Dissection of the gene of the bifunctional PGK-TIM fusion protein from the hyperthermophilic bacterium Thermotoga maritima: design and characterization of the separate triosephosphate isomerase.

Triosephosphate isomerase (TIM), from the hyperthermophilic bacterium Thermotoga maritima, has been shown to be covalently linked to phosphoglycerate kinase (PGK) forming a bifunctional fusion protein with TIM as the C-terminal portion of the subunits of the tetrameric protein (Schurig et al., EMBO J 14:442-451, 1995). To study the effect of the anomalous state of association on the structure, stability, and function of Thermotoga TIM, the isolated enzyme was cloned and expressed in Escherichia coli, and compared with its wild-type structure in the PGK-TIM fusion protein. After introducing a start codon at the beginning of the tpi open reading frame, the gene was expressed in E.c.BL21(DE3)/ pNBTIM. The nucleotide sequence was confirmed and the protein purified as a functional dimer of 56.5 kDa molecular mass. Spectral analysis, using absorption, fluorescence emission, near- and far-UV circular dichroism spectroscopy were used to compare the separated Thermotoga enzyme with its homologs from mesophiles. The catalytic properties of the enzyme at approximately 80 degrees C are similar to those of its mesophilic counterparts at their respective physiological temperatures, in accordance with the idea that under in vivo conditions enzymes occupy corresponding states. As taken from chaotropic and thermal denaturation transitions, the separated enzyme exhibits high intrinsic stability, with a half-concentration of guanidinium-chloride at 3.8 M, and a denaturation half-time at 80 degrees C of 2 h. Comparing the properties of the TIM portion of the PGK-TIM fusion protein with those of the isolated recombinant TIM, it is found that the fusion of the two enzymes not only enhances the intrinsic stability of TIM but also its catalytic efficiency.     

A novel bifunctional aspartate kinase-homoserine dehydrogenase from the hyperthermophilic bacterium,Thermotoga maritima

ABSTRACT

The orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH. L-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thaliana AK-HseDH. However, the concentration required to inhibit the activity was much lower (K_0.5 = 37 μM) than that needed to inhibit the A. thaliana enzyme (K_0.5 = 500 μM). In contrast to A. thaliana AK-HseDH, Hse oxidation of the T. maritima enzyme was almost impervious to inhibition by L-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritima AK-HseDH is likely responsible for the unique sensitivity to L-threonine.

Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK–HseDH: bifunctional aspartate kinase–homoserine dehydrogenase; AsaDH: aspartate–β–semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T).     

Thermostable chemotaxis proteins from the hyperthermophilic bacterium Thermotoga maritima.

An expressed sequence tag homologous to cheA was previously isolated by random sequencing of Thermotoga maritima cDNA clones (C. W. Kim, P. Markiewicz, J. J. Lee, C. F. Schierle, and J. H. Miller, J. Mol. Biol. 231: 960-981, 1993). Oligonucleotides complementary to this sequence tag were synthesized and used to identify a clone from a T. maritima lambda library by using PCR. Two partially overlapping restriction fragments were subcloned from the lambda clone and sequenced. The resulting 5,251-bp sequence contained five open reading frames, including cheA, cheW, and cheY. In addition to the chemotaxis genes, the fragment also encodes a putative protein isoaspartyl methyltransferase and an open reading frame of unknown function. Both the cheW and cheY genes were individually cloned into inducible Escherichia coli expression vectors. Upon induction, both proteins were synthesized at high levels. T. maritima CheW and CheY were both soluble and were easily purified from the bulk of the endogenous E. coli protein by heat treatment at 80 degrees C for 10 min. CheY prepared in this way was shown to be active by the demonstration of Mg(2+)-dependent autophosphorylation with [32P]acetyl phosphate. In E. coli, CheW mediates the physical coupling of the receptors to the kinase CheA. The availability of a thermostable homolog of CheW opens the possibility of structural characterization of this small coupling protein, which is among the least well characterized proteins in the bacterial chemotaxis signal transduction pathway.     

Characterization of a tetrameric inositol monophosphatase from the hyperthermophilic bacterium Thermotoga maritima.

Inositol monophosphatase (I-1-Pase) catalyzes the dephosphorylation step in the de novo biosynthetic pathway of inositol and is crucial for all inositol-dependent processes. An extremely heat-stable tetrameric form of I-1-Pase from the hyperthermophilic bacterium Thermotoga maritima was overexpressed in Escherichia coli. In addition to its different quaternary structure (all other known I-1-Pases are dimers), this enzyme displayed a 20-fold higher rate of hydrolysis of D-inositol 1-phosphate than of the L isomer. The homogeneous recombinant T. maritima I-1-Pase (containing 256 amino acids with a subunit molecular mass of 28 kDa) possessed an unusually high V(max) (442 micromol min(-1) mg(-1)) that was much higher than the V(max) of the same enzyme from another hyperthermophile, Methanococcus jannaschii. Although T. maritima is a eubacterium, its I-1-Pase is more similar to archaeal I-1-Pases than to the other known bacterial or mammalian I-1-Pases with respect to substrate specificity, Li(+) inhibition, inhibition by high Mg(2+) concentrations, metal ion activation, heat stability, and activation energy. Possible reasons for the observed kinetic differences are discussed based on an active site sequence alignment of the human and T. maritima I-1-Pases.     

The hyperthermophilic bacterium Thermotoga neapolitana possesses two isozymes of the 3-phosphoglycerate kinase/triosephosphate isomerase fusion protein

Abstract The phosphoglycerate kinase ( pgk ), triosephosphate isomerase ( tpi ), and enolase ( eno ) genes from Thermotoga neapolitana have been cloned and expressed in Escherichia coli . In high copy number, the pgk gene complemented an E. coli pgk ⁻ strain. In T. neapolitana , the pgk and tpi genes appear to be fused and eno is near those genes. Like T. maritima , T. neapolitana produces phosphoglycerate kinase as both an individual enzyme and a fusion protein with triosephosphate isomerase, and triosephosphate isomerase activity is not found without associated phosphoglycerate kinase activity. Unlike T. maritima , which forms only a 70-kDa fusion protein, T. neapolitana expresses both 73-kDa and 81-kDa isozymes of this fusion protein. These isozymes are present in both T. neapolitana cells and in E. coli cells expressing T. neapolitana genes.     

Characterization of acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima

Acetohydroxyacid synthase (AHAS) is the key enzyme in branched chain amino acid biosynthesis pathway. The enzyme activity and properties of a highly thermostable AHAS from the hyperthermophilic bacterium Thermotoga maritima is being reported. The catalytic and regulatory subunits of AHAS from T. maritima were over-expressed in Escherichia coli. The recombinant subunits were purified using a simplified procedure including a heat-treatment step followed by chromatography. A discontinuous colorimetric assay method was optimized and used to determine the kinetic parameters. AHAS activity was determined to be present in several Thermotogales including T. maritima. The catalytic subunit of T. maritima AHAS was purified approximately 30-fold, with an AHAS activity of approximately 160±27 U/mg and native molecular mass of 156±6 kDa. The regulatory subunit was purified to homogeneity and showed no catalytic activity as expected. The optimum pH and temperature for AHAS activity were 7.0 and 85 °C, respectively. The apparent K_m and V_max for pyruvate were 16.4±2 mM and 246±7 U/mg, respectively. Reconstitution of the catalytic and regulatory subunits led to increased AHAS activity. This is the first report on characterization of an isoleucine, leucine, and valine operon (ilv operon) enzyme from a hyperthermophilic microorganism and may contribute to our understanding of the physiological pathways in Thermotogales. The enzyme represents the most active and thermostable AHAS reported so far.     

Properties of a thermostable 4Fe-ferredoxin from the hyperthermophilic bacterium Thermotoga maritima

Abstract A ferredoxin has been purified from one of the most ancient and the most thermophilic bacteria known, Thermotoga maritima , which grous up to 90°C. The reduced protein ( M _r approx. 6300) contains a single S = 1 2 [4Fe 4S]¹⁺ cluster with complete cysteinyl ligation, and was unaffected after incubation at 95°C for 12 h. It functioned as an electron carrier for T. maritima pyruvate oxidoreductase. Remarkably, the properties and amino acid sequence of this hyperthermophilic bacterial protein are much more similar to those of ferredoxins from hyperthermophilic archaea, rather than ferredoxins from mesophilic and moderately thermophilic bacteria.     

Stability and folding of dihydrofolate reductase from the hyperthermophilic bacterium Thermotoga maritima.

Dihydrofolate reductase (DHFR) has been a well-established model system for protein folding. The enzyme DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR) displays distinct adaptations toward high temperatures at the level of both structure and stability. The enzyme represents an extremely stable dimer; no isolated structured monomers could be detected in equilibrium or during unfolding. The equilibrium unfolding strictly follows the two-state model for a dimer (N(2) right harpoon over left harpoon 2U), with a free energy of stabilization of DeltaG = -142 +/- 10 kJ/mol at 15 degrees C. The two-state model is applicable over the whole temperature range (5-70 degrees C), yielding a DeltaG vs T profile with maximum stability at around 35 degrees C. There is no flattening of the stability profile. Instead, the enhanced thermostability is characterized by shifts toward higher overall stability and higher temperature of maximum stability. TmDHFR unfolds in a highly cooperative manner via a nativelike transition state without intermediates. The unfolding reaction is much slower (ca. 10(8) times) compared to DHFR from Escherichia coli (EcDHFR). In contrast to EcDHFR, no evidence for heterogeneity of the native state is detectable. Refolding proceeds via at least two intermediates and a burst-phase of rather low amplitude. Reassociation of monomeric intermediates is not rate-limiting on the folding pathway due to the high association constant of the dimer.     

Solution NMR structure of the cold-shock protein from the hyperthermophilic bacterium Thermotoga maritima.

Cold-shock proteins (Csps) are a subgroup of the cold-induced proteins preferentially expressed in bacteria and other organisms on reduction of the growth temperature below the physiological temperature. They are related to the cold-shock domain found in eukaryotes and are some of the most conserved proteins known. Their exact function is still not known, but translational regulation, possibly via RNA chaperoning, has been discussed. Here we present the structure of a hyperthermophilic member of the Csp family. The NMR solution structure of TmCsp from Thermotoga maritima, the hyperthermophilic member of this class of proteins, was solved on the basis of 1015 conformational constraints. It contains five beta strands combined in two antiparallel beta sheets making up a beta barrel structure, in which beta strands 1-4 are arranged in a Greek-key topology. The side chain of R2, which is exclusively found in thermophilic members of the Csp family, probably participates in a peripheral ion cluster involving residues D20, R2, E47 and K63, suggesting that the thermostability of TmCsp is based on the peripheral ion cluster around the side chain of R2.     

Unique metal dependency of cytosolic alpha-mannosidase from Thermotoga maritima,a hyperthermophilic bacterium

A putative cytosolic alpha-mannosidase gene from a hyperthermophilic marine bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. The purified recombinant enzyme appeared to be a homodimer of a 110-kDa subunit. The enzyme showed metal-dependent ability to hydrolyze p-nitrophenyl-alpha-D-mannopyranoside. In the absence of a metal, the enzyme was inactive. Cobalt and cadmium supported high activity (60 U/mg at 70 degrees C), while the activity with zinc and chromium was poor. Cobalt (0.8 mol) bound to 1 mol monomer with a K(d) of 70 microM. The optimum pH and temperature were 6.0 and 80 degrees C, respectively. The activity was inhibited by swainsonine, but not by 1-deoxymannojirimycin, which is in agreement with the features of cytosolic alpha-mannosidase.     

Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: stability and binding properties

Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75 degrees C. As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C. Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at approximately 40 degrees C. At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields DeltaG(N-->U)=100(+/-5) kJ mol(-1 )for the free energy of stabilization. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol(-1). At temperatures around the optimal growth temperature of T. maritima (t(opt) approximately 80 degrees C), the yield of refolding does not exceed 80 %; the residual 20 % are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E. coli and the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified. Binding of maltose was found to be endothermic and entropy-driven, with DeltaH(b)=+47 kJ mol(-1) and DeltaS(b)=+257 J mol(-1) K(-1). Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) approximately 0.3 microM. This result is in agreement with data reported for the MBPs from E. coli and T. litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states.     

Characterization of a thermostable L-arabinose (D-galactose) isomerase from the hyperthermophilic eubacterium Thermotoga maritima

The araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni(2+) affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90 degrees C and pH 7.5 under the assay conditions used. Its apparent K(m) values for L-arabinose and D-galactose were 31 and 60 mM, respectively; the apparent V(max) values (at 90 degrees C) were 41.3 U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 74.8 mM(-1).min(-1) (L-arabinose) and 8.5 mM(-1).min(-1) (D-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn(2+) and/or Co(2+) than in the absence of these ions. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield of 56% for 6 h at 80 degrees C.     

Characterization of the genes and proteins of a two-component system from the hyperthermophilic bacterium Thermotoga maritima.

As a step towards studying representative members of the two-component family of signal transduction proteins, we have cloned genes encoding a histidine protein kinase and a response regulator from the hyperthermophilic bacterium Thermotoga maritima. The genes have been designated HpkA and drrA, respectively. The deduced HpkA sequence contains all five characteristic histidine protein kinase motifs with the same relative order and spacing found in the mesophilic bacterial proteins. A hydropathy profile indicates that HpkA possesses only one membrane-spanning segment located at the extreme N terminus. The N-terminal region of DrrA exhibits all of the characteristics of the conserved domains of mesophilic bacterial response regulators, and the C-terminal region shows high similarity to the OmpR-PhoB subfamily of DNA-binding proteins. Recombinant T. maritima proteins, truncated HpkA lacking the putative membrane-spanning N- terminal amino acids and DrrA, were expressed in Escherichia coli. Partial purification of T. maritima proteins was achieved by heat denaturation of E. coli host proteins. In an in vitro assay, truncated HpkA protein was autophosphorylated in the presence of ATP. Thus, the N-terminal hydrophobic region is not required for kinase activity. Phosphotransfer between truncated HpkA and DrrA was demonstrated in vitro with the partially purified proteins. The phosphorylation reactions were strongly temperature dependent. The results indicate that the recombinant T. maritima two-component proteins overexpressed in E. coli are stable as well as enzymatically active at elevated temperatures.     

Glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: molecular characterization and phylogenetic implications

The hyperthermophilic bacterium Thermotoga maritima, which grows at up to 90°C, contains an L-glutamate dehydrogenase (GDH). Activity of this enzyme could be detected in T. maritima crude extracts, and appeared to be associated with a 47-kDa protein which cross-reacted with antibodies against purified GDH from the hyperthermophilic archaeon Pyrococcus woesei. The single-copy T. maritima gdh gene was cloned by complementation in a glutamate auxotrophic Escherichia coli strain. The nucleotide sequence of the gdh gene predicts a 416-residue protein with a calculated molecular weight of 45852. The gdh gene was inserted in an expression vector and expressed in E. coli as an active enzyme. The T. maritima GDH was purified to homogeneity. The NH₂-terminal sequence of the purified enzyme was PEKSLYEMAVEQ, which is identical to positions 2–13 of the peptide sequence derived from the gdh gene. The purified native enzyme has a size of 265 kDa and a subunit size of 47 kDa, indicating that GDH is a homohexamer. Maximum activity of the enzyme was measured at 75°C and the pH optima are 8.3 and 8.8 for the anabolic and catabolic reaction, respectively. The enzyme was found to be very stable at 80°C, but appeared to lose activity quickly at higher temperatures. The T. maritima GDH shows the highest rate of activity with NADH (V _max of 172U/mg protein), but also utilizes NADPH (V _max of 12U/mg protein). Sequence comparisons showed that the T. maritima GDH is a member of the family II of hexameric GDHs which includes all the GDHs isolated so far from hyperthermophiles. Remarkably, phylogenetic analysis positions all these hyperthermophilic GDHs in the middle of the GDH family II tree, with the bacterial T. maritima GDH located between that of halophilic and thermophilic euryarchaeota. Received: 15 July 1996 / Accepted: 12 October 1996     

Characterization and mode of action of an exopolygalacturonase from the hyperthermophilic bacterium Thermotoga maritima

An intracellular pectinolytic enzyme, PelB (TM0437), from the hyperthermophilic bacterium Thermotoga maritima was functionally produced in Escherichia coli and purified to homogeneity. PelB belongs to family 28 of the glycoside hydrolases, consisting of pectin-hydrolysing enzymes. As one of the few bacterial exopolygalacturonases, it is able to remove monogalacturonate units from the nonreducing end of polygalacturonate. Detailed characterization of the enzyme showed that PelB is highly thermo-active and thermostable, with a melting temperature of 105 degrees C and a temperature optimum of 80 degrees C, the highest described to date for hydrolytic pectinases. PelB showed increasing activity on oligosaccharides with an increasing degree of polymerization. The highest activity was found on the pentamer (1000 U.mg(-1)). In addition, the affinity increased in conjunction with the length of the oligoGalpA chain. PelB displayed specificity for saturated oligoGalpA and was unable to degrade unsaturated or methyl-esterified oligoGalpA. Analogous to the exopolygalacturonase from Aspergillus tubingensis, it showed low activity with xylogalacturonan. Calculations on the subsite affinity revealed the presence of four subsites and a high affinity for GalpA at subsite +1, which is typical of exo-active enzymes. The physiological role of PelB and the previously characterized exopectate lyase PelA is discussed.     

Discovery and characterization of a novel ATP/polyphosphate xylulokinase from a hyperthermophilic bacterium Thermotoga maritima

Xylulokinase (XK, E.C. 2.7.1.17) is one of the key enzymes in xylose metabolism and it is essential for the activation of pentoses for the sustainable production of biocommodities from biomass sugars. The open reading frame (TM0116) from the hyperthermophilic bacterium Thermotoga maritima MSB8 encoding a putative xylulokinase were cloned and expressed in Escherichia coli BL21 Star (DE3) in the Luria–Bertani and auto-inducing high-cell-density media. The basic biochemical properties of this thermophilic XK were characterized. This XK has the optimal temperature of 85 °C. Under a suboptimal condition of 60 °C, the k _cat was 83 s^?1, and the K _m values for xylulose and ATP were 1.24 and 0.71 mM, respectively. We hypothesized that this XK could work on polyphosphate possibly because this ancestral thermophilic microorganism utilizes polyphosphate to regulate the Embden–Meyerhof pathway and its substrate-binding residues are somewhat similar to those of other ATP/polyphosphate-dependent kinases. This XK was found to work on low-cost polyphosphate, exhibiting 41 % of its specific activity on ATP. This first ATP/polyphosphate XK could have a great potential for xylose utilization in thermophilic ethanol-producing microorganisms and cell-free biosystems for low-cost biomanufacturing without the use of ATP.     

Xylanase attachment to the cell wall of the hyperthermophilic bacterium Thermotoga maritima

The cellular localization and processing of the endo-xylanases (1,4-beta-D-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile Thermotoga maritima were investigated, in particular with respect to the unusual outer membrane ("toga") of this gram-negative bacterium. XynB (40 kDa) was detected in the periplasmic fraction of T. maritima cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cell-bound XynA was localized mainly in the outer membranes of T. maritima cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of T. maritima via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.     

Characterization of an exceedingly active NADH oxidase from the anaerobic hyperthermophilic bacterium Thermotoga maritima

An NADH oxidase from the anaerobic hyperthermophilic bacterium Thermotoga maritima was purified. The enzyme was very active in catalyzing the reduction of oxygen to hydrogen peroxide with an optimal pH value of 7 at 80 degrees C. The V(max) was 230 +/- 14 mumol/min/mg (k(cat)/K(m) = 548,000 min(-1) mM(-1)), and the K(m) values for NADH and oxygen were 42 +/- 3 and 43 +/- 4 muM, respectively. The NADH oxidase was a heterodimeric flavoprotein with two subunits with molecular masses of 54 kDa and 46 kDa. Its gene sequences were identified, and the enzyme might represent a new type of NADH oxidase in anaerobes. An NADH-dependent peroxidase with a specific activity of 0.1 U/mg was also present in the cell extract of T. maritima.     

Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: structure and stability of the recombinant enzyme and its isolated cellulose-binding domain.

The hyperthermophilic bacterium Thermotoga maritima is capable of gaining metabolic energy utilizing xylan. XynA, one of the corresponding hydrolases required for its degradation, is a 120-kDa endo-1,4-D-xylanase exhibiting high intrinsic stability and a temperature optimum approximately 90 degrees C. Sequence alignments with other xylanases suggest the enzyme to consist of five domains. The C-terminal part of XynA was previously shown to be responsible for cellulose binding (Winterhalter C, Heinrich P, Candussio A, Wich G, Liebl W. 1995. Identification of a novel cellulose-binding domain within the multi-domain 120 kDa Xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol Microbiol 15:431-444). In order to characterize the domain organization and the stability of XynA and its C-terminal cellulose-binding domain (CBD), the two separate proteins were expressed in Escherichia coli. CBD, because of its instability in its ligand-free form, was expressed as a glutathione S-transferase fusion protein with a specific thrombin cleavage site as linker. XynA and CBD were compared regarding their hydrodynamic and spectral properties. As taken from analytical ultracentrifugation and gel permeation chromatography, both are monomers with 116 and 22 kDa molecular masses, respectively. In the presence of glucose as a ligand, CBD shows high intrinsic stability. Denaturation/renaturation experiments with isolated CBD yield > 80% renaturation, indicating that the domain folds independently. Making use of fluorescence emission and far-UV circular dichroism in order to characterize protein stability, guanidine-induced unfolding of XynA leads to biphasic transitions, with half-concentrations c1/2 (GdmCl) approximately 4 M and > 5 M, in accordance with the extreme thermal stability. At acid pH, XynA exhibits increased stability, indicated by a shift of the second guanidine-transition from 5 to 7 M GdmCl. This can be tentatively attributed to the cellulose-binding domain. Differences in the transition profiles monitored by fluorescence emission and dichroic absorption indicate multi-state behavior of XynA. In the case of CBD, a temperature-induced increase in negative ellipticity at 217 nm is caused by alterations in the environment of aromatic residues that contribute to the far-UV CD in the native state.