Characterization of the maltooligosyl trehalose synthase from the thermophilic archaeon Sulfolobus acidocaldarius

We report the molecular characterization and the detailed study of the recombinant maltooligosyl trehalose synthase mechanism from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The mts gene encoding a maltooligosyl trehalose synthase was overexpressed in Escherichia coli using the T7-expression system. The purified recombinant enzyme exhibited optimum activity at 75 degrees C and pH 5 with citrate-phosphate buffer and retained 60% of residual activity after 72 h of incubation at 80 degrees C. The recombinant enzyme was active on maltooligosaccharides such as maltotriose, maltotetraose, maltopentaose and maltoheptaose. Investigation of the enzyme action on maltooligosaccharides has brought much insight into the reaction mechanism. Results obtained from thin-layer chromatography suggested a possible mechanism of action for maltooligosyl trehalose synthase: the enzyme, after converting the alpha-1,4-glucosidic linkage to an alpha-1,1-glucosidic linkage at the reducing end of maltooligosaccharide glc(n) is able to release glucose and maltooligosaccharide glc(n-1) residues. And then, the intramolecular transglycosylation and the hydrolytic reaction continue, with the maltooligosaccharide glc(n-1) until the initial maltooligosaccharide is reduced to maltose. An hypothetical mechanism of maltooligosyl trehalose synthase acting on maltooligosaccharide is proposed.     

A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus

The phosphotriesterase PTE, identified in the soil bacterium Pseudomonas diminuta, is thought to have evolved in the last several decades to degrade the pesticide paraoxon with proficiency approaching the limit of substrate diffusion (k(cat)/K(M) of 4 x 10(7)M(-1)s(-1)). It belongs to the amidohydrolase superfamily, but its evolutionary origin remains obscure. The enzyme has important potentiality in the field of the organophosphate decontamination. Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus solfataricus, showing low but significant and extremely thermostable paraoxonase activity (k(cat)/K(M) of 4 x 10(3)M(-1)s(-1)). Looking for other thermostable phosphotriesterases we assayed, among others, crude extracts of Sulfolobus acidocaldarius and detected activity. Since the genome of S. acidocaldarius has been recently reported, we identified there an open reading frame highly related to the S. solfataricus enzyme. The gene was cloned, the protein overexpressed in Escherichia coli, purified, and proven to have paraoxonase activity. A comparative analysis detected some significant differences between the two archaeal enzymes.     

Glycogen-bound polyphosphate kinase from the archaebacterium Sulfolobus acidocaldarius.

Glycogen-bound polyphosphate kinase has been isolated from a crude extract of Sulfolobus acidocaldarius by isopycnic centrifugation in CsCl. Divalent cations (Mn2+ greater than Mg2+) stimulated the reaction. The enzyme does not require the presence of histones for its activity; it is inhibited strongly by phosphate and slightly by fluoride. The protein from the glycogen complex migrated in a sodium dodecyl sulfate-polyacrylamide gel as a 57-kilodalton protein band; after isoelectric focusing it separated into several spots in the pH range of 5.6 to 6.7.     

Two distinct human cloned T cell lines that exhibit natural killer-like and anti-human effector activities

Cloned T cell lines from mixed lymphocyte cultures stimulated with autologous Epstein Barr virus- (EBV) transformed lymphoblastoid cell line (LCL) cells were established using a limiting dilution technique in the presence of T cell growth factor (TCGF). The T cell lines included two distinct clones of cytotoxic T cells (Tc) in addition to EBV-specific Tc. A cytotoxic profile of one cloned line was similar to that of endogenous NK cells in peripheral blood. The other cloned Tc line showed an anti-human cytotoxicity. The susceptible targets for this latter Tc line were various human cells, including autologous LCL and peripheral blood lymphocytes (PBL), stimulated with pokeweed mitogen, along with NK-sensitive and NK-resistant cell lines. Weak cytotoxic activity was detected against various xenogeneic cell lines. Furthermore, autologous and allogeneic cloned T cell lines were resistant to killing by the anti-human effector clone. These t wo distinct cloned Tc lines expressed the Leu-1 and Leu-2a antigens, which are markers of suppressor/cytotoxic T cells.     

Two Holliday junction resolving enzymes in Sulfolobus solfataricus

Holliday junction resolving enzymes bind specifically to four-way DNA junctions created by the process of homologous recombination, cleaving them to yield recombinant duplex DNA products. Homologous recombination is known to occur in the third domain of life, the archaea, and may constitute a simplified model for the corresponding eucaryal pathway, but has not been well characterised. Identification of a gene encoding an archaeal Holliday junction resolving enzyme, Hjc, has recently been reported in the euryarchaea, and an activity has been observed in the hyperthermophilic crenarchaeote Sulfolobus solfataricus. Here we report the identification, heterologous expression and characterisation of the Hjc protein from Sulfolobus. We demonstrate that Sulfolobus has two distinct junction resolving enzymes, Hjc and Hje, with differing substrate specificities.     

嗜酸热硫化叶菌麦芽寡糖基海藻糖合酶基因的克隆和表达

麦芽寡糖基海藻糖合酶( Maltodigosyltrehalose synthase;MTSase)是近年来在一些微生物中发现的新型分子内糖苷转移酶,能将淀粉或淀粉部分水解物(大于3个葡糖基)的糖链还原末端的α-1,4糖苷键转化为α-1,1糖苷键,生成具海藻糖末端结构的产物[1],如下所示: Gn为聚合度n(n＞3)的麦芽寡糖,Gn-2-T为麦芽寡糖基海藻糖,-T为海藻糖基.     

嗜酸热硫化叶菌麦芽寡粉基海藻糖合酶基因的克隆和表达

The gene of MTSase (maltooligosyltrehalose synthase) from Sulfolobus acidocaldarius ATCC49426 was amplified by PCR. The primers were designed according to the published sequence of homologous gene from Sulfolobus acidocaldarius ATCC33909. This gene was inserted into the plasmid pBV220 and the resultant recombinant plasmid pBV220-GT was transformed to E. coli DH5 alpha. The activity of recombinant enzyme was about 10 u/g(wet cell). In order to improve the expression level of target protein, some nucleotides in the 3' and 5' of the gene were modified to optimize the second structure of mRNA by PCR amplification using the new primers devised according to the biosoftware GOLDKEY2.0. As a result, the activity of recombinant enzyme increase to 19.8 u/g(wet cell). Then, the helping plasmid pUBS520 which carried the gene encoding the tRNA of rare codons AGG and AGA was transformed to the recombinant strain. But it took little effect.     

Characterization and purification of a membrane-bound archaebacterial pyrophosphatase from Sulfolobus acidocaldarius.

Plasma membranes of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius (DSM 639) display a pyrophosphate-hydrolyzing activity [M. Lübben & G. Sch?fer (1987) Eur. J. Biochem. 164, 533-540]. In our present work, we solubilized and purified this pyrophosphatase to homogeneity. It consists of a single subunit with a molecular mass of 17-18 kDa, forming an oligomer of 70 kDa under native conditions. Edman degradation revealed 30 amino acids of the N-terminus. The enzyme cleaves phosphoric-acid-anhydride bonds independently of monovalent or divalent cations. Temperature and pH optima of 75 degrees C and 3.5-3.7, respectively, characterize it as an ectoenzyme. Membrane lipids of Sulfolobus stimulate the activity. The dolichol-pyrophosphate-complexing peptide-antibiotic bacitracin inhibited growth of Sulfolobus. A possible function of the acid pyrophosphatase is the hydrolysis of dolichol pyrophosphate in connection with glycosylation reactions of membrane proteins.     

Purification and characterization of DNA polymerase from the archaebacterium Sulfolobus acidocaldarius.

DNA polymerase has been purified about 25,000-fold from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. On SDS-PAGE the enzyme was observed to have a molecular weight of 100 kDa and to be about 90% pure. The native molecular weight was 108 kDa indicating that the enzyme is composed of a single polypeptide. Activity gel analysis showed an active polypeptide of about 100 kDa. Under conditions promoting proteolysis this polypeptide was degraded to a slightly smaller form of 98 kDa. The enzyme has been characterized in respect to optimal assay conditions, template specificity, sensitivity to inhibitors and associated nuclease activities. The high temperature optimum of 65 degrees C should be emphasized. No substantial similarities have been found with other prokaryotic and eukaryotic DNA polymerases, although the enzyme bears certain resemblances to prokaryotic non-replicative polymerases.     

Archaebacterial malate dehydrogenases. The enzymes from the thermoacidophilic organisms Sulfolobus acidocaldarius and Thermoplasma acidophilum show A-side stereospecificity for NAD+.

The stereoselective transfer of hydrogen from NADH to oxaloacetate catalysed by malate dehydrogenases (EC 1.1.1.37) from the thermoacidophilic archaebacteria Sulfolobus acidocaldarius and Thermoplasma acidophilum was studied by the p.m.r. method described by Zhou & Wong [(1981) J. Biochem. Biophys. Methods 4, 329-338]. Both enzymes are A-side (pro-R) stereospecific for NADH.     

The Glycogen-Bound Polyphosphate Kinase from Sulfolobus acidocaldarius Is Actually a Glycogen Synthase

Inorganic polyphosphate (polyP) is obtained by the polymerization of the terminal phosphate of ATP through the action of the enzyme polyphosphate kinase (PPK). Despite the presence of polyP in every living cell, a gene homologous to that of known PPKs is missing from the currently sequenced genomes of Eukarya, Archaea, and several bacteria. To further study the metabolism of polyP in Archaea, we followed the previously published purification procedure for a glycogen-bound protein of 57 kDa with PPK as well as glycosyl transferase (GT) activities from Sulfolobus acidocaldarius (R. Skórko, J. Osipiuk, and K. O. Stetter, J. Bacteriol. 171:5162–5164, 1989). In spite of using recently developed specific enzymatic methods to analyze polyP, we could not reproduce the reported PPK activity for the 57-kDa protein and the polyP presumed to be the product of the reaction most likely corresponded to glycogen-bound ATP under our experimental conditions. Furthermore, no PPK activity was found associated to any of the proteins bound to the glycogen-protein complex. We cloned the gene corresponding to the 57-kDa protein by using reverse genetics and functionally characterized it. The predicted product of the gene did not show similarity to any described PPK but to archaeal and bacterial glycogen synthases instead. In agreement with these results, the recombinant protein showed only GT activity. Interestingly, the GT from S. acidocaldarius was phosphorylated in vivo. In conclusion, our results convincingly demonstrate that the glycogen-protein complex of S. acidocaldarius does not contain a PPK activity and that what was previously reported as being glycogen-bound PPK is a bacterial enzyme-like thermostable glycogen synthase.     

Discrete cytosolic macromolecular BRAF complexes exhibit distinct activities and composition

As a central element within the RAS/ERK pathway, the serine/threonine kinase BRAF plays a key role in development and homeostasis and represents the most frequently mutated kinase in tumors. Consequently, it has emerged as an important therapeutic target in various malignancies. Nevertheless, the BRAF activation cycle still raises many mechanistic questions as illustrated by the paradoxical action and side effects of RAF inhibitors. By applying SEC‐PCP‐SILAC, we analyzed protein–protein interactions of hyperactive BRAF^V600E and wild‐type BRAF (BRAF^WT). We identified two macromolecular, cytosolic BRAF complexes of distinct molecular composition and phosphorylation status. Hyperactive BRAF^V600E resides in large complexes of higher molecular mass and activity, while BRAF^WT is confined to smaller, slightly less active complexes. However, expression of oncogenic K‐Ras^G12V, either by itself or in combination with RAF dimer promoting inhibitors, induces the incorporation of BRAF^WT into large, active complexes, whereas pharmacological inhibition of BRAF^V600E has the opposite effect. Thus, the quaternary structure of BRAF complexes is shaped by its activation status, the conformation of its kinase domain, and clinically relevant inhibitors.     

Partial Purification and Characterization of Thermostable Acid Phosphatase from Thermoacidophilic Archaeon Sulfolobus acidocaldarius

Thermostable acid phosphatase (APase) from thermoacidophilic archaeon Sulfolobus acidocaldarius was isolated, partially purified, and characterized. The optimum pH and temperature of the enzyme for p-nitrophenylphosphate (pNPP) as a substrate were 5.0 and 70°C, respectively. The apparent K _m value was 1.9 mM. This APase showed a native molecular mass of 20 kDa on a gel filtration chromatography. Of the APase activity, 60% remained after 60 min of heat treatment at 75°C. To confirm whether the APase is active in the monomeric form, we attempted to elute the enzyme from SDS-polyacrylamide gels with Disk electrophoresis apparatus and renature the enzyme. The APase activity was recovered up to 50% in the 14- to 35-kDa range, and maximum around 25 kDa. These results suggest that this APase is monomeric protein. Received: 8 July 1999 / Accepted: 9 August 1999     

Purification and properties of NADH dehydrogenase from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius

An NADH dehydrogenase was purified to electrophoretical homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium optimally growing at pH 2-3 and 75 degrees C. A 2,100-fold purification was achieved. The purified enzyme is an acidic protein with an isoelectric point of 5.6 and a molecular weight of 95,000, consisting of two 50,000-dalton subunits. The enzyme showed an absorption spectrum characteristic of flavoproteins, with maxima at 272, 372, and 448 nm. The enzyme is highly thermostable, is specific for NADH as an electron donor, and is capable of using 2,6-dichlorophenolindophenol, ferricyanide, benzoquinone, and naphthoquinone as electron acceptors. Though at a low rate, caldariellaquinone, a unique and sole benzothiophenequinone in the genus Sulfolobus, was also reduced by the enzyme, suggesting that the enzyme is a possible member of the respiratory chain of the thermoacidophilic archaebacterium.     

The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization

Recombinant pyrophosphatase from the hyperthermophilic archaebacterium Sulfolobus acidocaldarius (S-PPase) has been heterologously expressed in Escherichia coli and could be purified in large quantities. S-PPase, previously described as a tetrameric enzyme, was shown to be a homohexameric protein that had catalytic activity with Mg2+ > Zn2+ > Co2+ > Mn2+ > Ni2+, Ca2+. CD and FTIR spectra demonstrate a similar overall fold for S-PPase and PPases from E. coli (E-PPase) and Thermus thermophilus (T-PPase). The relative proportions of secondary structure elements in S-PPase are close to those of a previously proposed model. S-PPase is extremely heat resistant. Even at 95 degrees C the half-life of catalytic activity is 2.5 h, which is dramatically increased in the presence of divalent cations. More than one Mg2+ per monomer is needed for catalysis, but no more than one Mg2+ per monomer is sufficient for thermal stabilization. The Tm values for S-PPase are 89 degrees C (+EDTA), 99 degrees C (+Mg2+), and >100 degrees C (+Mn2+), compared to 58 degrees C (+EDTA), 84 degrees C (+Mg2+), and 93 degrees C (+Mn2+) for E-PPase and 86 degrees C (+EDTA), 99 degrees C (+Mg2+), and 96 degrees C (+Mn2+) for T-PPase. The guanidium hydrochloride-induced unfolding follows an unknown mechanism with a biphasic kinetic and an unstable intermediate. Unfolding curves of the S-, E-, and T-PPase are independent of the method applied (CD spectroscopy and fluorescence) and show a sigmoidal and monophasic transition, indicating a change in global structure during unfolding, which can be described by a two-state process comprising dissociation and denaturation of the folded hexamer into six monomers. The respective DeltaGN-->D(25 degrees C) values of the three PPases vary from 220 to 290 kJ/mol for the overall process and are not significantly higher for the two thermophilic PPases. The stabilizing effect of Mg2+ DeltaDeltaG(25 degrees C) is 16 kJ/mol for E-PPase and 5.5-8 kJ/mol for S-PPase and T-PPase.     

Enzymes from Sulfolobus shibatae for the production of trehalose and glucose from starch

Enzymes that convert starch and dextrins to α,α-trehalose and glucose were found in cell homogenates of the hyperthermophilic acidophilic archaeon Sulfolobus shibatae DMS 5389. Three enzymes were purified and characterized. The first, the S. shibatae trehalosyl dextrin-forming enzyme (SsTDFE), transformed starch and dextrins to the corresponding trehalosyl derivatives with an intramolecular transglycosylation process that converted the glucosidic linkage at the reducing end from α-1,4 to α-1,1. The second, the S. shibatae trehalose-forming enzyme (SsTFE), hydrolyzed the α-1,4 linkage adjacent to the α-1,1 bond of trehalosyl dextrins, forming trehalose and lower molecular weight dextrins. These two enzymes had molecular masses of 80 kDa and 65 kDa, respectively, and showed the highest activities at pH 4.5. The apparent optimal temperature for activity was 70°C for SsTDFE and 85°C for SsTFE. The third enzyme identified was an α-glycosidase (SsαGly), which catalyzed the hydrolysis of the α-1,4 glucosidic linkages in starch and dextrins, releasing glucose in a stepwise manner from the nonreducing end of the polysaccharide chain. The enzyme had a molecular mass of 313 kDa and showed the highest activity at pH 5.5 and at 85°C. Received: October 29, 1997 / Accepted: April 29, 1998     

Purification and characterization of the first archaeal aconitase from the thermoacidophilic Sulfolobus acidocaldarius.

The first archaeal aconitase was isolated from the cytosol of the thermoacidophilic Sulfolobus acidocaldarius. Interestingly, the enzyme was copurified with an isocitrate lyase. This enzyme, directly converting isocitrate, the reaction product of the aconitase reaction, was also unknown in crenarchaeota, thus far. Both proteins could only be separated by SDS gel electrophoresis yielding apparent molecular masses of 96 kDa for the aconitase and 46 kDa for the isocitrate lyase. Despite of its high oxygen sensitivity, the aconitase could be enriched 27-fold to a specific activity of approximately 55 micromol x min(-1) x mg(-1), based on the direct aconitase assay system. Maximal enzyme activities were measured at pH 7.4 and the temperature optimum for the archaeal enzyme was recorded at 75 degrees C, slightly under the growth optimum of S. acidocaldarius around 80 degrees C. Thermal inactivation studies of the aconitase revealed the enzymatic activity to be uninfluenced after one hour incubation at 80 degrees C. Even at 95 degrees C, a half-life of approximately 14 min was determined, clearly defining it as a thermostable protein. The apparent K(m) values for the three substrates cis-aconitate, citrate and isocitrate were found as 108 microM, 2.9 mM and 370 microM, respectively. The aconitase reaction was inhibited by the typical inhibitors fluorocitrate, trans-aconitate and tricarballylate. Amino-acid sequencing of three internal peptides of the S. acidocaldarius aconitase revealed the presence of highly conserved residues in the archaeal enzyme. By amino-acid sequence alignments, the S. acidocaldarius sequence was found to be highly homologous to either other putative archaeal or known eukaryal and bacterial sequences. As shown by EPR-spectroscopy, the enzyme hosts an interconvertible [3Fe--4S] cluster.     

Electron transport and energy conservation in the archaebacterium Sulfolobus acidocaldarius

Abstract The bioenergetic properties of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius are reviewed and discussed under the aspect whether this archaebacterium conserved energy by oxidative phosphorylation and how the involved catalysts are related to those from eubacteria and eukaryotes. The thermodynamic parameters contributing to the proton-motive force and the efficiency of proton pumping are presented. The major components of the electron transport system are identified and a novel type of heme- aa ₃ containing terminal oxidase is described, oxidizing reduced caldariella quinone. The properties of an F ₁-analogous ATPase and of a DCCD-binding proteolipid from the plasmamembrane of Sulfolobus are discussed as likely components of an F ₀ F ₁-analogous ATP-synthase. The structural and functional properties of this and other archaebacterial ATPases are compared to each other and with respect to evolutionary relations.     

Biochemical characterization and helix stabilizing properties of HSNP-C' from the thermoacidophilic archaeon Sulfolobus acidocaldarius

Helix stabilizing nucleoid protein HSNP-C' from the thermophilic archaeon Sulfolobus acidocaldarius has been characterized with respect to its interactions with nucleic acids by gel retardation assay, affinities to immobilized matrices, electron microscopy, and fluorescence titration. The amino acids implicated in the DNA binding site of the protein have been shown by selectively modifying specific amino acyl functional groups and looking at their effects on the DNA binding properties of the protein. Lysine, arginine, tryptophan, and tyrosine residues of the protein HSNP-C' were modified with pyridoxal-5-phosphate; 2,3-butanedione; BNPS-skatole; and tetranitromethane, respectively. The modification of residues was assessed according to standard procedures. The effect of the chemical modification on the function of the protein HSNP-C' with respect to DNA protein interactions was studied and the results indicate the definite involvement of tyrosines and also the significant involvement of the flanking tryptophan residues in the DNA binding domain on the protein.