A hexokinase with broad sugar specificity from a thermophilic bacterium

A recombinant thermophilic Thermus caldophilus GK24 hexokinase, one of the ROK-type (repressor protein, open reading frames, and sugar kinase) proteins, exists uniquely as a 120 kDa molecule with four subunits (31 kDa), in contrast to eukaryotic and bacterial sugar kinases which are monomers or dimers. The optimal temperature and pH for the enzyme reaction are 70-80 degrees C and 7.5, respectively. This enzyme shows broad specificity toward glucose, mannose, glucosamine, allose, 2-deoxyglucose, and fructose. To understand the sugar specificity at a structural level, the enzyme-ATP/Mg2+-sugar binding complex models have been constructed. It has been shown that the sugar specificity is probably dependent on the interaction energy occurred by the positional proximity of sugars bound in the active site of the enzyme, which exhibits a tolerance to modification at C2 or C3 of glucose.     

Some properties of a beta-galactosidase from an extremely thermophilic bacterium

An inducible beta-galactosidase from an extremely thermophilic organism, Thermus strain 4-1A, has been isolated and partially purified. There were significant dissimilarities to T. aquaticus beta-galactosidase. It had a pl of 4.5, was inhibited by sulphydryl inhibitors and a number of transition metals, and was activated by EDTA and SH-containing reagents. The beta-galactosidase showed strong product inhibition, and weaker inhibition by some other mono- and disaccharides. It was very stable up to 90 degrees C at pH 8. On immobilization by diazonium linkage to porous glass, the pH optimum (6.0), the K(M) with ONPG (5mM) and the product inhibition were not altered.     

A novel thermostable branching enzyme from an extremely thermophilic bacterial species, Rhodothermus obamensis

A branching enzyme (EC 2.4.1.18) gene was isolated from an extremely thermophilic bacterium, Rhodothermus obamensis. The predicted protein encodes a polypeptide of 621 amino acids with a predicted molecular mass of 72 kDa. The deduced amino acid sequence shares 42-50% similarity to known bacterial branching enzyme sequences. Similar to the Bacillus branching enzymes, the predicted protein has a shorter N-terminal amino acid extension than that of the Escherichia coli branching enzyme. The deduced amino acid sequence does not appear to contain a signal sequence, suggesting that it is an intracellular enzyme. The R. obamensis branching enzyme was successfully expressed both in E. coli and a filamentous fungus, Aspergillus oryzae. The enzyme showed optimum catalytic activity at pH 6.0-6.5 and 65 degrees C. The enzyme was stable after 30 min at 80 degrees C and retained 50% of activity at 80 degrees C after 16 h. Branching activity of the enzyme was higher toward amylose than toward amylopectin. This is the first thermostable branching enzyme isolated from an extreme thermophile.     

Carbon monoxide pathway enzyme activities in a thermophilic anaerobic bacterium grown acetogenically and in a syntrophic acetate-oxidizing coculture

We recently isolated an acetate-oxidizing rodshaped eubacterium (AOR) which was capable of oxidizing acetate to CO₂ when grown in coculture with the hydrogenotrophic methanogen Methanobacterium sp. strain THF. The AOR was also capable of growing axenically on H₂CO₂ which it converted to acetate. Previous results for the acetate oxidizing coculture showed isotopic exchange between acetate and CO₂, suggesting that the AOR was using a pathway for acetate oxidation resembling a reveral of the acetogenic (carbon monoxide) pathway. In this study, it was found that production of ¹⁴CO₂ from ¹⁴CH₃COO^- by the coculture was inhibited by 200 M cyanide, while methanogenesis from H₂–CO₂ was unaffected, implying the involvement of carbon monoxide dehydrogenase (CODH) in acetate oxidation. CODH was present at 0.055 mol methyl viologen reduced min^-1 mg^-1 protein in extracts of Methanobacterium sp. strain THF, but was present in higher levels in the acetate oxidizing coculture and in the AOR grown axenically and on H₂–CO₂ (2.0 and 6.4 mol min^-1 mg^-1 protein respectively). Anaerobic activity stains for CODH in native polyacrylamide gels from the AOR coculture showed components co-migrating with bands from both organisms, as well as an additional band in extracts of the coculture. Formate dehydrogenase (FDH) was present in both the AOR coculture and monoculture but not in extracts of H₂–CO₂ grown cells of Methanobacterium sp. strain THF. Formyltetrahydrofolate (FTHF) synthetase was not detectable in extracts of the AOR monoculture or coculture, although it was found in high amounts in extracts of H₂–CO₂ grown cells of the thermophilic acetogen Acetogenium kivui. Extracts of H₂–CO₂ grown cells of the AOR showed a fluorescence spectrum typical of pterin derivatives. Bioassay for folates showed levels to be at anabolic rather than catabolic levels. It is possible that the AOR uses pterins distinct from folate for catabolism. Isocitrate dehydrogenase, a citric acid cycle enzyme, was also present in the AOR, but at anabolic levels and -ketoglutarate dehydrogenase was not detectable.Abbreviations (AOR) acetate-oxidizing rod - (CODH) carbon monoxide dehydrogenase - (FDH) formate dehydrogenase - (FTHF) formyltetrahydrofolate     

Purification and properties of a stable beta-glucosidase from an extremely thermophilic anaerobic bacterium.

The expression of a high-Mr sialogalactoprotein (gp580) on rat 13762NF mammary adenocarcinoma cells was identified and correlated with spontaneous metastatic potential to colonize lung [Steck & Nicolson (1983) Exp. Cell Res. 147, 255-267]. Using a highly metastatic tumour-cell clone, MTLn3, we isolated and characterized gp580 from cells growing in vitro and in vivo in the mammary fat-pads of Fischer 344 rats. The glycoprotein was extracted with 4 M-guanidinium chloride/4% Zwittergent 3-12 solution in the presence of proteinase inhibitors. The extracts were then subjected to dissociative CsCl-density-gradient centrifugation, gel filtration on Sepharose CL-2B columns and ion-exchange chromatography on DEAE-Sephacel. The isolated glycoprotein possessed low electrophoretic mobility in SDS/polyacrylamide gels, and after desialylation bound 125I-labelled peanut agglutinin. Electrophoresis of gp580 in polyacrylamide-gradient gels resulted in a diffuse but homogeneous migrating band of Mr approx. 55,000. After removal of carbohydrate, gp580 was demonstrated to have a protein core of Mr approx. 150,000. The gp580 had a high density (1.430 g/ml) on isopycnic centrifugation in 4 M-guanidinium chloride and was resistant to most proteinases and other degradative enzymes, suggesting a mucin-like structure. Amino acid and carbohydrate analyses revealed that gp580 has high contents of serine, threonine, glutamic acid, aspartic acid, glucosamine and galactosamine; several acidic and neutral oligosaccharides were obtained from alkaline-borohydride digests. Cellular localization studies suggested that gp580 is associated mainly with the cell-surface and extracellular-matrix fractions of MTLn3 cells.     

A chaperonin from a thermophilic bacterium, Thermus thermophilus, that controls refoldings of several thermophilic enzymes.

A chaperonin has been purified from a thermophilic bacterium, Thermus thermophilus. It consists of two kinds of proteins with approximate Mr 58,000 and 10,000 and shows a 7-fold rotational symmetry from the top view and a "football"-like shape from the side view under the electron microscopic view. Its weak ATPase activity is inhibited by sulfite and activated by bicarbonate. ATP causes change of its mobility in nondenaturating polyacrylamide gel electrophoresis. The T. thermophilus chaperonin can promote in vitro refolding of several guanidine HCl-denatured enzymes from thermophilic bacteria. At high temperatures above 60 degrees C, where the native enzymes are stable but their spontaneous refoldings upon dilution of guanidine HCl fail, the chaperonin induces productive refolding in an ATP-dependent manner. No or very poor refolding is induced when the chaperonin is added to the solution aged after dilution. An excess amount of the chaperonin is inhibitory for refolding. At middle temperatures (30-50 degrees C), where spontaneous refoldings of the enzymes occur, the chaperonin arrests refolding in the absence of ATP and refolding is induced when ATP is supplemented. At temperatures below 20 degrees C, where spontaneous refoldings also occur, the chaperonin arrests the refolding but ATP does not induce refolding.     

A thermophilic amyloglucosidase fromHalobacterium sodomense,a halophilic bacterium from the Dead Sea

Halobacterium sodomense, a halophilic bacterium from the Dead Sea, degraded starch to glucose by means of an extracellular amyloglucosidase with a temperature optimum of around 65°C in the presence of 1.4 M NaCl, and around 75°C in the presence of 3.9 M NaCl. The enzyme required salt concentrations higher than 1 M for optimal activity, NaCl, KCl, and MgCl₂ being equally suitable as activators. The optimum pH was 7.5.H. sodomense culture supernatants showed only a very low maltose degrading activity. H. sodomense excreted amyloglucosidase constitutively, and relatively high activities were found in cultures grown in the absence of starch; when glucose was added to the growth medium, the amount of enzyme excreted into the medium decreased.     

A complete shikimate pathway in Toxoplasma gondii: an ancient eukaryotic innovation

The shikimate pathway is essential for survival of the apicomplexan parasites Plasmodium falciparum, Toxoplasma gondii and Cryptosporidium parvum. As it is absent in mammals it is a promising therapeutic target. Herein, we describe the genes encoding the shikimate pathway enzymes in T. gondii. The molecular arrangement and phylogeny of the proteins suggests homology with the eukaryotic fungal enzymes, including a pentafunctional AROM. Current rooting of the eukaryotic evolutionary tree infers that the fungi and apicomplexan lineages diverged deeply, suggesting that the arom is an ancient supergene present in early eukaryotes and subsequently lost or replaced in a number of lineages.     

Gluconeogenesis: An ancient biochemical pathway with a new twist

Synthesis of sugars from simple carbon sources is critical for survival of animals under limited nutrient availability. Thus, sugar-synthesizing enzymes should be present across the entire metazoan spectrum. Here, we explore the evolution of glucose and trehalose synthesis using a phylogenetic analysis of enzymes specific for the two pathways. Our analysis reveals that the production of trehalose is the more ancestral biochemical process, found in single cell organisms and primitive metazoans, but also in insects. The gluconeogenic-specific enzyme glucose-6-phosphatase (G6Pase) first appears in Cnidaria, but is also present in Echinodermata, Mollusca and Vertebrata. Intriguingly, some species of nematodes and arthropods possess the genes for both pathways. Moreover, expression data from Drosophila suggests that G6Pase and, hence, gluconeogenesis, initially had a neuronal function. We speculate that in insects—and possibly in some vertebrates—gluconeogenesis may be used as a means of neuronal signaling.     

A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI-1.

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.     

Development of a 2-pyrrolidone biosynthetic pathway in Corynebacterium glutamicum by engineering an acetyl-CoA balance route

Amino Acids - 2-Pyrrolidone is widely used in the textile and pharmaceutical industries. Here, we established a 2-pyrrolidone biosynthesis pathway in Corynebacterium glutamicum, by expressing...     

A 31P-NMR study on multilamellar liposomes formed from the lipids of a thermophilic bacterium

The membrane lipids of a thermophilic bacterium, Thermus SPS11, isolated from thermal springs in S?o Pedro do Sul, Portugal, were fractionated by chromatography on silica gel. The total lipid extract was found to contain one major phospholipid (PL), which accounts for about 90% of the total lipid phosphorous, and one major glycolipid (GL), which accounts for about 95% of the total carbohydrate in the non-phospholipid fraction. The membranes also contain about 11% by weight of a complex mixture of carotenoids (CA). Multilamellar liposomes, in excess water, formed from PL and mixtures of PL with GL and CA in proportions found in the natural membrane were investigated by proton-decoupled 31P-nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. All mixtures examined were found to be in a lamellar phase with disordered hydrophobic chains with no evidence for "non-bilayer structures" between 23 degrees and 85 degrees C. Compared to bilayers formed from pure PL or mixtures of PL and CA, significantly larger values for the chemical shift anisotropy of the 31P-NMR powder patterns were obtained from bilayers formed from mixtures of PL and GL, at temperatures above 75 degrees C, and mixtures of PL, GL and CA at all temperatures examined. These differences are interpreted in terms of changes in the order of the bilayer and/or changes in the orientation of the phosphate moiety of PL. The significance of these results to the thermophily of the bacterium is discussed.     

Characterization of a thermostable DNA photolyase from an extremely thermophilic bacterium, Thermus thermophilus HB27.

The photolyase gene from Thermus thermophilus was cloned and sequenced. The characteristic absorption and fluorescence spectra of the purified T. thermophilus photolyase suggested that the protein has flavin adenine dinucleotide as a chromophore. The second chromophore binding site was not conserved in T. thermophilus photolyase. The purified enzyme showed light-dependent photoreactivation activity in vitro at 35 and 65 degrees C and was stable when subjected to heat and acidic pH.     

Gene cloning and overexpression of a geranylgeranyl diphosphate synthase of an extremely thermophilic bacterium, Thermus thermophilus

A geranylgeranyl diphosphate (GGPP) synthase gene of an extremely thermophilic bacterium, Thermus thermophilus, was cloned and sequenced. T. thermophilus GGPP synthase, overexpressed in Escherichia coli cells as a glutathione S-transferase fusion protein, was purified and characterized. The fusion protein, retaining thermostability, formed a homodimer, and showed higher specific activity than did a partially purified thermostable enzyme previously reported. Optimal reaction conditions and kinetic parameters were also examined. The deduced amino acid sequence indicated that T. thermophilus GGPP synthase was excluded from the group of bacterial type GGPP synthases and lacked the insertion amino acid residues in the first aspartate-rich motif as do archaeal and eukaryotic short-chain prenyltransferases.     

Cloning and sequence of a type I pullulanase from an extremely thermophilic anaerobic bacterium,Caldicellulosiruptor saccharolyticus

A gene coding for a pullulanase from the obligately anaerobic, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus has been cloned in Escherichia coli. It consists of an open reading frame (pulA) of 2478 bp which codes for an enzyme of 95 732 Da and is flanked by two other open reading frames. A truncated version of the gene which lacks 381 bp of 5′-sequence also has pullulanase activity and it appears that the amino-terminal portion of the gene is not essential for either activity or thermostability. Amino acid sequence comparisons with other published amylases and pullulanases showed that it possesses homology to the four key regions common to these enzymes.     

Kosmotoga pacifica sp. nov., a thermophilic chemoorganoheterotrophic bacterium isolated from an East Pacific hydrothermal sediment

A novel strictly anaerobic thermophilic heterotrophic bacterium, strain SLHLJ1^T, was isolated from a Pacific hydrothermal sediment. Cells were Gram-negative coccobacilli (approximately 1.0 × 0.6 μm) with a toga. It grew at temperatures between 33 and 78 °C (optimum 70 °C). Elemental sulphur and l-cystine stimulated its growth. It contained C_16:0, C_16:1 ω11c, C_18:0 and C_18:1 ω9c as major fatty acids (>5 %), 3 phospholipids and 2 glycolipids as polar lipids. Its DNA G+C content was 43.7 mol%. Phylogenetic analyses based on 16S rRNA gene sequences placed strain SLHLJ1^T within the family Thermotogaceae. The novel isolate was most closely related to Kosmotoga arenicorallina (97.93 % 16S rRNA gene sequence similarity), K. olearia (92.43 %) and K. shengliensis (92.17 %). On the basis of phenotypic, chemotaxonomic and phylogenetic comparisons with its closest relatives, we propose its assignment to a novel species of the genus Kosmotoga. The name Kosmotoga pacifica sp. nov. is proposed with strain SLHLJ1^T (=DSM 26965^T = JCM 19180^T = UBOCC 3254^T) as the type species.     

A cell-associated oligo-1,6-alpha-glucosidase from an extremely thermophilic anaerobic bacterium, Thermoanaerobium Tok6-B1.

Cell-associated oligo-1,6-alpha-glucosidase (EC 3.2.1.10) was isolated from Thermoanaerobium Tok6-B1 grown on starch-containing medium. Activity was purified 11.4-fold by salt precipitation, gel filtration, hydroxyapatite and anion-exchange chromatography. Molecular mass was determined as 30,000 by SDS/polyacrylamide-gel electrophoresis and 33,000 by analytical gel filtration. The probable order of specificity was p-nitrophenyl-alpha D-glucose greater than-isomaltose greater than-isomaltotriose greater than-panose greater than-nigerose and no activity was shown against malto-oligosaccharides, melezitose, melibiose, raffinose, cellobiose, sophorose, gentiobiose, lactose, pullulan, dextran or amylose. The optima for activity and stability were between pH 5.6 and 7.0 and the half-life at pH 6.5 was 1000 min at 70 degrees C and 20 min at 76 degrees C. Activity was stabilized by substrate, Mg2+, Mn2+ and Ca2+, but was destabilized by Zn2+ and EDTA. N-Ethylmaleimide, glucose and 1-O-methyl-alpha D-glucose were inhibitory but 1-O-methyl-beta D-glucose stimulated activity. The activation energy (Ea) was 109 kJ/mol.