Dihydrodipicolinate synthase from Thermotoga maritima

DHDPS (dihydrodipicolinate synthase) catalyses the branch point in lysine biosynthesis in bacteria and plants and is feedback inhibited by lysine. DHDPS from the thermophilic bacterium Thermotoga maritima shows a high level of heat and chemical stability. When incubated at 90 degrees C or in 8 M urea, the enzyme showed little or no loss of activity, unlike the Escherichia coli enzyme. The active site is very similar to that of the E. coli enzyme, and at mesophilic temperatures the two enzymes have similar kinetic constants. Like other forms of the enzyme, T. maritima DHDPS is a tetramer in solution, with a sedimentation coefficient of 7.2 S and molar mass of 133 kDa. However, the residues involved in the interface between different subunits in the tetramer differ from those of E. coli and include two cysteine residues poised to form a disulfide bond. Thus the increased heat and chemical stability of the T. maritima DHDPS enzyme is, at least in part, explained by an increased number of inter-subunit contacts. Unlike the plant or E. coli enzyme, the thermophilic DHDPS enzyme is not inhibited by (S)-lysine, suggesting that feedback control of the lysine biosynthetic pathway evolved later in the bacterial lineage.     

Novel inositol catabolic pathway in Thermotoga maritima

myo‐inositol (MI) is a key sugar alcohol component of various metabolites, e.g. phosphatidylinositol‐based phospholipids that are abundant in animal and plant cells. The seven‐step pathway of MI degradation was previously characterized in various soil bacteria including Bacillus subtilis. Through a combination of bioinformatics and experimental techniques we identified a novel variant of the MI catabolic pathway in the marine hyperthermophilic bacterium Thermotoga maritima. By using in vitro biochemical assays with purified recombinant proteins we characterized four inositol catabolic enzymes encoded in the TM0412–TM0416 chromosomal gene cluster. The novel catabolic pathway in T. maritima starts as the conventional route using the myo‐inositol dehydrogenase IolG followed by three novel reactions. The first 2‐keto‐myo‐inositol intermediate is oxidized by another, previously unknown NAD‐dependent dehydrogenase TM0412 (named IolM), and a yet unidentified product of this reaction is further hydrolysed by TM0413 (IolN) to form 5‐keto‐l ‐gluconate. The fourth step involves epimerization of 5‐keto‐l ‐gluconate to d ‐tagaturonate by TM0416 (IolO). T. maritima is unable to grow on myo‐inositol as a single carbon source. The determined in vitro specificity of the InoEFGK (TM0418–TM0421) transporter to myo‐inositol‐phosphate suggests that the novel pathway in Thermotoga utilizes a phosphorylated derivative of inositol.     

Cloning and characterization of Thermotoga maritima beta-glucuronidase

The putative beta-glucuronidase from Thermotoga maritima, comprising 563 amino acid residues conjugated with a Hisx6 tag, was cloned and expressed in Escherichia coli. The enzyme has a moderately broad specificity, hydrolysing a range of p-nitrophenyl glycoside substrates, but has greatest activity on p-nitrophenyl beta-D-glucosiduronic acid (kcat=68 s(-1), kcat/K(M)= 4.5x10(5) M(-1) s(-1)). The enzyme also shows a relatively broad pH-dependence with activity from pH4.5 to 7.5 and a maximum at pH6.5. As expected the enzyme is stable towards heat denaturation, with a half life of 3h at 85 degrees C, in contrast to the mesophilic E. coli enzyme, which has a half life of 2.6h at 50 degrees C. The identity of the catalytic nucleophile was confirmed as Glu476 within the sequence VTEFGAD by trapping the glycosyl-enzyme intermediate using the mechanism-based inactivator, 2-deoxy-2-fluoro-beta-D-glucosyluronic acid fluoride and identifying the labeled peptide in peptic digests by HPLC-MS/MS methodologies. Consistent with this, the Glu476Ala mutant was shown to be hydrolytically inactive. The acid/base catalyst was confirmed as Glu383 by generation and kinetic analysis of enzyme mutants modified at that position, Glu383Ala and Glu383Gln. The demonstration of activity rescue by azide is consistent with the proposed role for this residue. This enzyme therefore appears suitable for use in enzymatic oligosaccharide synthesis in either the transglycosylation mode or by use of glycosynthase and thioglycoligase approaches.     

Characterization of RNase P from Thermotoga maritima

The protein subunit of RNase P from a thermophilic bacterium, Thermotoga maritima, was overexpressed in and purified from Escherichia coli. The cloned protein was reconstituted with the RNA subunit transcribed in vitro. The temperature optimum of the holoenzyme is near 50°C, with no enzymatic activity at 65°C or above. This finding is in sharp contrast to the optimal growth temperature of T.maritima, which is near 80°C. However, in heterologous reconstitution experiments in vitro with RNase P subunits from other species, we found that the protein subunit from T.maritima was responsible for the comparative thermal stability of such complexes.     

Mutational analysis of endonuclease V from Thermotoga maritima

Endonuclease V nicks damaged DNA at the second phosphodiester bond 3' to inosine, uracil, mismatched bases, or abasic (AP) sites. Alanine scanning mutagenesis was performed in nine conserved positions of Thermotoga maritima endonuclease V to identify amino acid residues involved in recognition or endonucleolytic cleavage of these diverse substrates. Alanine substitution at D43, E89, and D110 either abolishes or substantially reduces inosine cleavage activity. These three mutants gain binding affinity for binding to double-stranded or single-stranded inosine substrates in the absence of a metal ion, suggesting that these residues may be involved in coordinating catalytic metal ion(s). Y80A, H116A, and, to a lesser extent, R88A demonstrate reduced affinities for double-stranded or single-stranded inosine substrates or nicked products. The lack of tight binding to a nicked inosine product accounts for the increased rate of turnover of inosine substrate since the product release is less rate-limiting. Y80A, R88A, and H116A fail to cleave AP site substrates. Their activities toward uracil substrates are in the following order: H116A > R88A > Y80A. These residues may play a role in substrate recognition. K139A maintains wild-type binding affinity for binding to double-stranded and single-stranded inosine substrate, but fails to cleave AP site and uracil substrate efficiently, suggesting that K139 may play a role in facilitating non-inosine substrate cleavage.     

Identification of methylation sites in Thermotoga maritima chemotaxis receptors

Adaptation in bacterial chemotaxis involves reversible methylation of specific glutamate residues within the cytoplasmic domains of methyl-accepting chemotaxis proteins. The specific sites of methylation in Salmonella enterica and Escherichia coli chemoreceptors, identified 2 decades ago, established a consensus sequence for methylation by methyltransferase CheR. Here we report the in vitro methylation of chemoreceptors from Thermotoga maritima, a hyperthermophile that has served as a useful source of chemotaxis proteins for structural analysis. Sites of methylation have been identified by liquid chromatography-mass spectrometry/mass spectrometry. Fifteen sites of methylation were identified within the cytoplasmic domains of four different T. maritima chemoreceptors. The results establish a consensus sequence for chemoreceptor methylation sites in T. maritima that is distinct from the previously identified consensus sequence for E. coli and S. enterica. These findings suggest that consensus sequences for posttranslational modifications in one organism may not be directly extrapolated to analogous modifications in other bacteria.     

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.     

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.     

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%).     

Diversity and Versatility of the Thermotoga maritima Sugar Kinome

Sugar phosphorylation is an indispensable committed step in a large variety of sugar catabolic pathways, which are major suppliers of carbon and energy in heterotrophic species. Specialized sugar kinases that are indispensable for most of these pathways can be utilized as signature enzymes for the reconstruction of carbohydrate utilization machinery from microbial genomic and metagenomic data. Sugar kinases occur in several structurally distinct families with various partially overlapping as well as yet unknown substrate specificities that often cannot be accurately assigned by homology-based techniques. A subsystems-based metabolic reconstruction combined with the analysis of genome context and followed by experimental testing of predicted gene functions is a powerful approach of functional gene annotation. Here we applied this integrated approach for functional mapping of all sugar kinases constituting an extensive and diverse sugar kinome in the thermophilic bacterium Thermotoga maritima. Substrate preferences of 14 kinases mainly from the FGGY and PfkB families were inferred by bioinformatics analysis and biochemically characterized by screening with a panel of 45 different carbohydrates. Most of the analyzed enzymes displayed narrow substrate preferences corresponding to their predicted physiological roles in their respective catabolic pathways. The observed consistency supports the choice of kinases as signature enzymes for genomics-based identification and reconstruction of sugar utilization pathways. Use of the integrated genomic and experimental approach greatly speeds up the identification of the biochemical function of unknown proteins and improves the quality of reconstructed pathways.     

Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity

Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na(+) but displayed the highest activity in the presence of millimolar levels of both Na(+) and K(+). Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na(+) binding sites and one K(+) binding site are involved in enzyme activation. The affinity of the site that binds Na(+) first is increased with increasing K(+) concentration. In contrast, only one Na(+) binding site (K(+)-dependent) and one K(+) binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K(+)-independent Na(+) binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PP(i)-synthesizing activity, which also required Na(+) but was inhibited by K(+). These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na(+)-dependent H(+)-PPase homologues and may be an analogue of Na(+),K(+)-ATPase.     

Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima

AIMS: Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima for the hydrolysis of lactose and the production of galacto-oligosaccharides. METHODS AND RESULTS: A putative beta-galactosidase gene of Thermotoga maritima was expressed in Escherichia coli as a carboxyl terminal His-tagged recombinant enzyme. The gene encoded a 1100-amino acid protein with a calculated molecular weight of 129,501. The expressed enzyme was purified by heat treatment, His-tag affinity chromatography, and gel filtration. The optimum temperatures for beta-galactosidase activity were 85 and 80 degrees C with oNPG and lactose, respectively. The optimum pH value was 6.5 for both oNPG and lactose. In thermostability experiments, the enzyme followed first-order kinetics of thermal inactivation and its half-life times at 80 and 90 degrees C were 16 h and 16 min, respectively. Mn2+ was the most effective divalent cation for beta-galactosidase activity on both oNPG and lactose. The Km and Vmax values of the thermostable enzyme for oNPG at 80 degrees C were 0.33 mm and 79.6 micromol oNP min(-1) mg(-1). For lactose, the Km and Vmax values were dependent on substrate concentrations; 1.6 and 63.3 at lower concentrations up to 10 mm of lactose and 27.8 mm and 139 micromol glucose min(-1) mg(-1) at higher concentrations, respectively. The enzyme displayed non-Michaelis-Menten reaction kinetics with substrate activation, which was explained by simultaneous reactions of hydrolysis and transgalactosylation. CONCLUSIONS: The results suggest that the thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galacto-oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of hydrolysis and transgalactosylation performed by beta-galactosidase of hyperthermophilic bacteria.     

Trends in Codon and Amino Acid Usage in Thermotoga maritima

The usage of synonymous codons and the frequencies of amino acids were investigated in the complete genome of the bacterium Thermotoga maritima using a multivariate statistical approach. The GC3 content of each gene was the most prominent source of variation of codon usage. Surprisingly the usage of UGU and UGC (synonymous triplets coding for Cys, the least frequent amino acid in this species) was detected as the second most prominent source of variation. However, this result is probably an artifact due to the very low frequency of Cys together with the nonbiased composition of this genome. The third trend was related to the preferential usage of a subset of codons among highly expressed genes, and these triplets are presumed to be translationally optimal. Concerning the amino acid usage, the hydropathy level of each protein (and therefore the frequency of charged residues) was the main trend, while the second factor was related to the frequency of usage of the smaller residues, suggesting that the cell economy strongly influences the architecture of the proteins. The third axis of the analysis discriminated the usage of Phe, Tyr, Trp (aromatic residues) plus Cys, Met, and His. These six residues have in common the property of being the preferential targets of reactive oxygen species, and therefore the anaerobic condition of T. maritima is an important factor for the amino acid frequencies. Finally, the Cys content of each protein was the fourth trend. Received: 22 June 2001 / Accepted: 1 October 2001     

极端耐热纤维素酶Cel74的表达、纯化与特性

从海栖热袍菌中克隆出编码热稳定性的纤维素酶基因,以热激载体pHsh为表达质粒,构建重组质粒phsh—Ceff4,并转化至大肠杆菌中进行表达。基因表达产物通过热处理和离子交换层析,重组酶纯度达电泳纯。对纯化的重组酶酶学性质研究表明,最适反应温度85℃,最适反应pH4．6,pH4．5—6．0之间酶的相对酶活在80％以上。Co^2＋对酶活性有促进作用,Ca^2＋、Mg^2＋、Zn^2＋不影响酶活性,而Cu^2＋、Ni^2＋、Mn^2＋对酶活性有抑制作用。     

Suppressive subtractive hybridization detects extensive genomic diversity in Thermotoga maritima

Comparisons between genomes of closely related bacteria often show large variations in gene content, even between strains of the same species. Such studies have focused mainly on pathogens; here, we examined Thermotoga maritima, a free-living hyperthermophilic bacterium, by using suppressive subtractive hybridization. The genome sequence of T. maritima MSB8 is available, and DNA from this strain served as a reference to obtain strain-specific sequences from Thermotoga sp. strain RQ2, a very close relative (approximately 96% identity for orthologous protein-coding genes, 99.7% identity in the small-subunit rRNA sequence). Four hundred twenty-six RQ2 subtractive clones were sequenced. One hundred sixty-six had no DNA match in the MSB8 genome. These differential clones comprise, in sum, 48 kb of RQ2-specific DNA and match 72 genes in the GenBank database. From the number of identical clones, we estimated that RQ2 contains 350 to 400 genes not found in MSB8. Assuming a similar genome size, this corresponds to 20% of the RQ2 genome. A large proportion of the RQ2-specific genes were predicted to be involved in sugar transport and polysaccharide degradation, suggesting that polysaccharides are more important as nutrients for this strain than for MSB8. Several clones encode proteins involved in the production of surface polysaccharides. RQ2 encodes multiple subunits of a V-type ATPase, while MSB8 possesses only an F-type ATPase. Moreover, an RQ2-specific MutS homolog was found among the subtractive clones and appears to belong to a third novel archaeal type MutS lineage. Southern blot analyses showed that some of the RQ2 differential sequences are found in some other members of the order Thermotogales, but the distribution of these variable genes is patchy, suggesting frequent lateral gene transfer within the group.     

Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima

Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium Thermotoga maritima has been purified and characterized (k_cat ~100 s^?1). It was found that the same enzyme also had the ability to catalyze the production of acetaldehyde and CO₂ from pyruvate, an activity of pyruvate decarboxylase (PDC) at a rate approximately 10% of its AHAS activity. Compared to the catalytic subunit, reconstitution of the individually expressed and purified catalytic and regulatory subunits of the AHAS stimulated both activities of PDC and AHAS. Both activities had similar pH and temperature profiles with an optimal pH of 7.0 and temperature of 85 °C. The enzyme kinetic parameters were determined, however, it showed a non-Michaelis-Menten kinetics for pyruvate only. This is the first report on the PDC activity of an AHAS and the second bifunctional enzyme that might be involved in the production of ethanol from pyruvate in hyperthermophilic microorganisms.     

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.     

Sequencing and expression of additional xylanase genes from the hyperthermophile Thermotoga maritima FjSS3B.1

Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking-PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87 degrees C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity to xynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.