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1.  Crystallization and preliminary neutron diffraction studies of ADP-ribose pyrophosphatase-I from Thermus thermophilus HB8 
ADP-ribose pyrophosphatase-I, a Nudix enzyme, from T. thermophilus was crystallized for neutron diffraction. Neutron and X-ray diffraction data sets were collected to 2.1 and 1.5 Å resolution, respectively.
ADP-ribose pyrophosphatase-I from Thermus thermophilus HB8 (TtADPRase-I) prevents the intracellular accumulation of ADP-ribose by hydrolyzing it to AMP and ribose 5′-phosphate. To understand the catalytic mechanism of TtADPRase-I, it is necessary to investigate the role of glutamates and metal ions as well as the coordination of water molecules located at the active site. A macroseeding method was developed in order to obtain a large TtADPRase-I crystal which was suitable for a neutron diffraction study to provide structural information. Neutron and X-ray diffraction experiments were performed at room temperature using the same crystal. The crystal diffracted to 2.1 and 1.5 Å resolution in the neutron and X-ray diffraction experiments, respectively. The crystal belonged to the primitive space group P3221, with unit-cell parameters a = b = 50.7, c = 119 Å.
doi:10.1107/S1744309111044551
PMCID: PMC3253833  PMID: 22232170
ADP-ribose pyrophosphatase; Ndx4; neutron diffraction; Thermus thermophilus
2.  Simultaneous Use of MutS and RecA for Suppression of Nonspecific Amplification during PCR 
Journal of Nucleic Acids  2013;2013:823730.
Thermus thermophilus MutS, a thermostable mismatch-recognizing protein, is utilized in PCR to suppress nonspecific amplification by preventing synthesis from mismatched primers. T. thermophilus RecA also decreases nonspecific amplification by promoting proper hybridization between the primer and template. We observed that MutS and RecA function under the same reaction conditions and that MutS and RecA do not preclude each other. Furthermore, there were some DNA sequences for which only one of the 2 proteins effectively suppressed nonspecific amplification. The simultaneous use of MutS and RecA is a more attractive error-suppressing technique than the use of either of the 2 proteins alone.
doi:10.1155/2013/823730
PMCID: PMC3736406  PMID: 23970956
3.  Transcriptional Repression Mediated by a TetR Family Protein, PfmR, from Thermus thermophilus HB8 
Journal of Bacteriology  2012;194(17):4630-4641.
PfmR is one of four TetR family transcriptional regulators found in the extremely thermophilic bacterium, Thermus thermophilus HB8. We identified three promoters with strong negative regulation by PfmR, both in vivo and in vitro. PfmR binds pseudopalindromic sequences, with the consensus sequence of 5′-TACCGACCGNTNGGTN-3′ surrounding the promoters. According to the amino acid sequence and three-dimensional structure analyses of the PfmR-regulated gene products, they are predicted to be involved in phenylacetic acid and fatty acid metabolism. In vitro analyses revealed that PfmR weakly cross-regulated with the TetR family repressor T. thermophilus PaaR, which controls the expression of the paa gene cluster putatively involved in phenylacetic acid degradation but not with another functionally identified TetR family repressor, T. thermophilus FadR, which is involved in fatty acid degradation. The X-ray crystal structure of the N-terminal DNA-binding domain of PfmR and the nucleotide sequence of the predicted PfmR-binding site are quite similar to those of the TetR family repressor QacR from Staphylococcus aureus. Similar to QacR, two PfmR dimers bound per target DNA. The bases recognized by QacR within the QacR-binding site are conserved in the predicted PfmR-binding site, and they were important for PfmR to recognize the binding site and properly assemble on it. The center of the PfmR molecule contains a tunnel-like pocket, which may be the ligand-binding site of this regulator.
doi:10.1128/JB.00668-12
PMCID: PMC3415517  PMID: 22753056
4.  Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR) 
Polymerase chain reaction (PCR)-related technologies are hampered mainly by two types of error: nonspecific amplification and DNA polymerase-generated mutations. Here, we report that both errors can be suppressed by the addition of a DNA mismatch-recognizing protein, MutS, from a thermophilic bacterium. Although it had been expected that MutS has a potential to suppress polymerase-generated mutations, we unexpectedly found that it also reduced nonspecific amplification. On the basis of this finding, we propose that MutS binds a mismatched primer-template complex, thereby preventing the approach of DNA polymerase to the 3′ end of the primer. Our simple methodology improves the efficiency and accuracy of DNA amplification and should therefore benefit various PCR-based applications, ranging from basic biological research to applied medical science.
doi:10.3390/ijms14036436
PMCID: PMC3634412  PMID: 23519109
polymerase chain reaction; DNA mismatch repair; mismatch-recognizing protein; Thermus thermophilus; Aquifex aeolicus
5.  Crystal structures, dynamics and functional implications of molybdenum-cofactor biosynthesis protein MogA from two thermophilic organisms 
Three crystal structures of the molybdenum-cofactor biosynthesis protein MogA from two highly thermophilic organisms have been determined at high resolution. Comparative analyses revealed the residues involved in oligomerization. In addition, molecular-dynamics and docking studies suggested the binding affinities of several small molecules towards MogA and homologous proteins.
Molybdenum-cofactor (Moco) biosynthesis is an evolutionarily conserved pathway in almost all kingdoms of life, including humans. Two proteins, MogA and MoeA, catalyze the last step of this pathway in bacteria, whereas a single two-domain protein carries out catalysis in eukaryotes. Here, three crystal structures of the Moco-biosynthesis protein MogA from the two thermophilic organisms Thermus thermophilus (TtMogA; 1.64 Å resolution, space group P21) and Aquifex aeolicus (AaMogA; 1.70 Å resolution, space group P21 and 1.90 Å resolution, space group P1) have been determined. The functional roles and the residues involved in oligomerization of the protein molecules have been identified based on a comparative analysis of these structures with those of homologous proteins. Furthermore, functional roles have been proposed for the N- and C-terminal residues. In addition, a possible protein–protein complex of MogA and MoeA has been proposed and the residues involved in protein–protein interactions are discussed. Several invariant water molecules and those present at the subunit interfaces have been identified and their possible structural and/or functional roles are described in brief. In addition, molecular-dynamics and docking studies with several small molecules (including the substrate and the product) have been carried out in order to estimate their binding affinities towards AaMogA and TtMogA. The results obtained are further compared with those obtained for homologous eukaryotic proteins.
doi:10.1107/S1744309110035037
PMCID: PMC3079962  PMID: 21206014
MogA; molybdenum-cofactor biosynthesis proteins
6.  Structures of hypoxanthine-guanine phosphoribosyltransferase (TTHA0220) from Thermus thermophilus HB8 
Structures of hypoxanthine-guanine phosphoribosyltransferase from T. thermophilus HB8 in the unliganded form, in complex with IMP and in complex with GMP are reported at 2.1, 1.9 and 2.2 Å resolution, respectively.
Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase), which is a key enzyme in the purine-salvage pathway, catalyzes the synthesis of IMP or GMP from α-d-phosphoribosyl-1-pyrophosphate and hypoxanthine or guanine, respectively. Structures of HGPRTase from Thermus thermophilus HB8 in the unliganded form, in complex with IMP and in complex with GMP have been determined at 2.1, 1.9 and 2.2 Å resolution, respectively. The overall fold of the IMP complex was similar to that of the unliganded form, but the main-chain and side-chain atoms of the active site moved to accommodate IMP. The overall folds of the IMP and GMP complexes were almost identical to each other. Structural comparison of the T. thermo­philus HB8 enzyme with 6-­oxopurine PRTases for which structures have been determined showed that these enzymes can be tentatively divided into groups I and II and that the T. thermophilus HB8 enzyme belongs to group I. The group II enzymes are characterized by an N-­terminal extension with additional secondary elements and a long loop connecting the second α-helix and β-strand compared with the group I enzymes.
doi:10.1107/S1744309110023079
PMCID: PMC2917284  PMID: 20693661
transferases; Rossmann fold; purine nucleotide biosynthetic pathway
7.  Structural Characterization of Neutral and Acidic Glycolipids from Thermus thermophilus HB8 
PLoS ONE  2012;7(7):e35067.
The structural characterization of glycolipids from Thermus thermophilus HB8 was performed in this study. Two neutral and one acidic glycolipids were extracted and purified by the modified TLC-blotting method, after which their chemical structures were determined by chemical composition analysis, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The structure of one of the neutral glycolipids, NGL-A, was Galp(α1-6)GlcpNacyl(β1-2)Glcp(α1-)acyl2Gro, and the other, NGL-C, was Galf(β1-2)Galp(α1-6)GlcpNacyl(β1-2)Glcp(α1-)acyl2Gro. The structure of NGL-C was identical to that reported previously [Oshima, M. and Ariga, T. (1976) FEBS Lett. 64, 440]. Both neutral glycolipids shared a common structural unit found in the Thermus species. The acyl groups found in NGL-A and NGL-C, iso-type pentadecanoxy and heptadecanoxy fatty acid, were also the same as those found in this species. In contrast, the acidic glycolipid, AGL-B, possessed the structure of N-(((GlcpNAc(α1-)acyl2Gro)P-2)GroA)alkylamine. The alkyl group in AGL-B was an iso-type heptadecanyl, suggesting that the iso-type structure of the long alkyl chain is responsible for the thermal stability of the bacteria.
doi:10.1371/journal.pone.0035067
PMCID: PMC3398001  PMID: 22815675
8.  Structure of ST0929, a putative glycosyl transferase from Sulfolobus tokodaii  
The structure of ST0929 provides insight into the structural basis of the increase in the hydrolase side reaction that is observed for mutants in which a phenylalanine residue is replaced by a tyrosine residue in the subsite +1 tyrosine cluster of Sulfolobus sp.
The Sulfolobus tokodaii protein ST0929 shares close structural homology with S. acidocaldarius maltooligosyl trehalose synthase (SaMTSase), suggesting that the two enzymes share a common enzymatic mechanism. MTSase is one of a pair of enzymes that catalyze trehalose biosynthesis. The relative geometries of the ST0929 and SaMTSase active sites were found to be essentially identical. ST0929 also includes the unique tyrosine cluster that encloses the reducing-end glucose subunit in Sulfolobus sp. MTSases. The current structure provides insight into the structural basis of the increase in the hydrolase side reaction that is observed for mutants in which a phenylalanine residue is replaced by a tyrosine residue in the subsite +1 tyrosine cluster of Sulfolobus sp.
doi:10.1107/S1744309110006354
PMCID: PMC2852329  PMID: 20383007
trehalose biosynthesis; glucosyl transferase activity; hydrolase side reaction; maltooligoside trehalose synthase
9.  Phenylacetyl Coenzyme A Is an Effector Molecule of the TetR Family Transcriptional Repressor PaaR from Thermus thermophilusHB8 ▿ †  
Journal of Bacteriology  2011;193(17):4388-4395.
Phenylacetic acid (PAA) is a common intermediate in the catabolic pathways of several structurally related aromatic compounds. It is converted into phenylacetyl coenzyme A (PA-CoA), which is degraded to general metabolites by a set of enzymes. Within the genome of the extremely thermophilic bacterium Thermus thermophilusHB8, a cluster of genes, including a TetR family transcriptional regulator, may be involved in PAA degradation. The gene product, which we named T. thermophilusPaaR, negatively regulated the expression of the two operons composing the gene cluster in vitro. T. thermophilusPaaR repressed the target gene expression by binding pseudopalindromic sequences, with a consensus sequence of 5′-CNAACGNNCGTTNG-3′, surrounding the promoters. PA-CoA is a ligand of PaaR, with a proposed binding stoichiometry of 1:1 protein monomer, and was effective for transcriptional derepression. Thus, PaaR is a functional homolog of PaaX, a GntR transcriptional repressor found in Escherichia coliand Pseudomonasstrains. A three-dimensional structure of T. thermophilusPaaR was predicted by homology modeling. In the putative structure, PaaR adopts the typical three-dimensional structure of the TetR family proteins, with 10 α-helices. A positively charged surface at the center of the molecule is similar to the acyl-CoA-binding site of another TetR family transcriptional regulator, T. thermophilusFadR, which is involved in fatty acid degradation. The CoA moiety of PA-CoA may bind to the center of the PaaR molecule, in a manner similar to the binding of the CoA moiety of acyl-CoA to FadR.
doi:10.1128/JB.05203-11
PMCID: PMC3165508  PMID: 21725002
10.  Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3 
SAICAR synthetase from P. horikoshii OT3 has been cloned, expressed, purified and crystallized.
The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05 M cadmium sulfate hydrate, 0.1 M HEPES buffer pH 7.5 and 1.0 M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P31, with unit-cell parameters a = b = 95.62, c = 149.13 Å. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (V M) of 2.3 Å3 Da−1, corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides.
doi:10.1107/S1744309109052026
PMCID: PMC2815687  PMID: 20124717
SAICAR synthetase; PH0239; Pyrococcus horikoshii OT3; purine biosynthesis
11.  Expression, purification and X-ray analysis of 1,3-­propanediol dehydrogenase (Aq_1145) from Aquifex aeolicus VF5 
1,3-Propanediol dehydrogenase (Aq_1145) from A. aeolicus VF5 has been overexpressed, purified and crystallized. The crystals diffracted to 2.4 Å resolution.
1,3-Propanediol dehydrogenase is an enzyme that catalyzes the oxidation of 1,3-­propanediol to 3-hydroxypropanal with the simultaneous reduction of NADP+ to NADPH. SeMet-labelled 1,3-propanediol dehydrogenase protein from the hyperthermophilic bacterium Aquifex aeolicus VF5 was overexpressed in Escherichia coli and purified to homogeneity. Crystals of this protein were grown from an acidic buffer with ammonium sulfate as the precipitant. Single-wavelength data were collected at the selenium peak to a resolution of 2.4 Å. The crystal belonged to space group P32, with unit-cell parameters a = b = 142.19, c = 123.34 Å. The structure contained two dimers in the asymmetric unit and was solved by the MR-SAD approach.
doi:10.1107/S1744309109052403
PMCID: PMC2815688  PMID: 20124718
1,3-propanediol dehydrogenase; Aquifex aeolicus VF5; Aq_1145
12.  Structure of hypothetical Mo-cofactor biosynthesis protein B (ST2315) from Sulfolobus tokodaii  
The structure of a protein involved in the molybdopterin and molybdenum co-factor biosynthesis pathways of Sulfolobus tokodaii has been solved to a resolution of 1.9 Å.
The structure of a probable Mo-cofactor biosynthesis protein B from Sulfolobus tokodaii, belonging to space group P6422 with unit-cell parameters a = b = 136.68, c = 210.52 Å, was solved by molecular replacement to a resolution of 1.9 Å and refined to an R factor and R free of 16.8% and 18.5%, respectively. The asymmetric unit contains a trimer, while the biologically significant oligomer is predicted to be a hexamer by size-exclusion chromatography. The subunit structure and fold of ST2315 are similar to those of other enzymes that are known to be involved in the molybdopterin- and molybdenum cofactor-biosynthesis pathways.
doi:10.1107/S1744309109043772
PMCID: PMC2802863  PMID: 20054111
ST2315; Sulfolobus tokodaii; Mo-cofactor biosynthesis protein B
13.  Structure of SurE protein from Aquifex aeolicus VF5 at 1.5 Å resolution 
The structure of the stationary phase survival protein SurE protein from the hyperthermophile Aquifex aeolicus has been solved to 1.5 Å resolution. The divalent-metal-ion-dependent phosphatase active-site pocket is occupied by sulfate ions from the crystallization medium.
SurE is a stationary-phase survival protein found in bacteria, eukaryotes and archaea that exhibits a divalent-metal-ion-dependent phosphatase activity and acts as a nucleotidase and polyphosphate phosphohydrolase. The structure of the SurE protein from the hyperthermophile Aquifex aeolicus has been solved at 1.5 Å resolution using molecular replacement with one dimer in the asymmetric unit and refined to an R factor of 15.6%. The crystal packing reveals that two dimers assemble to form a tetramer, although gel-filtration chromatography showed the presence of only a dimer in solution. The phosphatase active-site pocket was occupied by sulfate ions from the crystallization medium.
doi:10.1107/S1744309109043814
PMCID: PMC2802864  PMID: 20054112
SurE; Aquifex aeolicus; stationary-phase survival
14.  Structure of d-lactate dehydrogenase from Aquifex aeolicus complexed with NAD+ and lactic acid (or pyruvate) 
The structure of d-lactate dehydrogenase from Aquifex aeolicus has been determined with each subunit of the homodimer in a ‘closed’ conformation and with the NAD+ cofactor and lactate (or pyruvate) bound at the inter-domain active-site cleft.
The crystal structure of d-lactate dehydrogenase from Aquifex aeolicus (aq_727) was determined to 2.12 Å resolution in space group P212121, with unit-cell parameters a = 90.94, b = 94.43, c = 188.85 Å. The structure was solved by molecular replacement using the coenzyme-binding domain of Lactobacillus helveticus d-lactate dehydrogenase and contained two homodimers in the asymmetric unit. Each subunit of the homodimer was found to be in a ‘closed’ conformation with the NADH cofactor bound to the coenzyme-binding domain and with a lactate (or pyruvate) molecule bound at the interdomain active-site cleft.
doi:10.1107/S1744309109044935
PMCID: PMC2802865  PMID: 20054113
d-lactate dehydrogenase; Aquifex aeolicus
15.  The structure of an archaeal ribose-5-phosphate isomerase from Methanocaldococcus jannaschii (MJ1603) 
The structure of ribose-5-phosphate isomerase from Methanocaldococcus jannaschii has been solved to 1.78 Å resolution, with the active site occupied by two molecules of propylene glycol mimicking the binding of a known arabinose-5-phosphate inhibitor.
Ribose-5-phosphate isomerase is a ubiquitous intracellular enzyme of bacterial, plant and animal origin that is involved in the pentose phosphate cycle, an essential component of cellular carbohydrate metabolism. Specifically, the enzyme catalyses the reversible conversion of ribose 5-phosphate to ribulose 5-­phosphate. The structure of ribose-5-phosphate isomerase from Methano­caldococcus jannaschii has been solved in space group P21 to 1.78 Å resolution using molecular replacement with one homotetramer in the asymmetric unit and refined to an R factor of 14.8%. The active site in each subunit was occupied by two molecules of propylene glycol in different orientations, one of which corresponds to the location of the phosphate moiety and the other to the location of the furanose ring of the inhibitor.
doi:10.1107/S1744309109044923
PMCID: PMC2802866  PMID: 20054114
ribose-5-phosphate isomerase; Methanocaldococcus jannaschii; pentose phosphate cycle
16.  Structure of a putative β-phosphoglucomutase (TM1254) from Thermotoga maritima  
The structure of a putative β-phosphoglucomutase from Thermotoga maritima belonging to the haloacid dehalogenase (HAD) hydrolase family has been determined to 1.74 Å resolution.
The structure of TM1254, a putative β-phosphoglucomutase from T. maritima, was determined to 1.74 Å resolution in a high-throughput structural genomics programme. Diffraction data were obtained from crystals belonging to space group P22121, with unit-cell parameters a = 48.16, b = 66.70, c = 83.80 Å, and were refined to an R factor of 19.2%. The asymmetric unit contained one protein molecule which is comprised of two domains. Structural homologues were found from protein databases that confirmed a strong resemblance between TM1254 and members of the haloacid dehalogenase (HAD) hydrolase family.
doi:10.1107/S1744309109046302
PMCID: PMC2802867  PMID: 20054115
β-phosphoglucomutase; Thermotoga maritima
17.  Structure of putative 4-amino-4-deoxychorismate lyase from Thermus thermophilus HB8 
The putative 4-amino-4-deoxychorismate lyase (TTHA0621) from T. thermophilus HB8 was cloned, overexpressed, purified and crystallized. Its crystal structure was determined by a combination of SAD and molecular-replacement methods and was refined to 1.93 Å resolution.
The pyridoxal 5′-phosphate-dependent enzyme 4-amino-4-deoxychorismate lyase converts 4-amino-4-deoxychorismate to p-aminobenzoate and pyruvate in one of the crucial steps in the folate-biosynthesis pathway. The primary structure of the hypothetical protein TTHA0621 from Thermus thermophilus HB8 suggests that TTHA0621 is a putative 4-amino-4-deoxychorismate lyase. Here, the crystal structure of TTHA0621 is reported at 1.93 Å resolution. The asymmetric unit contained four NCS molecules related by 222 noncrystallographic symmetry, in which the formation of intact dimers may be functionally important. The cofactor pyridoxal 5′-phosphate (PLP) binds to the protein in the large cleft formed by the N-terminal and C-terminal domains of TTHA0621. The high structural similarity and the conservation of the functional residues in the catalytic region compared with 4-amino-4-deoxychorismate lyase (PabC; EC 4.1.3.38) from Escherichia coli suggest that the TTHA0621 protein may also possess 4-amino-4-deoxychorismate lyase activity.
doi:10.1107/S1744309109050052
PMCID: PMC2802870  PMID: 20054118
4-amino-4-deoxychorismate lyase; pyridoxal 5′-phosphate; folate biosynthesis; Thermus thermophilus HB8
18.  A commensal symbiotic interrelationship for the growth of Symbiobacterium toebii with its partner bacterium, Geobacillus toebii 
BMC Research Notes  2011;4:437.
Background
Symbiobacterium toebii is a commensal symbiotic thermophile that absolutely requires its partner bacterium Geobacillus toebii for growth. Despite development of an independent cultivation method using cell-free extracts, the growth of Symbiobacterium remains unknown due to our poor understanding of the symbiotic relationship with its partner bacterium. Here, we investigated the interrelationship between these two bacteria for growth of S. toebii using different cell-free extracts of G. toebii.
Results
Symbiobacterium toebii growth-supporting factors were constitutively produced through almost all growth phases and under different oxygen tensions in G. toebii, indicating that the factor may be essential components for growth of G. toebii as well as S. toebii. The growing conditions of G. toebii under different oxygen tension dramatically affected to the initial growth of S. toebii and the retarded lag phase was completely shortened by reducing agent, L-cysteine indicating an evidence of commensal interaction of microaerobic and anaerobic bacterium S. toebii with a facultative aerobic bacterium G. toebii. In addition, the growth curve of S. toebii showed a dependency on the protein concentration of cell-free extracts of G. toebii, demonstrating that the G. toebii-derived factors have nutrient-like characters but not quorum-sensing characters.
Conclusions
Not only the consistent existence of the factor in G. toebii during all growth stages and under different oxygen tensions but also the concentration dependency of the factor for proliferation and optimal growth of S. toebii, suggests that an important biosynthetic machinery lacks in S. toebii during evolution. The commensal symbiotic bacterium, S. toebii uptakes certain ubiquitous and essential compound for its growth from environment or neighboring bacteria that shares the equivalent compounds. Moreover, G. toebii grown under aerobic condition shortened the lag phase of S. toebii under anaerobic and microaerobic conditions, suggests a possible commensal interaction that G. toebii scavengers ROS/RNS species and helps the initial growth of S. toebii.
doi:10.1186/1756-0500-4-437
PMCID: PMC3208422  PMID: 22023733
Symbiobacterium toebii; Geobacillus toebii; Bacterial symbiosis; Growth-supporting factor; Commensalism
19.  New structural insights and molecular-modelling studies of 4-methyl-5-β-hydroxyethylthiazole kinase from Pyrococcus horikoshii OT3 (PhThiK) 
The first crystal structure of 4-methyl-5-β-hydroxyethylthiazole kinase from an archaeon (P. horikoshii OT3) has been determined at 1.85 Å resolution. Comparative analyses of sequences and structures and modelling studies are presented.
4-Methyl-5-β-hydroxyethylthiazole kinase (ThiK) catalyses the phosphorylation of the hydroxyl group of 4-methyl-5-β-hydroxyethylthiazole. This work reports the first crystal structure of an archaeal ThiK: that from Pyrococcus horikoshii OT3 (PhThiK) at 1.85 Å resolution with a phosphate ion occupying the position of the β-phosphate of the nucleotide. The topology of this enzyme shows the typical ribokinase fold of an α/β protein. The overall structure of PhThiK is similar to those of Bacillus subtilis ThiK (BsThiK) and Enterococcus faecalis V583 ThiK (EfThiK). Sequence analysis of ThiK enzymes from various sources indicated that three-quarters of the residues involved in interfacial regions are conserved. It also revealed that the amino-acid residues in the nucleotide-binding, magnesium ion-binding and substrate-binding sites are conserved. Binding of the nucleotide and substrate to the ThiK enzyme do not influence the quaternary association (trimer) as revealed by the crystal structure of PhThiK.
doi:10.1107/S1744309109036033
PMCID: PMC2765881  PMID: 19851002
4-methyl-5-β-hydroxyethylthiazole kinase; Pyrococcus horikoshii OT3; molecular modelling
20.  Crystallization and preliminary X-ray analysis of 4-­pyridoxolactonase from Mesorhizobium loti  
Recombinant 4-pyridoxolactonase from M. loti MAFF303099 was crystallized in two forms and diffraction data were collected to 2.0 and 1.9 Å resolution, respectively.
4-Pyridoxolactonase from Mesorhizobium loti MAFF303099 has been overexpressed in Escherichia coli. The recombinant enzyme was purified and was crystallized by the sitting-drop vapour-diffusion method using PEG 4000 and ammonium sulfate as precipitants. Crystals of the free enzyme (form I) and of the 5-pyridoxolactone-bound enzyme (form II) grew under these conditions. Crystals of form I diffracted to 2.0 Å resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 77.93, b = 38.88, c = 81.60 Å, β = 117.33°. Crystals of form II diffracted to 1.9 Å resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 86.24, b = 39.35, c = 82.68 Å, β = 118.02°. The calculated V M values suggested that the asymmetric unit contains one molecule in both crystal forms.
doi:10.1107/S1744309109028772
PMCID: PMC2795592  PMID: 19724124
4-pyridoxolactonase; Mesorhizobium loti; pyridoxine-degradation pathway
21.  Structure and Function of the Small MutS-Related Domain 
MutS family proteins are widely distributed in almost all organisms from bacteria to human and play central roles in various DNA transactions such as DNA mismatch repair and recombinational events. The small MutS-related (Smr) domain was originally found in the C-terminal domain of an antirecombination protein, MutS2, a member of the MutS family. MutS2 is thought to suppress homologous recombination by endonucleolytic resolution of early intermediates in the process. The endonuclease activity of MutS2 is derived from the Smr domain. Interestingly, sequences homologous to the Smr domain are abundant in a variety of proteins other than MutS2 and can be classified into 3 subfamilies. Recently, the tertiary structures and endonuclease activities of all 3 Smr subfamilies were reported. In this paper, we review the biochemical characteristics and structures of the Smr domains as well as cellular functions of the Smr-containing proteins.
doi:10.4061/2011/691735
PMCID: PMC3200294  PMID: 22091410
22.  Inactivation of the DNA Repair Genes mutS, mutL or the Anti-Recombination Gene mutS2 Leads to Activation of Vitamin B1 Biosynthesis Genes 
PLoS ONE  2011;6(4):e19053.
Oxidative stress generates harmful reactive oxygen species (ROS) that attack biomolecules including DNA. In living cells, there are several mechanisms for detoxifying ROS and repairing oxidatively-damaged DNA. In this study, transcriptomic analyses clarified that disruption of DNA repair genes mutS and mutL, or the anti-recombination gene mutS2, in Thermus thermophilus HB8, induces the biosynthesis pathway for vitamin B1, which can serve as an ROS scavenger. In addition, disruption of mutS, mutL, or mutS2 resulted in an increased rate of oxidative stress-induced mutagenesis. Co-immunoprecipitation and pull-down experiments revealed previously-unknown interactions of MutS2 with MutS and MutL, indicating that these proteins cooperatively participate in the repair of oxidatively damaged DNA. These results suggested that bacterial cells sense the accumulation of oxidative DNA damage or absence of DNA repair activity, and signal the information to the transcriptional regulation machinery for an ROS-detoxifying system.
doi:10.1371/journal.pone.0019053
PMCID: PMC3084264  PMID: 21552516
23.  Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems 
Journal of Nucleic Acids  2010;2010:179594.
DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8.
doi:10.4061/2010/179594
PMCID: PMC2957137  PMID: 20981145
24.  Structure of d-alanine-d-alanine ligase from Thermus thermophilus HB8: cumulative conformational change and enzyme–ligand interactions 
The structures of five forms of d-alanine-d-alanine ligase from T. thermophilus HB8 showed a cumulative conformational change of the molecular structure through the induced rotation of the central domain in concert with a local conformational change of three loops. The active-site structures shed light on the catalytic mechanism and the roles of the conformational change.
d-Alanine-d-alanine ligase (Ddl) is one of the key enzymes in peptidoglycan biosynthesis and is an important target for drug discovery. The enzyme catalyzes the condensation of two d-­Ala molecules using ATP to produce d-Ala-d-Ala, which is the terminal peptide of a peptidoglycan monomer. The structures of five forms of the enzyme from Thermus thermophilus HB8 (TtDdl) were determined: unliganded TtDdl (2.3 Å resolution), TtDdl–adenylyl imidodiphosphate (2.6 Å), TtDdl–ADP (2.2 Å), TtDdl–ADP–d-Ala (1.9 Å) and TtDdl–ATP–d-Ala-d-Ala (2.3 Å). The central domain rotates as a rigid body towards the active site in a cumulative manner in concert with the local conformational change of three flexible loops depending upon substrate or product binding, resulting in an overall structural change from the open to the closed form through semi-open and semi-closed forms. Reaction-intermediate models were simulated using TtDdl-complex structures and other Ddl structures previously determined by X-ray methods. The catalytic process accompanied by the cumulative conformational change has been elucidated based on the intermediate models in order to provide new insights regarding the details of the catalytic mechanism.
doi:10.1107/S0907444909029710
PMCID: PMC2756165  PMID: 19770507
d-alanine-d-alanine ligase; Thermus thermophilus HB8; catalytic mechanism; conformational change
25.  Structure of a putative molybdenum-cofactor biosynthesis protein C (MoaC) from Sulfolobus tokodaii (ST0472) 
The crystal structure of a putative molybdenum-cofactor biosynthesis protein C (MoaC) from S. tokodaii (ST0472) was determined at 2.2 Å resolution.
The crystal structure of a putative molybdenum-cofactor (Moco) biosynthesis protein C (MoaC) from Sulfolobus tokodaii (ST0472) was determined at 2.2 Å resolution. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 123.31, b = 78.58, c = 112.67 Å, β = 118.1°. The structure was solved by molecular replacement using the structure of Escherichia coli MoaC as the probe model. The asymmetric unit is composed of a hexamer arranged as a trimer of dimers with noncrystallographic 32 symmetry. The structure of ST0472 is very similar to that of E. coli MoaC; however, in the ST0472 protein an additional loop formed by the insertion of seven residues participates in intermonomer interactions and the new structure also reveals the formation of an interdimer β-sheet. These features may contribute to the stability of the oligomeric state.
doi:10.1107/S174430910801590X
PMCID: PMC2443982  PMID: 18607082
molybdenum cofactors; Moco-biosynthesis protein; Sulfolobus tokodaii

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