analysis of the KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 
The KaiC-like protein PH0187 from the hyperthermophilic archaeon P. horikoshii OT3 was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal of PH0187 diffracted X-rays to 2.75 Å resolution.
KaiC is the central protein in the circadian rhythm in cyanobacteria. The 28 kDa KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of PH0187 were obtained using a reservoir solution consisting of 1.0 M ammonium phosphate monobasic and 0.1 M sodium citrate tribasic pH 5.3 (the final pH value of the reservoir solution was 4.8) and diffracted X-rays to 2.75 Å resolution. The crystal of PH0187 belonged to space group P6322, with unit-cell parameters a = b = 239.1, c = 106.5 Å. The crystal contained four PH0187 molecules in the asymmetric unit.
doi:10.1107/S1744309110048426
PMCID: PMC3079995  PMID: 21206047
PH0187; KaiC; circadian rhythm
15.  Production, crystallization and preliminary X-ray analysis of CTP:inositol-1-phosphate cytidylyltransferase from Archaeoglobus fulgidus  
The expression, purification, crystallization and preliminary X-ray diffraction analysis of CTP:inositol-1-phosphate cytidylyltransferase from A. fulgidus is described.
Archaeoglobus fulgidus, a hyperthermophilic archaeon, accumulates di-myo-inositol phosphate (DIP) in response to heat stress. Recently, the pathway for biosynthesis of DIP has been elucidated in this organism and involves a bifunctional enzyme that contains two domains: CTP:inositol-1-phosphate cytidylyltransferase (IPCT) as a soluble domain and di-myo-inositol-1,3′-phosphate-1-phosphate synthase (DIPPS) as a membrane domain. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis of the IPCT domain from A. fulgidus in the apo form are reported. The crystals diffracted to 2.4 Å resolution using a synchrotron source and belonged to the orthorhombic space group P21212, with unit-cell parameters a = 154.7, b = 83.9, c = 127.7 Å.
doi:10.1107/S1744309110032677
PMCID: PMC3001648  PMID: 21045295
CTP:inositol-1-phosphate cytidylyltransferase; Archaeoglobus fulgidus; compatible solutes; CDP-inositol; di-myo-inositol phosphate
16.  Extracellular overproduction and preliminary crystallographic analysis of a family I.3 lipase 
A family I.3 lipase from Pseudomonas sp. MIS38 was secreted from Escherichia coli cells to the external medium, purified and crystallized and preliminary crystallographic studies were performed.
A family I.3 lipase from Pseudomonas sp. MIS38 was secreted from Escherichia coli cells to the external medium, purified and crystallized and preliminary crystallographic studies were performed. The crystal was grown at 277 K by the hanging-drop vapour-diffusion method. Native X-ray diffraction data were collected to 1.7 Å resolution using synchrotron radiation at station BL38B1, SPring-8. The crystal belongs to space group P21, with unit-cell parameters a = 48.79, b = 84.06, c = 87.04 Å. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V M was calculated to be 2.73 Å3 Da−1 and the solvent content was 55%.
doi:10.1107/S1744309107004575
PMCID: PMC2330184  PMID: 17329810
family I.3 lipases
17.  Preliminary crystallographic studies of glucose dehydrogenase from the promiscuous Entner–Doudoroff pathway in the hyperthermophilic archaeon Sulfolobus solfataricus  
A glucose dehydrogenase from the hyperthermophilic archaeon S. solfataricus has been crystallized and subjected to preliminary crystallographic analysis.
The hyperthermophilic archaeon Sulfolobus solfataricus grows optimally above 353 K and can metabolize glucose and its C4 epimer galactose via a non-phosphorylative variant of the Entner–Doudoroff pathway involving catalytically promiscuous enzymes that can operate with both sugars. The initial oxidation step is catalysed by glucose dehydrogenase (SsGDH), which can utilize both NAD and NADP as cofactors. The enzyme operates with glucose and galactose at similar catalytic efficiency, while its substrate profile also includes a range of other five- and six-carbon sugars. Crystals of the 164 kDa SsGDH homotetramer have been grown under a variety of conditions. The best crystals to date diffract to 1.8 Å on a synchrotron source, have orthorhombic symmetry and belong to space group P21212. Attempts are being made to solve the structure by MAD and MR.
doi:10.1107/S174430910403101X
PMCID: PMC1952374  PMID: 16508107
Sulfolobus solfataricus; glucose dehydrogenase; Entner–Doudoroff pathway
18.  Crystallization and preliminary X-ray analysis of PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins 
PH1010, a DUF54-family protein from the hyperthermophilic archaeon P. horikoshii OT3, was crystallized and X-ray diffraction data were collected to 1.90 Å resolution.
PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins, was expressed, purified and crystallized. Crystallization was performed by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.90 Å resolution using a synchrotron-radiation source. The space group of the crystal was determined to be P212121, with unit-cell parameters a = 46.9, b = 49.5, c = 132.7 Å. The crystal contained two PH1010 molecules in the asymmetric unit (V M = 2.4 Å3 Da−1) and had a solvent content of 48%.
doi:10.1107/S1744309107024487
PMCID: PMC2335084  PMID: 17554180
DUF54 family; PH1010; Pyrococcus horikoshii
19.  Crystallization and preliminary X-ray crystallographic studies of the [NiFe] hydrogenase maturation proteins HypC and HypD 
The [NiFe] hydrogenase maturation proteins HypC and HypD were purified and crystallized. Crystals of HypC and HypD suitable for data collection diffracted to 1.80 and 2.07 Å resolution, respectively.
HypC and HypD proteins are required for the insertion of the Fe atom with diatomic ligands into the large subunit of [NiFe] hydrogenases, an important step in the maturation process of this type of hydrogenase. The crystallization and preliminary crystallographic analysis of HypC and HypD from Thermococcus kodakaraensis KOD1 are reported. Crystals of HypC grew in two different forms. Monoclinic crystals of HypC in space group C2 with unit-cell parameters a = 78.2, b = 59.1, c = 54.0 Å, β = 109.0° were obtained using PEG 4000 and ammonium sulfate or sodium bromide as precipitants. They diffracted X-rays to 1.8 Å resolution and were suitable for structure determination. Crystals of HypD were also obtained in two different forms. The monoclinic crystals obtained using PEG 4000 and magnesium chloride diffracted X-rays to beyond 2.1 Å resolution, despite growing as clusters. They belong to space group P21, with unit-cell parameters a = 42.3, b = 118.4, c = 81.2 Å, β = 100.9°, and are suitable for data collection.
doi:10.1107/S1744309107023391
PMCID: PMC2335088  PMID: 17554182
[NiFe] hydrogenase; maturation; metal cluster; iron–sulfur protein
20.  Crystallization and preliminary X-ray analysis of a putative sensor histidine kinase domain: the C-­terminal domain of HksP4 from Aquifex aeolicus VF5 
The putative sensor histidine kinase domain of the cytoplasmic protein HksP4 from the hyperthermophilic bacterium A. aeolicus VF5 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained in the presence of ATP and AMPPNP; they were found to belong to the same space group P212121 and diffracted X-rays to 3.1 and 2.9 Å resolution, respectively.
The histidine kinase domain of the cytoplasmic protein HksP4 from the hyperthermophilic bacterium Aquifex aeolicus VF5, located in the C-terminal half of the protein, was expressed, purified and crystallized. Diffraction-quality crystals were obtained in the presence of adenosine triphosphate (ATP) or adenosine 5′-­(β,γ-imido)triphosphate (AMPPNP) by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals obtained in the presence of ATP and AMPPNP diffracted X-rays to 3.1 and 2.9 Å resolution, respectively, on BL-5A at Photon Factory (Ibaraki, Japan) and were found to belong to the same space group P212121, with unit-cell parameters a = 80.2, b = 105.5, c = 122.0 Å and a = 81.5, b = 105.5, c = 130.9 Å, respectively. Their Matthews coefficients (V M = 2.74 and 2.51 Å3 Da−1, respectively) indicated that both crystals contained four protein molecules per asymmetric unit.
doi:10.1107/S1744309111018434
PMCID: PMC3144801  PMID: 21795799
histidine kinase domains; HksP4; Aquifex aeolicus VF5
21.  Crystallization and preliminary X-ray diffraction analysis of a chitin-binding domain of hyperthermophilic chitinase from Pyrococcus furiosus  
The expression, purification and preliminary X-ray diffraction studies of a chitin-binding domain of the chitinase from P. furiosus are reported.
The crystallization and preliminary X-ray diffraction analysis of the chitin-binding domain of chitinase from a hyperthermophilic archaeon, Pyrococcus furiosus, are reported. The recombinant protein was prepared using an Escherichia coli overexpression system and was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected to 1.70 Å resolution. The crystal belonged to space group P43212 or P41212. The unit-cell parameters were determined to be a = b = 48.8, c = 85.0 Å.
doi:10.1107/S1744309105010110
PMCID: PMC1952313  PMID: 16511072
hyperthermophilic chitinase; chitin-binding domain; archaea; Pyrococcus furiosus
22.  Crystallization and X-ray diffraction analysis of a catalytic domain of hyperthermophilic chitinase from Pyrococcus furiosus  
The expression, purification and preliminary X-ray diffraction analysis of a catalytic domain of a chitinase from P. furiosus is reported.
The crystallization and preliminary X-ray diffraction analysis of a catalytic domain of chitinase (PF1233 gene) from the hyperthermophilic archaeon Pyrococcus furiosus is reported. The recombinant protein, prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at the undulator beamline BL44XU at SPring-8 to a resolution of 1.50 Å. The crystals belong to space group P212121, with unit-cell parameters a = 90.0, b = 92.8, c = 107.2 Å.
doi:10.1107/S1744309106026157
PMCID: PMC2242910  PMID: 16880559
hyperthermophilic chitinases; catalytic domain; archaea; Pyrococcus furiosus
23.  Expression, purification, crystallization and preliminary X-ray analysis of a nucleoside kinase from the hyperthermophile Methanocaldococcus jannaschii  
Nucleoside kinase from the hyperthermophilic archaeon M. jannaschii is a member of the PFK-B family which belongs to the ribokinase superfamily. Here, its expression, purification, crystallization and preliminary X-ray analysis are described.
Methanocaldococcus jannaschii nucleoside kinase (MjNK) is an ATP-dependent non-allosteric phosphotransferase that shows high catalytic activity for guanosine, inosine and cytidine. MjNK is a member of the phosphofructokinase B family, but participates in the biosynthesis of nucleoside monophosphates rather than in glycolysis. MjNK was crystallized as the apoenzyme as well as in complex with an ATP analogue and Mg2+. The latter crystal form was also soaked with fructose-6-phosphate. Synchrotron-radiation data were collected to 1.70 Å for the apoenzyme crystals and 1.93 Å for the complex crystals. All crystals exhibit orthorhombic symmetry; however, the apoenzyme crystals contain one monomer per asymmetric unit whereas the complex crystals contain a dimer.
doi:10.1107/S1744309105015642
PMCID: PMC1952333  PMID: 16511104
Methanocaldococcus jannaschii; phosphofructokinase B family; ribokinase superfamily; nucleoside kinases; hyperthermophiles; archaea
24.  Purification, crystallization and X-ray crystallographic analysis of Archaeoglobus fulgidus neelaredoxin 
Diffraction-quality crystals of A. fulgidus neelaredoxin have been produced. The expression, purification, crystallization and X-ray crystallographic analysis of A. fulgidus neelaredoxin are reported.
Neelaredoxins are a type of superoxide reductase (SOR), which are blue 14 kDa metalloproteins with a catalytic nonhaem iron centre coordinated by four histidines and one cysteine in the ferrous form. Anaerobic organisms such as Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing archaeon, have developed defence mechanisms against toxic oxygen species in which superoxide reductases play a key role. SOR is responsible for scavenging toxic superoxide anion radicals (O2 ·−), catalysing the one-electron reduction of superoxide to hydrogen peroxide. Crystals of recombinant A. fulgidus neela­redoxin in the oxidized form (13.7 kDa, 125 residues) were obtained using polyethylene glycol and ammonium sulfate. These crystals diffracted to 1.9 Å resolution and belonged to the tetragonal space group P41212, with unit-cell parameters a = b = 75.72, c = 185.44 Å. Cell-content analysis indicated the presence of a tetramer in the asymmetric unit, with a Matthews coefficient (V M) of 2.36 Å3 Da−1 and an estimated solvent content of 48%. The three-dimensional structure was determined by the MAD method and is currently under refinement.
doi:10.1107/S1744309110000916
PMCID: PMC2833046  PMID: 20208170
superoxide reductases; Archaeoglobus fulgidus; oxygen detoxification; neelaredoxin
25.  Crystallization and preliminary X-ray analysis of flap endonuclease 1 (FEN1) from Desulfurococcus amylolyticus  
Flap endonuclease 1 from D. amylolyticus was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 2.00 Å resolution.
Flap endonuclease 1 (FEN1) is a structure-specific nuclease that removes 5′-­overhanging flaps in DNA repair and removes the RNA/DNA primer during maturation of the Okazaki fragment in lagging-strand DNA replication. FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with monoammonium dihydrogen phosphate as the precipitant at pH 8.3. X-ray diffraction data were collected to 2.00 Å resolution. The space group of the crystal was determined as the primitive hexagonal space group P321, with unit-cell parameters a = b = 103.76, c = 84.58 Å. The crystal contained one molecule in the asymmetric unit.
doi:10.1107/S1744309109031248
PMCID: PMC2795602  PMID: 19724134
FEN1; DNA repair; DNA replication

Results 1-25 (375886)