Thermotoga maritima MazG protein has both nucleoside triphosphate pyrophosphohydrolase and pyrophosphatase activities

MazG proteins form a widely conserved family among bacteria, but their cellular function is still unknown. Here we report that Thermotoga maritima MazG protein (Tm-MazG), the product of the TM0913 gene, has both nucleoside triphosphate pyrophosphohydrolase (NTPase) and pyrophosphatase activities. Tm-MazG catalyzes the hydrolysis of all eight canonical ribo- and deoxyribonucleoside triphosphates to their corresponding nucleoside monophosphates and PPi and subsequently hydrolyzes the resultant PPi to Pi. The NTPase activity with deoxyribonucleoside triphosphates as substrate is higher than corresponding ribonucleoside triphosphates. dGTP is the best substrate among the deoxyribonucleoside triphosphates, and GTP is the best among the ribonucleoside triphosphates. Both NTPase and pyrophosphatase activities were enhanced at higher temperatures and blocked by the alpha,beta-methyleneadenosine triphosphate, which cannot be hydrolyzed by Tm-MazG. Furthermore, PPi is an inhibitor for the Tm-MazG NTPase activity. Significant decreases in the NTPase activity and concomitant increases in the pyrophosphatase activity were observed when mutations were introduced at the highly conserved amino acid residues in Tm-MazG N-terminal region (E41Q/E42Q, E45Q, E61Q, R97A/R98A, and K118A). These results demonstrated that Tm-MazG has dual enzymatic functions, NTPase and pyrophosphatase, and that these two enzymatic activities are coordinated.     

A thermostable non-xylanolytic alpha-glucuronidase of Thermotoga maritima MSB8

A putative alpha-glucosidase belonging to glycosyl hydrolase family 4 of Thermotoga maritima (TM0752) was expressed in Escherichia coli and it was found that the recombinant protein (Agu4B) was a p-nitrophenyl alpha-D-glucuronopyranoside hydrolyzing alpha-glucuronidase, not alpha-glucosidase. It did not hydrolyze 4-O-methyl-D-glucuronoxylan or its fragment oligosaccharides. Agu4B was thermostable with an optimum temperature of 80 degrees C. It strictly required Mn(2+) and thiol compounds for its activity. The presence of NAD(+) slightly activated the enzyme. The amino acid sequence of Agu4B showed higher identity with Agu4A (another alpha-glucuronidase of T. maritima, 61%) than with AglA (alpha-glucosidase of T. maritima, 48%).     

Structural characterization of the putative ABC-type 2 transporter from Thermotoga maritima MSB8

This study describes the structure of the putative ABC-type 2 transporter TM0543 from Thermotoga maritima MSB8 determined at a resolution of 2.3 Å. In comparative sequence-clustering analysis, TM0543 displays similarity to NatAB-like proteins, which are components of the ABC-type Na⁺ efflux pump permease. However, the overall structure fold of the predicted nucleotide-binding domain reveals that it is different from any known structure of ABC-type efflux transporters solved to date. The structure of the putative TM0543 domain also exhibits different dimer architecture and topology of its presumed ATP binding pocket, which may indicate that it does not bind nucleotide at all. Structural analysis of calcium ion binding sites found at the interface between TM0543 dimer subunits suggests that protein may be involved in ion-transporting activity. A detailed analysis of the protein sequence and structure is presented and discussed.     

Crystal structure of TM1457 from Thermotoga maritima

The crystal structure of a hypothetical protein, TM1457, from Thermotoga maritima has been determined at 2.0A resolution. TM1457 belongs to the DUF464 family (57 members) for which there is no known function. The structure shows that it is composed of two helices in contact with one side of a five-stranded beta-sheet. Two identical monomers form a pseudo-dimer in the asymmetric unit. There is a large cleft between the first alpha-helix and the second beta-strand. This cleft may be functionally important, since the two highly conserved motifs, GHA and VCAXV(S/T), are located around the cleft. A structural comparison of TM1457 with known protein structures shows the best hit with another hypothetical protein, Ybl001C from Saccharomyces cerevisiae, though they share low structural similarity. Therefore, TM1457 still retains a unique topology and reveals a novel fold.     

Local variability of the phosphoglycerate kinase-triosephosphate isomerase fusion protein from Thermotoga maritima MSB 8

The pgk-tpi gene locus of Thermotoga maritima encodes both phosphoglycerate kinase (PGK) and a bienzyme complex consisting of a fusion protein of PGK with triosephosphate isomerase (TIM). No separate tpi gene for TIM is present in T. maritima. A frame-shift at the end of the pgk gene has been previously proposed as a mechanism to regulate the expression of the two protein variants [Schurig et al., EMBO J. 14 (1995), 442-451]. Surprisingly, the complete T. maritima genome was found to contain a pgk-tpi sequence not requiring the proposed frameshift mechanism. To clarify the apparent discrepancy, a variety of DNA sequencing techniques were applied, disclosing an anomalous local variability in the pgk-tpi fusion region. The comparison of different DNA samples and the mass spectrometric analysis of the amino acid sequence of the natural fusion protein from T. maritima MSB8 confirmed the local variability of the DNA variants. Since not all peptide masses could be assigned, further variations are conceivable, suggesting an even higher heterogeneity of the T. maritima MSB8 strain.     

Two Extremely Thermostable Xylanases of the Hyperthermophilic Bacterium Thermotoga maritima MSB8

During growth with xylose or xylan as the source of carbon, xylanase production by Thermotoga maritima MSB8 was enhanced about 10-fold compared with growth with glucose or starch. Two extremely thermostable endoxylanases (1,4-(beta)-d-xylan-xylanohydrolase, EC 3.2.1.8), designated XynA and XynB, were identified and purified from cells of this organism. XynA and XynB occurred as proteins with apparent molecular masses of about 120 and 40 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximum activity at the optimal pH (pH 6.2 and pH 5.4 for XynA and XynB, respectively) was measured at about 92(deg)C for XynA (10-min assay) and at about 105(deg)C for XynB (5-min assay). XynB activity was stimulated twofold by the addition of 500 mM NaCl, while XynA displayed maximum activity without the addition of salt. Both xylanases were tolerant of relatively high salt concentrations. At 2 M (about 12% wt/vol) NaCl, XynA and XynB retained 49 and 65%, respectively, of their maximum activities. In contrast to XynB, XynA was able to adsorb to microcrystalline cellulose. Antibodies raised against a recombinant truncated XynA protein cross-reacted with XynB, indicating that the enzymes may have sequence or structural similarities. Part of the xylanase activity appeared to be associated with the outer membrane of T. maritima cells, since more than 40% of the total xylanase activity present in the crude cellular extract was found in the membrane fraction after high-speed centrifugation. Most of the membrane-bound activity appeared to be due to the 120-kDa xylanase XynA.     

Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80 degrees C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70 degrees C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) kcat followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the kcat of 8.0 s(-1) had the highest (1.1 x 10(4) M(-1) s(-1)) kcat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.     

Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1-phosphate. The K _m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k _cat was 5.4 s^-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s^-1) k _cat followed by 6-deoxy-D-glucose (17 s^-1) and 2-deoxy-D-glucose (16 s^-1). The natural substrate, D-glucose with the k _cat of 8.0 s^-1 had the highest (1.1×10⁴ M^-1 s^-1) k _cat/K _m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.     

Crystal structure of the flagellar rotor protein FliN from Thermotoga maritima

FliN is a component of the bacterial flagellum that is present at levels of more than 100 copies and forms the bulk of the C ring, a drum-shaped structure at the inner end of the basal body. FliN interacts with FliG and FliM to form the rotor-mounted switch complex that controls clockwise-counterclockwise switching of the motor. In addition to its functions in motor rotation and switching, FliN is thought to have a role in the export of proteins that form the exterior structures of the flagellum (the rod, hook, and filament). Here, we describe the crystal structure of most of the FliN protein of Thermotoga maritima. FliN is a tightly intertwined dimer composed mostly of beta sheet. Several well-conserved hydrophobic residues form a nonpolar patch on the surface of the molecule. A mutation in the hydrophobic patch affected both flagellar assembly and switching, showing that this surface feature is important for FliN function. The association state of FliN in solution was studied by analytical ultracentrifugation, which provided clues to the higher-level organization of the protein. T. maritima FliN is primarily a dimer in solution, and T. maritima FliN and FliM together form a stable FliM(1)-FliN(4) complex. Escherichia coli FliN forms a stable tetramer in solution. The arrangement of FliN subunits in the tetramer was modeled by reference to the crystal structure of tetrameric HrcQB(C), a related protein that functions in virulence factor secretion in Pseudomonas syringae. The modeled tetramer is elongated, with approximate dimensions of 110 by 40 by 35 Angstroms, and it has a large hydrophobic cleft formed from the hydrophobic patches on the dimers. On the basis of the present data and available electron microscopic images, we propose a model for the organization of FliN subunits in the C ring.     

Crystal structure of the cell division protein FtsA from Thermotoga maritima

Bacterial cell division requires formation of a septal ring. A key step in septum formation is polymerization of FtsZ. FtsA directly interacts with FtsZ and probably targets other proteins to the septum. We have solved the crystal structure of FtsA from Thermotoga maritima in the apo and ATP-bound form. FtsA consists of two domains with the nucleotide-binding site in the interdomain cleft. Both domains have a common core that is also found in the actin family of proteins. Structurally, FtsA is most homologous to actin and heat-shock cognate protein (Hsc70). An important difference between FtsA and the actin family of proteins is the insertion of a subdomain in FtsA. Movement of this subdomain partially encloses a groove, which could bind the C-terminus of FtsZ. FtsZ is the bacterial homologue of tubulin, and the FtsZ ring is functionally similar to the contractile ring in dividing eukaryotic cells. Elucidation of the crystal structure of FtsA shows that another bacterial protein involved in cytokinesis is structurally related to a eukaryotic cytoskeletal protein involved in cytokinesis.     

Characterization of multi-functional properties and conformational analysis of MutS2 from Thermotoga maritima MSB8

The MutS2 homologues have received attention because of their unusual activities that differ from those of MutS. In this work, we report on the functional characteristics and conformational diversities of Thermotoga maritima MutS2 (TmMutS2). Various biochemical features of the protein were demonstrated via diverse techniques such as scanning probe microscopy (SPM), ATPase assays, analytical ultracentrifugation, DNA binding assays, size chromatography, and limited proteolytic analysis. Dimeric TmMutS2 showed the temperature-dependent ATPase activity. The non-specific nicking endonuclease activities of TmMutS2 were inactivated in the presence of nonhydrolytic ATP (ADPnP) and enhanced by the addition of TmMutL. In addition, TmMutS2 suppressed the TmRecA-mediated DNA strand exchange reaction in a TmMutL-dependent manner. We also demonstrated that small-angle X-ray scattering (SAXS) analysis of dimeric TmMutS2 exhibited nucleotide- and DNA-dependent conformational transitions. Particularly, TmMutS2-ADPnP showed the most compressed form rather than apo-TmMutS2 and the TmMutS2-ADP complex, in accordance with the results of biochemical assays. In the case of the DNA-binding complexes, the stretched conformation appeared in the TmMutS2-four-way junction (FWJ)-DNA complex. Convergences of biochemical- and SAXS analysis provided abundant information for TmMutS2 and clarified ambiguous experimental results.     

Crystal structure of full length topoisomerase I from Thermotoga maritima

DNA topoisomerases are a family of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. Bacterial and archeal type IA topoisomerases, including topoisomerase I, topoisomerase III, and reverse gyrase, are crucial in regulation of DNA supercoiling and maintenance of genetic stability. The crystal structure of full length topoisomerase I from Thermotoga maritima was determined at 1.7A resolution and represents an intact and fully active bacterial topoisomerase I. It reveals the torus-like structure of the conserved transesterification core domain comprising domains I-IV and a tightly associated C-terminal zinc ribbon domain (domain V) packing against domain IV of the core domain. The previously established zinc-independence of the functional activity of T.maritima topoisomerase I is further supported by its crystal structure as no zinc ion is bound to domain V. However, the structural integrity is preserved by the formation of two disulfide bridges between the four Zn-binding cysteine residues. A functional role of domain V in DNA binding and recognition is suggested and discussed in the light of the structure and previous biochemical findings. In addition, implications for bacterial topoisomerases I are provided.     

Identification and characterization of a novel intracellular alkaline alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima MSB8

The gene for a novel alpha-amylase, designated AmyC, from the hyperthermophilic bacterium Thermotoga maritima was cloned and heterologously overexpressed in Escherichia coli. The putative intracellular enzyme had no amino acid sequence similarity to glycoside hydrolase family (GHF) 13 alpha-amylases, yet the range of substrate hydrolysis and the product profile clearly define the protein as an alpha-amylase. Based on sequence similarity AmyC belongs to a subgroup within GHF 57. On the basis of amino acid sequence similarity, Glu185 and Asp349 could be identified as the catalytic residues of AmyC. Using a 60-min assay, the maximum hydrolytic activity of the purified enzyme, which was dithiothreitol dependent, was found to be at 90 degrees C. AmyC displayed a remarkably high pH optimum of pH 8.5 and an unusual sensitivity towards both ATP and EDTA.     

海栖热袍菌极耐高温木聚糖酶基因xynB64在大肠杆菌中的融合表达

来源于极端嗜热菌海栖热袍菌(Thermotoga maritima MSB8)的木聚糖酶B具有极高的热稳定性,在饲料、造纸、能源和食品医药行业具有巨大应用潜力。携带酶基因xynB64的pET28a(+)重组载体在宿主大肠杆菌BL21(DE3)中诱导表达,重组酶活力较低。更换宿主为携带稀有tRNA基因的大肠杆菌:BL21-CodonPlus(DE3)-RIPL和Rosetta(DE3)后,酶活力分别提高了197%和277%,但是后者中的表达会形成部分包涵体。宿主菌为大肠杆菌Rosetta(DE3),更换载体为4种融合表达载体pET32a(+)、pET42a(+)、pET43.1a(+)和pMAL-c2X进行表达,重组酶分别融合了Trx、GST、Nus和MBP标签。其中Rosetta(DE3)/pMAL-c2X-xynB64表达酶活力最高,相当于Rosetta(DE3)/pET28a-xynB64表达酶的88%,而且目的酶表达量占全细胞蛋白的40%,几乎不形成包涵体。     

Crystal structure and substrate-binding mode of cellulase 12A from Thermotoga maritima

Cellulases have been used in many applications to treat various carbohydrate-containing materials. Thermotoga maritima cellulase 12A (TmCel12A) belongs to the GH12 family of glycoside hydrolases. It is a β-1,4-endoglucanase that degrades cellulose molecules into smaller fragments, facilitating further utilization of the carbohydrate. Because of its hyperthermophilic nature, the enzyme is especially suitable for industrial applications. Here the crystal structure of TmCel12A was determined by using an active-site mutant E134C and its mercury-containing derivatives. It adopts a β-jellyroll protein fold typical of the GH12-family enzymes, with two curved β-sheets A and B and a central active-site cleft. Structural comparison with other GH12 enzymes shows significant differences, as found in two longer and highly twisted β-strands B8 and B9 and several loops. A unique Loop A3-B3 that contains Arg60 and Tyr61 stabilizes the substrate by hydrogen bonding and stacking, as observed in the complex crystals with cellotetraose and cellobiose. The high-resolution structures allow clear elucidation of the network of interactions between the enzyme and its substrate. The sugar residues bound to the enzyme appear to be more ordered in the -2 and -1 subsites than in the +1, +2 and -3 subsites. In the E134C crystals the bound -1 sugar at the cleavage site consistently show the α-anomeric configuration, implicating an intermediate-like structure.     

Structural basis of the substrate subsite and the highly thermal stability of xylanase 10B from Thermotoga maritima MSB8

The crystal structure of xylanase 10B from Thermotoga maritima MSB8 (TmxB), a hyperthermostable xylanase, has been solved in its native form and in complex with xylobiose or xylotriose at 1.8 A resolution. In order to gain insight into the substrate subsite and the molecular features for thermal stability, we compared TmxB with family 10 xylanase structures from nine microorganisms. As expected, TmxB folds into a (beta/alpha)8-barrel structure, which is common among the glycoside hydrolase family 10. The enzyme active site and the environment surrounding the xylooligosaccharide of TmxB are highly similar to those of family 10 xylanases. However, only two xylose moieties were found in its binding pocket from the TmxB-xylotriose complex structure. This finding suggests that TmxB could be a potential biocatalyst for the large-scale production of xylobiose. The result of structural analyses also indicated that TmxB possesses some additional features that account for its thermostability. In particular, clusters of aromatic residues together with a lack of exposed hydrophobic residues are characteristic of the TmxB structure. TmxB has also a significant number of ion pairs on the protein surface that are not found in other thermophilic family 10 xylanases.     

Crystal structure of the protease domain of a heat-shock protein HtrA from Thermotoga maritima

HtrA (high temperature requirement A), a periplasmic heat-shock protein, functions as a molecular chaperone at low temperatures, and its proteolytic activity is turned on at elevated temperatures. To investigate the mechanism of functional switch to protease, we determined the crystal structure of the NH(2)-terminal protease domain (PD) of HtrA from Thermotoga maritima, which was shown to retain both proteolytic and chaperone-like activities. Three subunits of HtrA PD compose a trimer, and multimerization architecture is similar to that found in the crystal structures of intact HtrA hexamer from Escherichia coli and human HtrA2 trimer. HtrA PD shares the same fold with chymotrypsin-like serine proteases, but it contains an additional lid that blocks access the of substrates to the active site. A corresponding lid found in E. coli HtrA is a long loop that also blocks the active site of another subunit. These results suggest that the activation of the proteolytic function of HtrA at elevated temperatures might occur by a conformational change, which includes the opening of the helical lid to expose the active site and subsequent rearrangement of a catalytic triad and an oxyanion hole.