An insight into the interaction mode between CheB and chemoreceptor from two crystal structures of CheB methylesterase catalytic domain

We have determined 2.2 Å resolution crystal structure of Thermotoga maritima CheB methylesterase domain to provide insight into the interaction mode between CheB and chemoreceptors. T. maritima CheB methylesterase domain has identical topology of a modified doubly-wound α/β fold that was observed from the previously reported Salmonella typhimurium counterpart, but the analysis of the electrostatic potential surface near the catalytic triad indicated considerable charge distribution difference. As the CheB demethylation consensus sites of the chemoreceptors, the CheB substrate, are not uniquely conserved between T. maritima and S. typhimurium, such surfaces with differing electrostatic properties may reflect CheB regions that mediate protein–protein interaction. Via the computational docking of the two T. maritima and S. typhimurium CheB structures to the respective T. maritima and Escherichia coli chemoreceptors, we propose a CheB:chemoreceptor interaction mode.     

Thermotoga maritima IscU. Structural characterization and dynamics of a new class of metallochaperone

Members of the IscU family of proteins are among the most conserved of all protein groups, extending across all three kingdoms of life. IscU serves as a scaffold for the assembly of intermediate iron-sulfur cluster centers and further mediates delivery to apo protein targets. Several proteins that mediate delivery of single metal ions to apo targets (termed metallochaperones) have recently been characterized structurally. Each displays a ferredoxin-like betaalphabetabetaalphabeta motif as a structural core. Assembly and delivery of a polynuclear iron-sulfur cluster is, however, a more complex pathway and presumably would demand a distinctive protein mediator. Here, we demonstrate Thermotoga maritima IscU (Tm IscU) to display unique structural and motional characteristics that distinguish it from other members of this class of proteins. In particular, IscU adopts a mobile, physiologically relevant, molten globule-like state that is vastly different from the previously identified ferredoxin-like fold that has thus far been characterized for other metallochaperones. The secondary structural content of Tm IscU is consistent with previous circular dichroism measurements on apo and holo protein, consisting of six alpha-helices and three beta-strands, the latter forming an anti-parallel beta-sheet. Extensive dynamics studies are consistent with a protein that has reasonably well defined secondary structural elements, but with a tertiary structure that is fluxional among widely different conformational arrangements. Analogous conformational flexibility does not exist in other structurally characterized metallochaperones; however, such a dynamic molecule may account for the lack of long-range NOEs, and allow both for the flexibility that is necessary for the multiple roles of Fe-S cluster assembly, and recognition and delivery of that cluster to a target protein. Additionally, the fluxionality of IscU is unique in that the protein appears to be more compact (based on 1H/2H exchange, R1, R2, and NOE data) but yet more fluid (lack of long-range NOEs) than typical molten globule proteins.     

Exopolygalacturonate lyase from Thermotoga maritima: cloning,characterization and organic synthesis application

A new exopolygalacturonate lyase (Pel) gene of the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli cells. A 42 kDa monomeric Pel was shown to undergo N-terminal processing by cleavage at a putative site between alanine and serine residues. The enzyme catalyzes selectively a beta-4,5 elimination at the third galacturonic unit from the reducing end of polygalacturonic acid by producing (4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid)-(1-->4)-(alpha-D-galactopyranosyluronic acid)-(1-->4)-alpha-D-galactopyranuronic acid (3) with a 60% yield. The optimum activity of the enzyme was detected at pH 9.5 and T> or=95 degrees C. The highly thermostable enzyme constitutes a useful catalyst for a simplified synthesis of 4,5-unsaturated trigalacturonic acid 3, a trisaccharide which is extremely difficult to obtain via chemical synthesis.     

Crystal structure of Thermotoga maritima 4-alpha-glucanotransferase and its acarbose complex: implications for substrate specificity and catalysis

4-alpha-Glucanotransferase (GTase) is an essential enzyme in alpha-1,4-glucan metabolism in bacteria and plants. It catalyses the transfer of maltooligosaccharides from an 1,4-alpha-D-glucan molecule to the 4-hydroxyl group of an acceptor sugar molecule. The crystal structures of Thermotoga maritima GTase and its complex with the inhibitor acarbose have been determined at 2.6A and 2.5A resolution, respectively. The GTase structure consists of three domains, an N-terminal domain with the (beta/alpha)(8) barrel topology (domain A), a 65 residue domain, domain B, inserted between strand beta3 and helix alpha6 of the barrel, and a C-terminal domain, domain C, which forms an antiparallel beta-structure. Analysis of the complex of GTase with acarbose has revealed the locations of five sugar-binding subsites (-2 to +3) in the active-site cleft lying between domain B and the C-terminal end of the (beta/alpha)(8) barrel. The structure of GTase closely resembles the family 13 glycoside hydrolases and conservation of key catalytic residues previously identified for this family is consistent with a double-displacement catalytic mechanism for this enzyme. A distinguishing feature of GTase is a pair of tryptophan residues, W131 and W218, which, upon the carbohydrate inhibitor binding, form a remarkable aromatic "clamp" that captures the sugar rings at the acceptor-binding sites +1 and +2. Analysis of the structure of the complex shows that sugar residues occupying subsites from -2 to +2 engage in extensive interactions with the protein, whereas the +3 glucosyl residue makes relatively few contacts with the enzyme. Thus, the structure suggests that four subsites, from -2 to +2, play the dominant role in enzyme-substrate recognition, consistent with the observation that the smallest donor for T.maritima GTase is maltotetraose, the smallest chain transferred is a maltosyl unit and that the smallest residual fragment after transfer is maltose. A close similarity between the structures of GTase and oligo-1,6-glucosidase has allowed the structural features that determine differences in substrate specificity of these two enzymes to be analysed.     

A novel amylolytic enzyme from Thermotoga maritima,resembling cyclodextrinase and alpha-glucosidase,that liberates glucose from the reducing end of the substrates

The gene previously designated as putative cyclodextrinase from Thermotoga maritima (TMG) was cloned and overexpressed in Escherichia coli. The recombinant TMG was partially purified and its enzymatic characteristics on various substrates were examined. The enzyme hydrolyzes various maltodextrins including maltotriose to maltoheptaose and cyclomaltodextrins (CDs) to mainly glucose and maltose. Although TMG could not degrade pullulan, it rapidly hydrolyzes acarbose, a strong amylase and glucosidase inhibitor, to acarviosine and glucose. Also, TMG initially hydrolyzes p-nitrophenyl-alpha-pentaoside to give maltopentaose and p-nitrophenol, implying that the enzyme specifically cleaves a glucose unit from the reducing end of maltooligosaccharides unlike to other glucosidases. Since its enzymatic activity is negligible if alpha-methylglucoside is present in the reducing end, the type of the residue at the reducing end of the substrate is important for the TMG activity. These results support the fact that TMG is a novel exo-acting glucosidase possessing the characteristics of both CD-/pullulan hydrolyzing enzyme and alpha-glucosidase.     

Structural and immunochemical relationship between the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 396/C-1 and Escherichia coli O126

The structure of the O-antigen polysaccharide (PS) from the enteroaggregative Escherichia coli strain 396/C-1 has been determined. Sugar and methylation analyses together with 1H and 13C NMR spectroscopy were the main methods used. Inter-residue correlations were determined by 1H,1H-NOESY, 1H,13C-heteronuclear multiple-bond correlation and dipole-dipole cross-correlated relaxation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [structure: see text]. Analysis of NMR data reveals that on average the PS consists of approximately 13 repeating units and indicates that the biological repeating unit contains an N-acetylglucosamine residue at its reducing end. This structure is different to that reported for the O-antigen polysaccharide from E. coli O126. Monospecific anti-E. coli O126 rabbit serum from The International Escherichia and Klebsiella Centre did not distinguish between the E. coli strain 396/C-1 and the E. coli O126 reference strain, neither in slide agglutination nor in an indirect enzyme immunoassay. Subsequent successful serotyping of the E. coli strain 396/C-1 showed it to be E. coli O126:K+:H27.     

Determination of the heterogeneous interactome between Edwardsiella tarda and fish gills

Edwardsiellosis caused by Edwardsiella tarda is a frequent occurrence throughout the world and has resulted in extensive losses in aquaculture. However, information regarding to protein-protein interaction between the pathogenic cells and host is not available although the portal of entry of the pathogen is determined. In this study, fish gill and bacterial pull-down approaches were used to isolate both bacterial outer membrane proteins that bind to gills and fish gill proteins that interact with bacterial cells, respectively. Eight interacting bacterial proteins and twelve interacting fish proteins were obtained. The genes of seven bacterial proteins were cloned and expressed for preparation of antibodies. The prepared antibodies were used to investigate protein-protein interactions between bacterial cells and fish gills. Five heterogeneous protein-protein interactions were determined. Moreover, the protective ability of three of the bacterial recombinant proteins, selected at random, was investigated in a mouse model where they showed significant protection. The gill proteins were highly homologous proteins with from humans and other animals where they are known to be involved in host immunity. These findings indicate that the heterogeneous interactome has significantly biological significance. Our results demonstrate a way to determine and understand the heterogeneous interaction between of E. tarda and gills.     

Structural relation of the antigenic polysaccharides of Escherichia coli O40, Shigella dysenteriae type 9, and E. coli K47

O-polysaccharides were isolated from the lipopolysaccharides of Escherichia coli O40 and Shigella dysenteriae type 9 and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of E. coli O40 was established: -->2)-beta-D-Galp-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1--> TheO-polysaccharide structure of S. dysenteriae type 9 established earlier was revised and found to be identical to the reported structure of the capsular polysaccharide of E. coli K47 and to differ from that of the E. coli O40 polysaccharide in the presence of a 3,4-linked pyruvic acid acetal having the (R)-configuration (RPyr): -->2)-beta-D-Galp3,4(RPyr)-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->     

Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM)

The genome of the hyperthermophilic bacterium Thermotoga maritima (Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously. The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino-terminus. The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide in Escherichia coli and found to be optimally active at pH 6, 90 degrees C, with a melting temperature of approximately 105 degrees C. The cel74 gene was previously found to be induced during T. maritima growth on a variety of polysaccharides, including barley glucan, carboxymethyl cellulose (CMC), glucomannan, galactomannan and starch. However, while Tm Cel74 was most active towards barley glucan and to a lesser extent CMC, glucomannan and tamarind (xyloglucan), no activity was detected on other glycans, including galactomannan, laminarin and starch. Also, Tm Cel74 did not contain a carbohydrate binding module (CBM), versions of which have been identified in the amino acid sequences of other family 74 enzymes. As such, a CBM associated with a chitinase in another hyperthermophile, Pyrococcus furiosus, was used to create a fusion protein that was active on crystalline cellulose; Tm Cel74 lacked activity on this substrate. Based on the cleavage pattern determined for Tm Cel74 on glucan-based substrates, this enzyme likely initiates recruitment of carbohydrate carbon and energy sources by creating oligosaccharides that are transported into the cell for further processing.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 522/C1 and the international type strain from Escherichia coli O 178

The structure of the O-antigenic polysaccharide (PS) from the enteroaggregative Escherichia coli strain 522/C1 has been determined. Component analysis and (1)H and (13)C NMR spectroscopy techniques were used to elucidate the structure. Inter-residue correlations were determined by (1)H,(1)H-NOESY and (1)H,(13)C-heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [ structure: see text]. Analysis of NMR data reveals that on average the PS consists of four repeating units and indicates that the biological repeating unit contains an N-acetylgalactosamine residue at its reducing end. Serotyping of the E. coli strain 522/C1 showed it to be E. coli O 178:H7. Determination of the structure of the O-antigen PS of the international type strain from E. coli O 178:H7 showed that the two polysaccharides have identical repeating units. In addition, this pentasaccharide repeating unit is identical to that of the capsular polysaccharide from E. coli O9:K 38, which also contains O-acetyl groups.     

Investigations into the design principles in the chemotactic behavior of Escherichia coli

Inspired by the recent studies on the analysis of biased random walk behavior of Escherichia coli[Passino, K.M., 2002. Biomimicry of bacterial foraging for distributed optimization and control. IEEE Control Syst. Mag. 22 (3), 52-67; Passino, K.M., 2005. Biomimicry for Optimization, Control and Automation. Springer-Verlag, pp. 768-798; Liu, Y., Passino, K.M., 2002. Biomimicry of social foraging bacteria for distributed optimization: models, principles, and emergent behaviors. J. Optim. Theory Appl. 115 (3), 603-628], we have developed a model describing the motile behavior of E. coli by specifying some simple rules on the chemotaxis. Based on this model, we have analyzed the role of some key parameters involved in the chemotactic behavior to unravel the underlying design principles. By investigating the target tracking capability of E. coli in a maze through computer simulations, we found that E. coli clusters can be controlled as target trackers in a complex micro-scale-environment. In addition, we have explored the dynamical characteristics of this target tracking mechanism through perturbation of parameters under noisy environments. It turns out that the E. coli chemotaxis mechanism might be designed such that it is sensitive enough to efficiently track the target and also robust enough to overcome environmental noises.     

Glucose fermentation to acetate,CO2 and H2 in the anaerobic hyperthermophilic eubacterium Thermotoga maritima: involvement of the Embden-Meyerhof pathway

The hyperthermophilic anaerobic eubacterium Thermotoga maritima was grown on glucose as carbon and energy source. During growth 1 mol glucose was fermented to 2 mol acetate, 2 mol CO₂ and 4 mol H₂. The molar growth yicld on glucose (Y_glucose) was about 45 g cell dry mass/mol glucose. In the presence of elemental sulfur growing cultures of T. maritima converted 1 mol glucose to 2 mol acetate, 2 mol CO₂ about 0.5 mol H₂ and about 3.5 mol H₂S. Y_glucose was about 45 g/mol. Cell extracts contained all enzymes of the Embden-Meyerhof pathway: hexokinase (0.29 U/mg, 50°C), glucose-6-phosphate isomerase (0.56 U/mg, 50°C), phosphofructokinase (0.19 U/mg, 50° C), fructose-1,6-bisphosphate aldolase (0.033 U/mg, 50°C), triosephosphate isomerase (6.3 U/mg, 50°C), glyceraldehyde-3-phosphate dehydrogenase (NAD⁺ reducing: 0.63 U/mg, 50°C), phosphoglycerate kinase (3.7 U/mg, 50°C), phosphoglycerate mutase (0.4 U/mg, 50°C); enolase (4 U/mg, 80°C), pyruvate kinase (0.05 U/mg, 50°C). Furthermore, cell extracts contained pyruvate: ferredoxin oxidoreductasee (0.43 U/mg, 60°C); NADH: ferredoxin oxidoreductase (benzylviologen reduction: 0.46 U/mg, 80°C); hydrogenase (benzylviologen reduction: 15 U/mg, 80°C), phosphate acetyltransferase (0.13 U/mg, 80°C), acetate kinase (1.2 U/mg, 55°C), lactate dehydrogenase (0.16 U/mg, 80°C) and pyruvate carboxylase (0.02 U/mg, 50°C). The findings indicate that the hyperthermophilic eubacterium T. maritima ferments sugars (glucose) to acetate, CO₂ and H₂ involving the Embden-Meyerhof pathway, phosphate acetyltransferase and acetate kinase. Thus, the organism differs from the hyperthermophilic archaeon Pyrococcus furiosus which ferments sugars to acetate, CO₂ and H₂ involving a modified non-phosphorylated Entner-Doudoroff pathway and acetyl-CoA synthetase (ADP forming).     

Structures of the O-polysaccharides of Salmonella enterica O59 and Escherichia coli O15

The O-polysaccharide of Salmonella enterica O59 was studied using sugar analysis and 2D ¹H and ¹³C NMR spectroscopy, and the following structure of the tetrasaccharide repeating unit was established:→2)-β-d-Galp-(1→3)-α-d-GlcpNAc-(1→4)-α-l-Rhap-(1→3)-β-d-GlcpNAc-(1→Accordingly, the O-antigen gene cluster of S. enterica O59 includes all genes necessary for the synthesis of this O-polysaccharide. Earlier, another structure has been reported for the O-polysaccharide of Salmonella arizonae (S. enterica IIIb) O59, which later was found to be identical to that of Citrobacter (Citrobacter braakii) O35 and, in this work, also to the O-polysaccharide of Escherichia coli O15.     

Characterization of the cheY genes from Leptospira interrogans and their effects on the behavior of Escherichia coli

The motility and chemotaxis system are critical for the virulence of pathogenic leptospire, which enable them to penetrate host tissue barriers during infection. The completed genome sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroups Icterohaemorrhagiae (L. interrogans strain Lai) suggested that there were multiple copies of putative chemotaxis homologues located at its large chromosome. In order to verify the function of these proteins, the putative cheY genes were cloned into pQE31 vector and then expressed, respectively, in wild-type Escherichia coli strain RP437 and cheY defective strain RP5232. The results showed that all the five cheYs could restore the swarming of RP5232 strain to some extend. Overexpression of CheYs in RP437 showed inhibited swarming of RP437. To investigate the mechanism of chemotaxis signaling in L. interrogans strain Lai, certain aspartates (Asp-53, Asp-61, Asp-70, Asp-62, and Asp-66 for L. interrogans strain Lai CheY1, CheY2, CheY3, CheY4, and CheY5, respectively) were mutated. Expression of these mutated cheYs manifested neither restoration of the swarming ability of RP5232 nor inhibition on swarming ability of RP437. Multiple amino acid sequence alignment predicted ternary structures and the result of mutation experiment suggested that these conserved aspartate residues of L. interrogans were analogous to that in E. coli CheY in function and structure. So, L. interrogans and E. coli may have similar mechanisms of activation of the chemotaxis phosphorelay pathway, but there are differences in their control by signal terminator.     

Close relation of the O-polysaccharide structure of Escherichia coli O168 and revised structure of the O-polysaccharide of Shigella dysenteriae type 4

The O-polysaccharide was isolated from the lipopolysaccharide of Escherichia coli O168 and studied by chemical analyses and Smith degradation along with (1)H and (13)C NMR spectroscopies. The following structure of the branched pentasaccharide repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text] where 6-O-acetylation of GlcNAc is partial. Reinvestigation of the O-polysaccharide of Shigella dysenteriae type 4 established earlier showed it to have the same structure except for that the lateral Fuc residue is nonstoichiometrically O-acetylated at each position.     

Structural insight into the mechanism of streptozotocin inhibition of O-GlcNAcase

Despite decades of its use in diabetes research, the mechanism of cytotoxicity of streptozotocin (STZ) toward pancreatic β-islet cells has remained a topic of discussion. Although STZ toxicity is likely a function of its capacity to promote DNA alkylation, it has been proposed that STZ induces pancreatic β-cell death through O-GlcNAcase inhibition. In this report, we explore the binding mode of STZ to a close homolog of human O-GlcNAcase, BtGH84 from Bacteroides thetaiotaomicron. Our results show that STZ binds in the enzyme active site in its intact form, without the formation of a covalent adduct, consistent with solution studies on BtGH84 and human O-GlcNAcase, as well as with structural work on a homolog from Clostridium perfringens. The active site of the BtGH84 is considerably deformed upon STZ binding and as a result the catalytic machinery is expelled from the binding cavity.     

Presence of presenilin 1/2 affects the invasion and replication of Salmonella typhimurium

In this study, we used four different cell lines, with or without presenilin-1 or -2, to investigate the hypothesis that the presence of presenilin, the most prominently mutated gene in Alzheimer’s patients, affects the infection rate of host cells by Salmonella. The invasion and replication of Salmonella in presenilin 1/2-deficient cells were significantly lower than those in presenilin 1/2-expressing cells. Among several presenilin-interacting proteins, the expression of filamin-A in presenilin 1/2-deficient cells was significantly lower than in presenilin 1/2-expressing cells. However, Salmonella infection of filamin-A-deficient M2 cells did not significantly differ from infection of filamin-A-containing A7 cells, ruling out the possibility that filamin-A is a major protein inhibiting Salmonella invasion and replication. It is of interest to note that Hes-1 expression, a downstream target of Notch signaling pathway, was significantly decreased by Salmonella infection. Our results demonstrate that the presence of presenilins affects the invasion and replication processes of Salmonella.