MiaB protein from Thermotoga maritima. Characterization of an extremely thermophilic tRNA-methylthiotransferase

In Escherichia coli, the MiaB protein catalyzes the methylthiolation of N-6-isopentenyl adenosine in tRNAs, the last reaction step during biosynthesis of 2-methylthio-N-6-isopentenyl adenosine (ms2i6A-37). For the first time the thermophilic bacterium Thermotoga maritima is shown here to contain such a MiaB tRNA-modifying enzyme, named MiaBTm, and to synthesize ms2i6A-37 as demonstrated by an analysis of modified nucleosides from tRNA hydrolysates. The corresponding gene (TM0653) was identified by sequence similarity to the miaB gene cloned and expressed in E. coli. MiaBTm was purified to homogeneity and thoroughly characterized by biochemical and spectroscopic methods. It is a monomer of 443 residues with a molecular mass of 50,710 kilodaltons. Its amino acid sequence shares the CysXXX-CysXXCys sequence with MiaB from E. coli as well as with biotin synthase and lipoate synthase. This sequence was shown to be essential for chelation of an iron-sulfur center and for activity in these enzymes. As isolated, MiaBTm contains both iron and sulfide and an apoprotein form can coordinate up to 4 iron and 4 sulfur atoms per polypeptide chain. UV-visible absorption, resonance Raman, variable temperature magnetic circular dichroism, and EPR spectroscopy of MiaBTm indicate the presence of a [4Fe-4S]+2/+1 cluster under reducing and anaerobic conditions, whereas [3Fe-4S]+1 and [2Fe-2S]+2 forms are generated under aerobic conditions. The redox potential of the [4Fe-4S]+2/+1 transition is -495 +/- 10 mV (versus the normal hydrogen electrode). Finally, the expression of MiaBTm from T. maritima in an E. coli mutant strain lacking functional miaB gene allowed production of ms2i6A-37. These results provide further information on the enzymes involved in methylthiolation of tRNAs.     

Topographical and enzymatic characterization of amylases from the extremely thermophilic eubacterium Thermotoga maritima.

The hyperthermophilic eubacterium Thermotoga maritima uses starch as a substrate, without releasing amylase activity into the culture medium. The enzyme is associated with the 'toga'. Its expression level is too low to allow the isolation of the pure enzyme. Using cycloheptaamylose and acarbose affinity chromatography and common chromatographic procedures, two enzyme fractions are obtained. They differ in specificity, pH-optimum, temperature dependence and stability. Substrate specificity and Ca2+ dependence indicate alpha-, beta- and gluco-amylase activity. Compared with alpha-amylase from Bacillus licheniformis (Tmax = 75 degrees C), the amylases from Thermotoga maritima show exceedingly high thermal stability with an upper temperature limit at 95 degrees C. Significant turnover occurs only between 70 and 100 degrees C, i.e. in the range of viability of the microorganism.     

Purification and characterization of a highly thermostable glucose isomerase produced by the extremely thermophilic eubacterium, Thermotoga maritima

Thermotoga maritima, among the most thermophilic eubacteria currently known, produces glucose isomerase when grow in the presence of xylose. The purified enzyme is a homotetramer with submit molecular Wight of about 45,000. It has a number of features in common with previously described glucose isomerases-pH optimum of 6.5 to 7.5, presence of activesite histidine, requirement for metal cations such as Co(2+) and Mg(2+), and preference for xylose as substrate. In addition, it has significant sequence/structural homology with other glucose isomerases, as shown by both N-terminal sequencing and immunological crossreactivity. The T. maritima enzyme is distinguished by its extreme thermostability-a temperature optimum of 105 to 110 degrees C, and an estimated half-life of 10 minutes at 120 degrees C, pH 7.0. The high degree of thermostability, coupled with a neutral to slightly acid pH optimum, reveal this enzyme to be a promising candidate for improvement of the industrial glucose isomerization process (c) 1993 Wiley & Sons, Inc.     

Structure and function of mutationally generated monomers of dimeric phosphoribosylanthranilate isomerase from Thermotoga maritima

BACKGROUND: Oligomeric proteins may have been selected for in hyperthermophiles because subunit association provides extra stabilization. Phosphoribosylanthranilate isomerase (PRAI) is monomeric and labile in most mesophilic microorganisms, but dimeric and stable in the hyperthermophile Thermotoga maritima (tPRAI). The two subunits of tPRAI are associated back-to-back and are locked together by a hydrophobic loop. The hypothesis that dimerization is important for thermostability has been tested by rationally designing monomeric variants of tPRAI. RESULTS: The comparison of tPRAI and PRAI from Escherichia coli (ePRAI) suggested that levelling the nonplanar dimer interface would weaken the association. The deletion of two residues in the loop loosened the dimer. Subsequent filling of the adjacent pocket and the exchange of polar for apolar residues yielded a weakly associating and a nonassociating monomeric variant. Both variants are as active as the parental dimer but far more thermolabile. The thermostability of the weakly associating monomer increased significantly with increasing protein concentration. The X-ray structure of the nonassociating monomer differed from that of the parental subunit only in the restructured interface. The orientation of the original subunits was maintained in a crystal contact between two monomers. CONCLUSIONS: tPRAI is dimeric for reasons of stability. The clearly separated responsibilities of the betaalpha loops, which are involved in activity, and the alphabeta loops, which are involved in protein stability, has permitted the evolution of dimers without compromising their activity. The preserved interaction in the crystal contacts suggests the most likely model for dimer evolution.     

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.     

The HU protein from Thermotoga maritima: recombinant expression, purification and physicochemical characterization of an extremely hyperthermophilic DNA-binding protein.

The histone-like protein TmHU from the hyperthermophilic eubacterium Thermotoga maritima was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and cation exchange chromatography. CD spectroscopical studies with secondary structure analysis as well as comparative modeling demonstrate that the dimeric TmHU has a tertiary structure similar to other homologous HU proteins. The Tm of the protein was determined to be 96 degrees C, and thermal unfolding is nearly completely reversible. Surface plasmon resonance measurements for TmHU show that the protein binds to DNA in a highly cooperative manner, with a KD of 73 nM and a Hill coefficient of 7.6 for a 56 bp DNA fragment. It is demonstrated that TmHU is capable to increase the melting point of a synthetic, double-stranded DNA (poly[d(A-T)]) by 47 degrees C, thus suggesting that DNA stabilization may be a major function of this protein in hyperthermophiles. The significant in vitro protection of double-helical DNA may be useful for biotechnological applications.     

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.     

Phosphoglycerate kinase and triosephosphate isomerase from the hyperthermophilic bacterium Thermotoga maritima form a covalent bifunctional enzyme complex.

Phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima has been purified to homogeneity. A second larger enzyme with PGK activity and identical N-terminal sequence was also found. Surprisingly, this enzyme displayed triosephosphate isomerase (TIM) activity. No other TIM is detectable in T. maritima crude extracts. As shown by ultracentrifugal analysis, PGK is a 43 kDa monomer, whereas the bifunctional PGK-TIM fusion protein is a homotetramer of 240-285 kDa. SDS-PAGE indicated a subunit size of 70 kDa for the fusion protein. Both enzymes show high thermostability. Measurements of the catalytic properties revealed no extraordinary results. pH optima, Km values and activation energies were found to be in the range observed for other PGKs and TIMs investigated so far. The corresponding pgk and tpi genes are part of the apparent gap operon of T. maritima. This gene segment contains two overlapping reading frames, where the 43 kDa PGK is encoded by the upstream open reading frame, the pgk gene. On the other hand, the 70 kDa PGK-TIM fusion protein is encoded jointly by the pgk gene and the overlapping downstream open reading frame of the tpi gene. A programmed frameshift may be responsible for this fusion. A comparison of the amino acid sequence of both the PGK and the TIM parts of the fusion protein with those of known PGKs and TIMs reveals high similarity to the corresponding enzymes from different procaryotic and eucaryotic organisms.     

The MurE synthetase from Thermotoga maritima is endowed with an unusual D-lysine adding activity

The peptidoglycan of Thermotoga maritima, an extremely thermophilic eubacterium, was shown to contain no diaminopimelic acid and approximate amounts of both enantiomers of lysine (Huber, R., Langworthy, T. A., K?nig, H., Thomm, M., Woese, C. R., Sleytr, U. B., and Stetter, K. O. (1986) Arch. Microbiol. 144, 324-333). To assess the possible involvement of the MurE activity in the incorporation of D-lysine, the murE gene from this organism was cloned in Escherichia coli, and the corresponding protein was purified as the C-terminal His6-tagged form. In vitro assays showed that D-lysine and meso-diaminopimelic acid were added to UDP-N-acetylmuramoyl-dipeptide with 25 and 10% efficiencies, respectively, relative to L-lysine. The purified enzyme was used to synthesize the L- and D-lysine-containing UDP-N-acetylmuramoyl-tripeptides; chemical analysis revealed an unusual structure for the D-lysine-containing nucleotide, namely acylation of the epsilon-amino function of D-lysine by the D-glutamyl residue. In vitro assays with MurF and MraY enzymes from T. maritima showed that this novel nucleotide was not a substrate for MurF but that it could be directly processed into tripeptide lipid I by MraY, thereby substantiating the role of MurE in the incorporation of D-lysine into peptidoglycan.     

ATP-dependent glucokinase from the hyperthermophilic bacterium Thermotoga maritima represents an extremely thermophilic ROK glucokinase with high substrate specificity

The gene (open reading frame (ORF) Tm1469, glk) encoding ATP-dependent ROK (repressors, ORFs, sugar kinases) glucokinase (ATP-GLK, EC 2.7.1.2) of the hyperthermophilic bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 80 kDa composed of 36-kDa subunits. Rate dependence (at 80 degrees C) on glucose and ATP followed Michaelis-Menten kinetics with apparent Km values of 1.0 and 0.36 mM, respectively; apparent Vmax values were about 370 U mg(-1). The enzyme was highly specific for glucose as phosphoryl acceptor. Besides glucose only 2-deoxyglucose was phosphorylated to some extent, whereas mannose and fructose were not used. With a temperature optimum of 93 degrees C the enzyme is the most thermoactive bacterial ATP-GLK described.     

Purification and characterization of an extremely stable glucose isomerase from Geobacillus thermodenitrificans TH2

The D-glucose/D-xylose isomerase was purified from a thermophilic bacterium, Geobacillus thermodenitrificans TH2, by precipitating with heat shock and using Q-Sepharose ion exchange column chromatography, and then characterized. The purified enzyme had a single band having molecular weight of 49 kDa on SDS-PAGE. In the presence of D-glucose as a substrate, the optimum temperature and pH of the enzyme were found to be 80°C and 7.5, respectively. The purified xylose isomerase of G. thermodenitrificans TH2 was extremely stable at pH 7.5 after 96 h incubation at 4°C and 50°C. When the thermal stability profile was analyzed, it was determined that the purified enzyme was extremely stable during incubation periods of 4 months and 4 days at 4°C and 50°C, respectively. The K _m and V _max values of the purified xylose isomerase from G. thermodenitrificans TH2 were calculated as 32 mM and 4.68 μmol/min per mg of protein, respectively. Additionally, it was detected that some metal ions affected the enzyme activity at different ratios. The enzyme was active and stable at high temperatures and nearly neutral pHs which are desirable for the usage in the food and ethanol industry.     

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.     

ATP-dependent 6-phosphofructokinase from the hyperthermophilic bacterium Thermotoga maritima: characterization of an extremely thermophilic, allosterically regulated enzyme

The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic bacterium Thermotoga maritimawas purified 730-fold to homogeneity. The enzyme is a 140-kDa homotetramer composed of 34 kDa subunits. Kinetic constants were determined for all substrates in both reaction directions at pH 7 and at 75 degrees C. Rate dependence (forward reaction) on fructose 6-phosphate (F-6-P) showed sigmoidal kinetics with a half-maximal saturation constant ( S(0.5)) of 0.7 mM and a Hill coefficient of 2.2. The apparent K(m) for ATP was 0.2 mM and the apparent V(max) value was about 360 U/mg. The enzyme also catalyzed in vitro the reverse reaction with an apparent K(m) for fructose 1,6-bisphosphate and ADP of 7.6 mM and 1.4 mM, respectively, and an apparent V(max) of about 13 U/mg. Divalent cations were required for maximal activity; Mg(2+), which was most effective, could partially be replaced by Mn(2+) and Fe(2+). Enzyme activity was allosterically regulated by classical effectors of ATP-PFKs of Eukarya and Bacteria; it was activated by ADP and inhibited by PEP. The enzyme had a temperature optimum of 93 degrees C and showed a significant thermostability up to 100 degrees C. Using the N-terminal amino acid sequence of the subunit, the pfk gene coding for ATP-PFK was identified and functionally overexpressed in Escherichia coli. The purified recombinant ATP-PFK had identical kinetic and allosteric properties as the native enzyme purified from T. maritima. The deduced amino acid sequence showed high sequence similarity to members of the PFK-A family. In accordance with its allosteric properties, ATP-PFK of T. maritima contained the conserved allosteric effector-binding sites for ADP and PEP.     

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.     

The extremely thermophilic eubacterium, Thermotoga maritima, contains a novel iron-hydrogenase whose cellular activity is dependent upon tungsten.

Thermotoga maritima is the most thermophilic eubacterium currently known and grows up to 90 degrees C by a fermentative metabolism in which H2, CO2, and organic acids are end products. It was shown that the production of H2 is catalyzed by a single hydrogenase located in the cytoplasm. The addition of tungsten to the growth medium was found to increase both the cellular concentration of the hydrogenase and its in vitro catalytic activity by up to 10-fold, but the purified enzyme did not contain tungsten. It is a homotetramer of Mr 280,000 and contains approximately 20 atoms of Fe and 18 atoms of acid-labile sulfide/monomer. Other transition metals, including nickel (and also selenium), were present in only trace amounts (less than 0.1 atoms/monomer). The hydrogenase was unstable at both 4 and 23 degrees C, even under anaerobic conditions, but no activity was lost in anaerobic buffer containing glycerol and dithiothreitol. Under these conditions the enzyme was also quite thermostable (t50% approximately 1 h at 90 degrees C) but extremely sensitive to irreversible inactivation by O2 (t50% approximately 10 s in air). The optimum pH ranges for H2 evolution and H2 oxidation were 8.6-9.5 and greater than or equal to 10.4, respectively, and the optimum temperature for catalytic activity was above 95 degrees C. In contrast to mesophilic Fe hydrogenases, the T. maritima enzyme had very low H2 evolution activity, did not use T. maritima ferredoxin as an electron donor for H2 evolution, was inhibited by acetylene but not by nitrite, and exhibited EPR signals typical of [2Fe-2S]1+ clusters. Moreover, the oxidized enzyme did not exhibit the rhombic EPR signal that is characteristic of the catalytic iron-sulfur cluster of mesophilic Fe hydrogenases. These data suggest that T. maritima hydrogenase has a different FeS site and/or mechanism for catalyzing H2 production. The potential role of tungsten in regulating the activity of this enzyme is discussed.     

The solution structure and interactions of CheW from Thermotoga maritima.

Using protein from the hyperthermophile Thermotoga maritima, we have determined the solution structure of CheW, an essential component in the formation of the bacterial chemotaxis signaling complex. The overall fold is similar to the regulatory domain of the chemotaxis kinase CheA. In addition, interactions of CheW with CheA were monitored by nuclear magnetic resonance (NMR) techniques. The chemical shift perturbation data show the probable contacts that CheW makes with CheA. In combination with previous genetic data, the structure also suggests a possible binding site for the chemotaxis receptor. These results provide a structural basis for a model in which CheW acts as a molecular bridge between CheA and the cytoplasmic tails of the receptor.     

Motility and thermotactic responses of Thermotoga maritima.

Thermotoga maritima, a thermophilic eubacterium, is motile at temperatures ranging from 50 to 105 degrees C. The cells are propelled by a single flagellum which most of the time spins clockwise. Changes in the swimming direction ("tumbles") are achieved by short reversals of the direction of filament rotation. The average speed of swimming cells depends on the temperature, reaching a maximum value of about 60 microns/s at 85 degrees C. The cells show a thermotactic response to temporal temperature changes. When the temperature is raised, the rate of tumbles is increased, while decreasing temperature decreases the tumbling rate.     

Functional identification of an 8-oxoguanine specific endonuclease from Thermotoga maritima

To date, no 8-oxoguanine-specific endonuclease-coding gene has been identified in Thermotoga maritima of the order Thermotogales, although its entire genome has been deciphered. However, the hypothetical protein Tm1821 from T. maritima, has a helix-hairpin-helix motif that is considered to be important for DNA binding and catalytic activity. Here, Tm1821 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration. Tm1821 protein was found to efficiently cleave an oligonucleotide duplex containing 8-oxoguanine, but Tm1821 had little effect on other substrates containing modified bases. Moreover, Tm1821 strongly preferred DNA duplexes containing an 8-oxoguanine:C pair among oligonucleotide duplexes containing 8-oxoguanine paired with four different bases (A, C, G, or T). Furthermore, Tm1821 showed AP lyase activity and Schiff base formation with 8-oxoguanine in the presence of NaBH4, which suggests that it is a bifunctional DNA glycosylase. Tm1821 protein shares unique conserved amino acids and substrate specificity with an 8-oxoguanine DNA glycosylase from the hyperthermophilic archaeon. Thus, the DNA recognition and catalytic mechanisms of Tm1821 protein are likely to be similar to archaeal repair protein, although T. maritima is an eubacterium.     

Directed evolution of the alpha-L-fucosidase from Thermotoga maritima into an alpha-L-transfucosidase

The alpha-L-fucosidase from Thermotoga maritima (Tm alpha fuc) was converted into alpha-L-transfucosidase variants by directed evolution. The wild-type enzyme catalyzes oligosaccharide synthesis by transfer of a fucosyl residue from a pNP-fucoside donor to pNP-fucoside (self-condensation) with alpha-(1-->3) regioselectivity or pNP-galactoside (transglycosylation) with alpha-(1-->2) regioselectivity at low yields (7%). The wild-type enzyme was submitted to one cycle of mutagenesis, followed by rational recombination of the selected mutations, which allowed identification of variants with improved transferase activity. The transferase and hydrolytic kinetics of all the mutants were assessed by NMR methods and capillary electrophoresis. It was shown that the best mutant exhibited a dramatic 32-fold increase in the transferase/hydrolytic kinetic ratio, while keeping 60% of the overall wild-type enzyme activity. Accordingly, the maximum yield of a specific transglycosylation product [pNP-Gal-alpha-(1-->2)-Fuc] reached more than 60% compared to 7% with WT enzyme at equimolar and low concentrations of donor and acceptor (10 mM). Such an improvement was obtained with only three mutations (T264A, Y267F, L322P), which were all located in the second amino acid shell of the fucosidase active site. Molecular modeling suggested that some of these mutations (T264A, Y267F) cause a reorientation of the amino acids that are in direct contact with the substrates, resulting in a better docking energy. Such mutants with high transglycosidase activity may constitute novel enzymatic tools for the synthesis of fucooligosaccharides.