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The first step in the molybdenum cofactor (Moco) biosynthesis pathway involves the conversion of guanosine triphosphate (GTP) to precursor Z by two proteins (MoaA and MoaC). MoaA belongs to the S-adenosylmethionine-dependent radical enzyme superfamily and is believed to generate protein and/or substrate radicals by reductive cleavage of S-adenosylmethionine using an Fe-S cluster. MoaC has been suggested to catalyze the release of pyrophosphate and the formation of the cyclic phosphate of precursor Z. However, structural evidence showing the binding of a substrate-like molecule to MoaC is not available. Here, apo and GTP-bound crystal structures of MoaC from Thermus thermophilus HB8 are reported. Furthermore, isothermal titration calorimetry experiments have been carried out in order to obtain thermodynamic parameters for the protein-ligand interactions. In addition, molecular-dynamics (MD) simulations have been carried out on the protein-ligand complex of known structure and on models of relevant complexes for which X-ray structures are not available. The biophysical, structural and MD results reveal the residues that are involved in substrate binding and help in speculating upon a possible mechanism.

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.

The aconitase family of hydro-lyase enzymes includes three classes of proteins that catalyze the isomerization of alpha-hydroxy acids to beta-hydroxy acids. Besides aconitase, isopropylmalate isomerase (IPMI) proteins specifically catalyze the isomerization of alpha,beta-dicarboxylates with hydrophobic gamma-chain groups, and homoaconitase (HACN) proteins catalyze the isomerization of tricarboxylates with variable chain length gamma-carboxylate groups. These enzymes' stereospecific hydro-lyase activities make them attractive catalysts to produce diastereomers from unsaturated precursors. However, sequence similarity and convergent evolution among these proteins lead to widespread misannotation and uncertainty about gene function. To find the substrate specificity determinants of homologous IPMI and HACN proteins from Methanocaldococcus jannaschii, the small-subunit HACN protein (MJ1271) was crystallized for X-ray diffraction. The structural model showed characteristic residues in a flexible loop region between alpha2 and alpha3 that distinguish HACN from IPMI and aconitase proteins. Site-directed mutagenesis of MJ1271 produced loop-region variant proteins that were reconstituted with wild-type MJ1003 large-subunit protein. The heteromers formed promiscuous hydro-lyases with reduced activity but broader substrate specificity. Both R26K and R26V variants formed relatively efficient IPMI enzymes, while the T27A variant had uniformly lower specificity constants for both IPMI and HACN substrates. The R26V T27Y variant resembles the MJ1277 IPMI small subunit in its flexible loop sequence but demonstrated the broad substrate specificity of the R26V variant. These mutations may reverse the evolution of HACN activity from an ancestral IPMI gene, demonstrating the evolutionary potential for promiscuity in hydro-lyase enzymes. Understanding these specificity determinants enables the functional reannotation of paralogous HACN and IPMI genes in numerous genome sequences. These structural and kinetic results will help to engineer new stereospecific hydro-lyase enzymes for chemoenzymatic syntheses.

RecJ is a single-stranded DNA (ssDNA)-specific 5'-3' exonuclease that plays an important role in DNA repair and recombination. To elucidate how RecJ achieves its high specificity for ssDNA, we determined the entire structures of RecJ both in a ligand-free form and in a complex with Mn(2+) or Mg(2+) by x-ray crystallography. The entire RecJ consists of four domains that form a molecule with an O-like structure. One of two newly identified domains had structural similarities to an oligonucleotide/oligosaccharide-binding (OB) fold. The OB fold domain alone could bind to DNA, indicating that this domain is a novel member of the OB fold superfamily. The truncated RecJ containing only the core domain exhibited much lower affinity for the ssDNA substrate compared with intact RecJ. These results support the hypothesis that these structural features allow specific binding of RecJ to ssDNA. In addition, the structure of the RecJ-Mn(2+) complex suggests that the hydrolysis reaction catalyzed by RecJ proceeds through a two-metal ion mechanism.

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 P3(1), with unit-cell parameters a = b = 95.62, c = 149.13 A. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (V(M)) of 2.3 A(3) 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.

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 A. The crystal belonged to space group P3(2), with unit-cell parameters a = b = 142.19, c = 123.34 A. The structure contained two dimers in the asymmetric unit and was solved by the MR-SAD approach.

Asymmetric diadenosine tetraphosphate (Ap(4)A) hydrolases degrade the metabolite Ap(4)A back into ATP and AMP. The three-dimensional crystal structure of Ap(4)A hydrolase (16 kDa) from Aquifex aeolicus has been determined in free and ATP-bound forms at 1.8 and 1.95 A resolution, respectively. The overall three-dimensional crystal structure of the enzyme shows an alphabetaalpha-sandwich architecture with a characteristic loop adjacent to the catalytic site of the protein molecule. The ATP molecule is bound in the primary active site and the adenine moiety of the nucleotide binds in a ring-stacking arrangement equivalent to that observed in the X-ray structure of Ap(4)A hydrolase from Caenorhabditis elegans. Binding of ATP in the active site induces local conformational changes which may have important implications in the mechanism of substrate recognition in this class of enzymes. Furthermore, two invariant water molecules have been identified and their possible structural and/or functional roles are discussed. In addition, modelling of the substrate molecule at the primary active site of the enzyme suggests a possible path for entry and/or exit of the substrate and/or product molecule.

The clustered regularly interspaced short palindromic repeat (CRISPR) systems composed of DNA direct repeats designated as CRISPRs and several CRISPR-associated (cas) genes, which are present in many prokaryotic genomes, make up a host defense system against invading foreign replicons such as phages. In order to investigate the altered expression profiles of the systems after phage infection using a model organism, Thermus thermophilus HB8, which has 12 CRISPR loci, genome-wide transcription profiling of the strain infected with lytic phage PhiYS40 was performed by DNA microarray analysis. Significant alteration of overall mRNA expression gradually increased during infection (i.e., from the eclipse period to the period of host cell lysis). Interestingly, the expression of most cAMP receptor protein (CRP)-regulated genes, including two CRISPR-associated (cas) operons, was most markedly up-regulated, especially around the beginning of host cell lysis, although up-regulation of the crp gene was not observed. The expression of the CRP-regulated genes was less up-regulated in a crp-deficient strain than in the wild type. Thus, it is suggested that cAMP is a signaling molecule that transmits information on phage infection to CRP to up-regulate these genes. On the other hand, the expression of several cas genes and that of CRISPRs were up-regulated independent of CRP, suggesting the involvement of unidentified regulatory factor(s) induced by phage infection. On analysis of the expression profile of the entire genome, we could speculate that upon phage infection, the signal was transmitted to the cells, with host response systems including CRISPR defense systems being activated, while the overall efficiencies of transcription, translation, and metabolism in the cells decreased. These findings will facilitate understanding of the host response mechanism following phage infection., (Copyright 2009 Elsevier Inc. All rights reserved.)

In many microorganisms, menaquinone is an essential lipid-soluble electron carrier. Recently, an alternative menaquinone biosynthetic pathway was found in some microorganisms [Hiratsuka, T., Furihata, K., Ishikawa, J., Yamashita, H., Itoh, N., Seto, H., Dairi, T., 2008. An alternative menaquinone biosynthetic pathway operating in microorganisms. Science 321, 1670-1673]. Here, we report the 1.55 A crystal structure of MqnD (TTHA1568) from Thermus thermophilus HB8, an enzyme within the alternative menaquinone biosynthetic pathway. The structure comprises two domains with alpha/beta structures, a large domain and a small domain. L(+)-Tartaric acid was bound to the pocket between the two domains, suggesting that this pocket is a putative active site. The conserved glycine residues at positions 78, 80 and 82 seem to act as hinges, allowing the substrate to access the pocket. Highly conserved residues, such as Asp14, Asp38, Asn43, Ser57, Thr107, Ile144, His145, Glu146, Leu176 and Tyr234, are located at this pocket, suggesting that these residues are involved in substrate binding and/or catalysis, and especially, His145 could function as a catalytic base. Since humans and their commensal intestinal bacteria, including lactobacilli, lack the alternative menaquinone biosynthetic pathway, this enzyme in pathogenic species, such as Helicobacter pylori and Campylobacter jejuni, is an attractive target for the development of chemotherapeutics. This high-resolution structure may contribute toward the development of its inhibitors.

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 P2(1) to 1.78 A 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.

The structure of a probable Mo-cofactor biosynthesis protein B from Sulfolobus tokodaii, belonging to space group P6(4)22 with unit-cell parameters a = b = 136.68, c = 210.52 A, was solved by molecular replacement to a resolution of 1.9 A 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.

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 A 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.

The crystal structure of D-lactate dehydrogenase from Aquifex aeolicus (aq_727) was determined to 2.12 A resolution in space group P2(1)2(1)2(1), with unit-cell parameters a = 90.94, b = 94.43, c = 188.85 A. 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.

The structure of TM1254, a putative beta-phosphoglucomutase from T. maritima, was determined to 1.74 A resolution in a high-throughput structural genomics programme. Diffraction data were obtained from crystals belonging to space group P22(1)2(1), with unit-cell parameters a = 48.16, b = 66.70, c = 83.80 A, 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.

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 A 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.

4-Methyl-5-beta-hydroxyethylthiazole kinase (ThiK) catalyses the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole. This work reports the first crystal structure of an archaeal ThiK: that from Pyrococcus horikoshii OT3 (PhThiK) at 1.85 A resolution with a phosphate ion occupying the position of the beta-phosphate of the nucleotide. The topology of this enzyme shows the typical ribokinase fold of an alpha/beta 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.

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 A resolution), TtDdl-adenylyl imidodiphosphate (2.6 A), TtDdl-ADP (2.2 A), TtDdl-ADP-D-Ala (1.9 A) and TtDdl-ATP-D-Ala-D-Ala (2.3 A). 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.

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 A resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 77.93, b = 38.88, c = 81.60 A, beta = 117.33 degrees. Crystals of form II diffracted to 1.9 A resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 86.24, b = 39.35, c = 82.68 A, beta = 118.02 degrees. The calculated V(M) values suggested that the asymmetric unit contains one molecule in both crystal forms.

TTHA1846 is a conserved hypothetical protein from Thermus thermophilus HB8 with a molecular mass of 15.1 kDa that belongs to the thioesterase superfamily (Pfam 03061). Here, the 1.9 A resolution crystal structure of TTHA1846 from T. thermophilus is reported. The crystal structure is a dimer of dimers. Each subunit adopts the so-called hot-dog fold composed of five antiparallel beta-strands flanked on one side by a rather long alpha-helix and shares structural similarity to a number of thioesterases. Unexpectedly, TTHA1846 binds one metal ion and one ligand per subunit. The ligand density was modelled as coenzyme A (CoA). Its structure was confirmed by MALDI-TOF mass spectrometry and electron-density mapping. X-ray absorption fine-structure (XAFS) measurement of the crystal unambiguously characterized the metal ion as zinc. The zinc ion is tetrahedrally coordinated by the side chains of Asp18, His22 and Glu50 and the CoA thiol group. This is the first structural report of the interaction of CoA with a zinc ion. From structural and database analyses, it was speculated that the zinc ion may play an inhibitory role in the enzymatic activity.

A major bacterial alarmone, guanosine 3',5'-bispyrophosphate (ppGpp), controls cellular growth under conditions of nutritional starvation. For most bacteria, intracellular ppGpp levels are tightly controlled by the synthesis/degradation cycle of RelA and SpoT activities. This study shows a novel ppGpp regulatory protein governing the cellular growth of Thermus thermophilus, Ndx8, a member of the Nudix pyrophosphatase family that degrades ppGpp to yield guanosine 3',5'-bisphosphate. The ndx8-null mutant strain exhibited early stage growth arrest accompanied by the stationary phase-specific morphologies and global transcriptional modulation under nutritionally defined conditions. Several possible substrate compounds of Ndx8, which specifically accumulated in the ndx8 mutant cells, were identified by employing a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach. Among them, the hydrolytic activity of Ndx8 for ppGpp was significant not only in vitro but also in vivo. Finally, the elimination of ppGpp synthetic activity suppressed the observed phenotype of the ndx8 mutation, suggesting that the function of Ndx8 as a growth regulator is involved in ppGpp accumulation, which is thought to act as a trigger of the growth phase transition. These results suggest a novel mechanism of ppGpp-mediated growth control by the functional relay between Ndx8 and SpoT activity as ppGpp scavengers.

Nucleotide hydrolases are known to hydrolyze not only noncanonical dNTPs to reduce the risk of mutation, but also canonical dNTPs to maintain the dNTP concentrations in the cell. dGTP triphosphohydrolase from Escherichia coli is known as an enzyme that hydrolyzes dGTP. Recently, we identified a triphosphohydrolase from Thermus thermophilus HB8 that hydrolyzes all canonical dNTPs through a complex activation mechanism. These dNTP triphosphohydrolases are widely distributed in eubacteria, but it is difficult to predict whether they possess hydrolytic activity for dGTP or dNTP. To obtain information concerning the structure-function relationships of this protein family, we characterized two dNTP triphosphohydrolases, PA1124 and PA3043, from Pseudomonas aeruginosa. Molecular phylogenic analysis showed that dNTP triphosphohydrolases can be classified into three groups. Experimentally, PA1124 had a preference for dGTP, similar to the E. coli enzyme, whereas PA3043 displayed a broad substrate specificity. Both enzymes hydrolyzed substrates in the absence of additional dNTP as an activating effector. These kinetic data suggest that PA3043 is a novel type distinct from both the E. coli and T. thermophilus enzymes. On the basis of these results, we propose that the dNTP triphosphohydrolase family should be classified into at least three subfamilies.

The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3'-5' exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg(2+)/Mn(2+)-dependent DNA/RNA polymerase, Mn(2+)-dependent 3'-5' exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn(2+)-dependent 3'-5' exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3'-5' exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'-5' exonuclease activity.

We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-L-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-L-tyrosine. The 1.7 A crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-L-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-L-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Angstroms resolutions by SAD phasing at CuK alpha and CrK alpha wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation.

The expression and solubilization of insoluble proteins have been facilitated by the introduction of protein tags. In our analyses of viral protein R (Vpr) of human immunodeficiency virus 1 (HIV-1), however, several conventional tag proteins enhanced its expression but failed to solubilize it. Therefore, we decided to explore whether proteins derived from Thermus thermophilus HB8 (T. th.), a highly heat-stable bacterium, could be used as tag proteins to enhance the solubilization of Vpr. Based on the data accumulated during the recent structural genomics project of T. th., we selected 15 T. th. proteins with high expression levels and solubilities. From this group, we identified a T. th. tag protein that expressed Vpr in a soluble form. Furthermore, two T. th. tag proteins, including the identified one, were found to solubilize the extremely insoluble membrane-spanning domain of the envelope protein of HIV-1. When green fluorescent protein (GFP) was used as a passenger protein of T. th. tags, the brightness and stability of GFP were similar to those of untagged GFP, suggesting that the T. th. tags do not negatively affect the function of the passenger protein. Thus, data of structural genomics can be applied to generate a customized versatile protein tag for protein analyses.

DNA recombination events need to be strictly regulated, because an increase in the recombinational frequency causes unfavorable alteration of genetic information. Recent studies revealed the existence of a novel anti-recombination enzyme, MutS2. However, the mechanism by which MutS2 inhibits homologous recombination has been unknown. Previously, we found that Thermus thermophilus MutS2 (ttMutS2) harbors an endonuclease activity and that this activity is confined to the C-terminal domain, whose amino acid sequence is widely conserved in a variety of proteins with unknown function from almost all organisms ranging from bacteria to man. In this study, we determined the crystal structure of the ttMutS2 endonuclease domain at 1.7-angstroms resolution, which resembles the structure of the DNase I-like catalytic domain of Escherichia coli RNase E, a sequence-nonspecific endonuclease. The N-terminal domain of ttMutS2, however, recognized branched DNA structures, including the Holliday junction and D-loop structure, a primary intermediate in homologous recombination. The full-length of ttMutS2 digested the branched DNA structures at the junction. These results indicate that ttMutS2 suppresses homologous recombination through a novel mechanism involving resolution of early intermediates.

The TTHB212 gene from extremely thermophilic bacterium Thermus thermophilus HB8 forms an operon with the upstream sigE gene encoding an extracytoplasmic function sigma factor, sigma(E), the sole alternative sigma factor of this strain, on megaplasmid pTT27. The TTHB212 gene encodes a poorly conserved protein, which has been predicted to be a transmembrane one with N-terminal intracellular and C-terminal extracytoplasmic domains. The N-terminal domain of TTHB212 protein (TTHB212N) prevented sigma(E) from binding to RNA polymerase (RNAP) core enzyme in vitro, and TTHB212N bound sigma(E) in a molar ratio of 1:1 when both proteins were co-expressed in Escherichia coli. Furthermore, TTHB212N inhibited the transcription activity of RNAP-sigma(E) holoenzyme, but not that of the RNAP-sigma(A) one, in vitro. The expression of several genes that are under the control of sigma(E) was increased in a TTHB212 gene-disruptant strain. Thus, TTHB212 protein was identified as an anti-sigma(E) factor. These findings indicate that T. thermophilus HB8 has a regulatory system involving sigma(E) and anti-sigma(E) factors.

The crystal structure of a hypothetical protein ST2348 (GI: 47118305) from the hyperthermophilic bacteria Sulfolobus tokodaii has been determined using X-ray crystallography. The protein consists of two CBS (cystathione beta synthase) domains, whose function has been analyzed and reported here. PSI-BLAST shows a conservation of this domain in about 100 proteins in various species. However, none of the close homologs of ST2348 have been functionally characterized so far. Structure and sequence comparison of ST2348 with human AMP-kinase gamma1 subunit and the CBS domain pair of bacterial IMP dehydrogenase is suggestive of its binding to AMP and ATP. A highly conserved residue Asp118, located in a negatively charged patch near the ligand binding cleft, could serve as a site for phosphorylation similar to that found in the chemotatic signal protein CheY and thereby ST2348 can function as a signal transduction molecule.

Carbonic anhydrases are zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. Three crystal structures of gamma-class carbonic anhydrase (one of which is bound to a bicarbonate molecule) from the aerobic OT3 strain of the hyperthermophilic archeon Pyrococcus horikoshii have been solved by molecular replacement in space group F4(1)32. The asymmetric unit contains a monomer of 173 amino acids and a catalytic Zn2+ ion. The protein fold is a regular prism formed by a left-handed beta-helix, similar to previously reported structures. The active-site Zn2+ ion located at the interface between the two monomers is bound to three histidyl residues and a water molecule in a tetrahedral fashion. In addition to the 20 beta-strands comprising the beta-helix, there is also a long C-terminal alpha-helix. For the first time, Ca2+ ions have been observed in addition to the catalytic Zn2+ ion. It is hypothesized that Tyr159 (which corresponds to the catalytically important Asn202 in previously reported structures) utilizes C-H...pi interactions to fulfill its functions. This study may shed light on the catalytic mechanism of the enzyme and throw open new questions on the mechanism of product removal in carbonic anhydrases.

Thermus thermophilus SdrP is one of four cyclic AMP receptor protein (CRP)/fumarate and nitrate reduction regulator (FNR) family proteins from the extremely thermophilic bacterium T. thermophilus HB8. Expression of sdrP mRNA increased in the stationary phase during cultivation at 70 degrees C. Although the sdrP gene was non-essential, an sdrP-deficient strain showed growth defects, particularly when grown in a synthetic medium, and increased sensitivity to disulphide stress. The expression of several genes was altered in the sdrP disruptant. Among them, we found eight SdrP-dependent promoters using in vitro transcription assays. A predicted SdrP binding site similar to that recognized by Escherichia coli CRP was found upstream of each SdrP-dependent promoter. In the wild-type strain, expression of these eight genes tended to increase upon entry into the stationary phase. Transcriptional activation in vitro was independent of any added effector molecule. The hypothesis that apo-SdrP is the active form of the protein was supported by the observation that the three-dimensional structure of apo-SdrP is similar to that of the DNA-binding form of E. coli CRP. Based on the properties of the SdrP-regulated genes found in this study, it is speculated that SdrP is involved in nutrient and energy supply, redox control, and polyadenylation of mRNA.

The major damage to DNA caused by alkylating agents involves the formation of O6-methylguanine (O6-meG). Almost all species possess O6-methylguanine-DNA-methyltransferase (Ogt) to repair such damage. Ogt repairs O6-meG lesions in DNA by stoichiometric transfer of the methyl group to a cysteine residue in its active site (PCHR). Thermus thermophilus HB8 has an Ogt homologue, TTHA1564, but in this case an alanine residue replaces cysteine in the putative active site. To reveal the possible function of TTHA1564 in processing O6-meG-containing DNA, we characterized the biochemical properties of TTHA1564. No methyltransferase activity for synthetic O6-meG-containing DNA could be detected, indicating TTHA1564 is an alkyltransferase-like protein. Nevertheless, gel shift assays showed that TTHA1564 can bind to DNA containing O6-meG with higher affinity (9-fold) than normal (unmethylated) DNA. Experiments using a fluorescent oligonucleotide suggested that TTHA1564 recognizes O6-meG in DNA using the same mechanism as other Ogts. We then investigated whether TTHA1564 functions as a damage sensor. Pull-down assays identified 20 proteins, including a nucleotide excision repair protein UvrA, which interacts with TTHA1564. Interaction of TTHA1564 with UvrA was confirmed using a surface plasmon resonance assay. These results suggest the possible involvement of TTHA1564 in DNA repair pathways.

The crystal structure of a putative molybdenum-cofactor (Moco) biosynthesis protein C (MoaC) from Sulfolobus tokodaii (ST0472) was determined at 2.2 A resolution. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 123.31, b = 78.58, c = 112.67 A, beta = 118.1 degrees . 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 beta-sheet. These features may contribute to the stability of the oligomeric state.

Seryl-tRNA synthetase (SerRS) catalyzes the ligation of serine to the 3'-end of serine tRNA (tRNA(Ser)), which is typical of the type-2 tRNAs characterized by a long extra arm. The SerRSs are divided into two types, the archaeal/eukaryal and bacterial types. In this study, we solved the crystal structures of the SerRS from the archaeon Pyrococcus horikoshii bound with 5'-O-[N-(L-seryl)-sulfamoyl]-adenosine at 2.6 A and with ATP at 2.8 A, as well as in the apo form at 3.0 A. P. horikoshii SerRS recognizes the seryl and adenylate moieties in a manner similar to those of the bacterial and mitochondrial SerRSs from Thermus thermophilus and Bos taurus, respectively, but different from that of the unusual SerRS from the methanogenic archaeon Methanosarcina barkeri. P. horikoshii SerRS efficiently aminoacylated not only P. horikoshii tRNA(Ser) but also bacterial tRNA(Ser)s from T. thermophilus and Escherichia coli. Models of P. horikoshii SerRS bound with the T. thermophilus and P. horikoshii tRNA(Ser)s suggested that the helical domain of P. horikoshii SerRS is involved in the extra arm binding. This region of P. horikoshii SerRS has additional basic residues as compared with T. thermophilus SerRS, and a Trp residue specific to the archaeal/eukaryal SerRSs. Mutational analyses revealed that the basic and Trp residues are important for tRNA aminoacylation. P. horikoshii SerRS has the archaea-specific insertion, which collaborates with the core domain to form a basic channel leading to the active site. Two sulfate ions are bound to the channel, suggesting that the tRNA 3' region might bind to the channel.

It was essential for the structural genomics of Thermus thermophilus HB8 to efficiently crystallize a number of proteins. To this end, three conventional robots, an HTS-80 (sitting-drop vapour diffusion), a Crystal Finder (hanging-drop vapour diffusion) and a TERA (modified microbatch) robot, were subjected to a crystallization condition screening test involving 18 proteins from T. thermophilus HB8. In addition, a TOPAZ (microfluidic free-interface diffusion) designed specifically for initial screening was also briefly examined. The number of diffraction-quality crystals and the time of appearance of crystals increased in the order HTS-80, Crystal Finder, TERA. With the HTS-80 and Crystal Finder, the time of appearance was short and the rate of salt crystallization was low. With the TERA, the number of diffraction-quality crystals was high, while the time of appearance was long and the rate of salt crystallization was relatively high. For the protein samples exhibiting low crystallization success rates, there were few crystallization conditions that were common to the robots used. In some cases, the success rate depended greatly on the robot used. The TOPAZ showed the shortest time of appearance and the highest success rate, although the crystals obtained were too small for diffraction studies. These results showed that the combined use of different robots significantly increases the chance of obtaining crystals, especially for proteins exhibiting low crystallization success rates. The structures of 360 of 944 purified proteins have been successfully determined through the combined use of an HTS-80 and a TERA.

Pyruvate dehydrogenase (PDH), branched-chain 2-oxo acid dehydrogenase (BCDH) and 2-oxoglutarate dehydrogenase (OGDH) are multienzyme complexes that play crucial roles in several common metabolic pathways. These enzymes belong to a family of 2-oxo acid dehydrogenase complexes that contain multiple copies of three different components (E1, E2 and E3). For the Thermus thermophilus enzymes, depending on its substrate specificity (pyruvate, branched-chain 2-oxo acid or 2-oxoglutarate), each complex has distinctive E1 (E1p, E1b or E1o) and E2 (E2p, E2b or E2o) components and one of the two possible E3 components (E3b and E3o). (The suffixes, p, b and o identify their respective enzymes, PDH, BCDH and OGDH.) Our biochemical characterization demonstrates that only three specific E3*E2 complexes can form (E3b*E2p, E3b*E2b and E3o*E2o). X-ray analyses of complexes formed between the E3 components and the peripheral subunit-binding domains (PSBDs), derived from the corresponding E2-binding partners, reveal that E3b interacts with E2p and E2b in essentially the same manner as observed for Geobacillus stearothermophilus E3*E2p, whereas E3o interacts with E2o in a novel fashion. The buried intermolecular surfaces of the E3b*PSBDp/b and E3o*PSBDo complexes differ in size, shape and charge distribution and thus, these differences presumably confer the binding specificities for the complexes.

Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR-neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH-pi interactions in the Ls-AChBP-CTD complex than in the Ls-AChBP-IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs.

DNA mismatch repair corrects mismatched base pairs mainly caused by replication error. Recent studies revealed that human MutL endonuclease, hPMS2, plays an essential role in the repair. However, there has been little biochemical analysis of the MutL endonuclease. In particular, it is unknown for what the MutL utilizes ATP binding and hydrolyzing activity. Here we report the detailed functional analysis of Thermus thermophilus MutL (ttMutL). ttMutL exhibited an endonuclease activity that decreased on alteration of Asp-364 in ttMutL to Asn. The biochemical characteristics of ttMutL were significantly affected on ATP binding, which suppressed nonspecific DNA digestion and promoted the mismatch- and MutS-dependent DNA binding. The inactivation of the cysteinyl residues in the C-terminal domain resulted in the perturbation in ATP-dependent regulation of the endonuclease activity, although the ATP-binding motif is located in the N-terminal domain. Complementation experiments revealed that the endonuclease activity of ttMutL and its regulation by ATP binding are necessary for DNA repair in vivo.

Novel groups of uncultivable anaerobic thermophiles were isolated from compost by enrichment cultivation in medium with a cell-free extract of Geobacillus toebii. The cell-free extract of G. toebii provided the medium with growth-supporting factors (GSF) needed to cultivate the previously uncultured microorganisms. Twenty-nine GSF-requiring candidates were successfully cultivated, and 16 isolated novel bacterial strains were classified into three different groups of uncultivable bacteria. The similarity among these 16 isolates and a phylogenetic analysis using 16S rRNA gene sequences revealed that these GSF-requiring strains represented novel groups within the family Clostridiaceae.

The YdjC-family protein is widely distributed, from human to bacteria, but so far no three-dimensional structure and functional analysis of this family of proteins has been reported. We determined the three-dimensional structure of the YdjC homolog TTHB029 at a resolution of 2.9A. The overall structure of the monomer consists of (betaalpha)-barrel fold forming a homodimer. Asp21, His60, and His127 residues coordinate to Mg(2+) as a possible active site. TTHB029 shows structural similarity to the peptidoglycan N-acetylglucosamine deacetylase from Streptococcus pneumoniae (SpPgdA). The active site groove of SpPgdA includes the Zn(2+) coordinated to Asp276, His326, and His330. Despite the low sequence identity, metal-binding residues of Asp-His-His were conserved among the two enzymes. There were definitive differences, however, in that one of the histidines of the metal-binding site was substituted for the other histidine located on the other loop. Moreover, these important metal-binding residues and the residues of the presumed active site are fully conserved in YdjC-family protein.

Deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase) from Thermus thermophilus HB8 (TTHB8) hydrolyzes wide variety of dNTPs to deoxyribonucleoside and inorganic triphosphate in magnesium-dependent manner. In this paper, we assess the specificity for various metal ions and of the dNTP triphosphohydrolase activity of the dNTPase from TTHB8. Manganese and cobalt ions more effectively induced the activity for dNTPs than magnesium and, unexpectedly, brought about the degradation of single kind of dNTP. Manganese and cobalt concentrations of 10 nM were enough to induce the activity, while magnesium of about 1 mM was required for the induction of the activity. To further evaluate metal ions inherent to dNTPase in TTHB8 cells, we measured intracellular concentrations of major metal ions in TTHB8 cells by inductively coupled plasma emission spectroscopy and compared them with the dependence of metal ion concentration on dNTPase activity. Though cobalt ion was below detectable level, magnesium and manganese ions were detected at sufficient level to induce dNTPase activity. These results suggest that both manganese and magnesium ions are likely to be functional under intracellular condition. In addition, the proposed model of dNTPase activity induced by magnesium and multiple dNTPs was discussed based on the results obtained in this study.

Symbiobacterium toebii is a commensal symbiotic thermophile that cannot grow without support from a partner bacterium. We investigated the properties of Symbiobacterium growth-supporting factors (SGSFs) produced by the partner bacterium Geobacillus toebii. SGSFs occurred in both the cell-free extract (CFE) and culture supernatant of G. toebii and might comprise multifarious materials because of their different biological properties. The heavy SGSF contained in the cytosolic component exhibited heat- and proteinase-sensitive proteinaceous properties and had a molecular mass of >50 kDa. In contrast, the light SGSF contained in the extracellular component exhibited heat-stable, proteinase-resistant, nonprotein properties and had a molecular mass of <10 kDa. Under morphological examination using light microscopy, S. toebii cultured with the culture supernatant of G. toebii was filamentous, whereas S. toebii cultured with the CFE of G. toebii was rod-shaped. These results strongly suggest that the SGSFs produced by G. toebii comprise two or more types that differ in their growth-supporting mechanisms, although all support the growth of S. toebii. Upon the examination of the distribution of SGSFs in other bacteria, both cytosolic and extracellular components of Geobacillus kaustophilus, Escherichia coli, and Bacillus subtilis had detectable growth-supporting effects for S. toebii, indicating that common SGSF materials are widely present in various bacterial strains.

ADP-ribose (ADPR) is one of the main substrates of Nudix proteins. Among the eight Nudix proteins of Thermus thermophilus HB8, we previously determined the crystal structure of Ndx4, an ADPR pyrophosphatase (ADPRase). In this study we show that Ndx2 of T. thermophilus also preferentially hydrolyzes ADPR and flavin adenine dinucleotide and have determined its crystal structure. We have determined the structures of Ndx2 alone and in complex with Mg2+, with Mg2+ and AMP, and with Mg2+ and a nonhydrolyzable ADPR analogue. Although Ndx2 recognizes the AMP moiety in a manner similar to those for other ADPRases, it recognizes the terminal ribose in a distinct manner. The residues responsible for the recognition of the substrate in Ndx2 are not conserved among ADPRases. This may reflect the diversity in substrate specificity among ADPRases. Based on these results, we propose the classification of ADPRases into two types: ADPRase-I enzymes, which exhibit high specificity for ADPR; and ADPRase-II enzymes, which exhibit low specificity for ADPR. In the active site of the ternary complexes, three Mg2+ ions are coordinated to the side chains of conserved glutamate residues and water molecules. Substitution of Glu90 and Glu94 with glutamine suggests that these residues are essential for catalysis. These results suggest that ADPRase-I and ADPRase-II enzymes have nearly identical catalytic mechanisms but different mechanisms of substrate recognition.

In view of the biological significance of understanding the ribosomal machinery of both prokaryotes and eukaryotes, the L30e ribosomal protein from Methanocaldococcus jannaschii was cloned, overexpressed, purified and crystallized using the microbatch-under-oil method with the crystallization conditions 40% PEG 400, 0.1 M MES pH 6.0 and 5% PEG 3000 at 291 K. A diffraction-quality crystal (0.20 x 0.20 x 0.35 mm) was obtained that belonged to the primitive tetragonal space group P4(3), with unit-cell parameters a = 46.1, b = 46.1, c = 98.5 A, and diffracted to a resolution of 1.9 A. Preliminary calculations reveal that the asymmetric unit contains two monomers with a Matthews coefficient (V(M)) of 2.16 A(3) Da(-1).