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At least 19 recordsLinked to original sources
PubMed2006

Identification of a novel alpha-galactosidase from the hyperthermophilic archaeon Sulfolobus solfataricus.

Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable alpha-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for alpha-linked galactosides, which are optimally hydrolyzed at pH 5 and 90 degrees C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable alpha-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.

Enzyme Activation↗
PubMed2003

Targeted disruption of the alpha-amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus.

Sulfolobus solfataricus secretes an acid-resistant alpha-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand alpha-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal alpha-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-alpha-amylase antibodies raised against the purified protein immunoprecipitated secreted alpha-amylase activity and verified the enzymatic identity of the sequenced protein. A new gene replacement method was used to disrupt the amyA coding sequence by insertion of a modified allele of the S. solfataricus lacS gene. PCR and DNA sequence analysis were used to characterize the altered amyA locus in the recombinant strain. The amyA::lacS mutant lost the ability to grow on starch, glycogen, or pullulan as sole carbon and energy sources. During growth on a non-catabolite-repressing carbon source with added starch, the mutant produced no detectable secreted amylase activity as determined by enzyme assay, plate assay, or Western blot analysis. These results clarify the biological role of the alpha-amylase and provide additional methods for the directed genetic manipulation of the S. solfataricus genome.

Amino Acid Sequence↗
PubMed2003

2-Propanol degradation by Sulfolobus solfataricus.

Sulfolobus solfataricus used 2-propanol and 2-propanone (acetone) when grown in static cultures at 78 degrees C with or without glucose at 10 g l-1. The presence of 3.92 g 2-propanol l-1 in both cases inhibited growth. However, acetone accumulation following 2-propanol depletion suggested that 2-propanol was co-metabolized via the acetone metabolic pathway. Glucose at 10 g l-1 increased 2-propanol and acetone utilization from 0.93 g l-1 to 1.77 g l-1 and from 0.11 g l-1 to 1.62 g l-1, respectively. Without glucose, immobilized S. solfataricus cells increased the 2-propanol removal rate to 0.035 g l-1 h-1, compared to 0.0012 g l-1 h-1 by its suspended counterpart. The results suggest the establishment of an immobilized reactor configuration is preferential for the treatment of high temperature solvent waste streams by this acidothermophilic Crenarchaeon.

2-Propanol↗
PubMed1984

Glucose metabolism in the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus.

Sulfolobus solfataricus is a thermophilic archaebacterium able to grow at 87 degrees C and pH 3.5 on glucose as sole carbon source. The organism metabolizes glucose by two main routes. The first route involves an ATP-dependent phosphorylation to give glucose 6-phosphate, which readily isomerizes to fructose 6-phosphate. In the second route, glucose is converted into gluconate by an NAD+-dependent dehydrogenation; gluconate is then dehydrated to 2-keto-3-deoxygluconate, which, in turn, is cleaved to pyruvate and glyceraldehyde. Each metabolic step has been tested in vitro at 70 degrees C on dialysed homogenates or partially purified fractions; minimal requirements of single enzymes have been evaluated. Identification of the intermediates is based on chromatographic, spectroscopic and/or synthetic evidence and on specific enzymic assays. The oxidative breakdown of glucose to pyruvate occurring in S. solfataricus differs from the Entner-Doudoroff pattern in that there is an absence of any phosphorylation step.

Adenosine Triphosphate↗
PubMed1996

Characterisation of glucose transport in the hyperthermophilic Archaeon Sulfolobus solfataricus.

Sulfolobus solfiataricus is a hyperthermophilic Archaeon growing at 80 degrees C, pH 3. The glucose transport system of this organism has been characterised kinetically at this temperature and pH using 2-deoxy-D-glucose: the sugar analogue is transported into the cells with a Km = 1.8 +/- 0.3 microM and a Vmax = 3.6 +/- 0.1 nmol min(-1) (mg protein)(-1), with an intracellular accumulation of up to 200-fold over the extracellular concentration. Transport was significantly reduced at pH 5. Inhibition of 2-deoxy-D-glucose transport was investigated using a variety of sugars and sugar analogues; D-glucose, D-galactose and D-mannose showed the highest affinity for the transporter, with D-glucose possessing a Ki = 120 +/- 20 nM.

Biological Transport↗
PubMed1993

Nucleotide sequence and molecular evolution of the gene coding for glyceraldehyde-3-phosphate dehydrogenase in the thermoacidophilic archaebacterium Sulfolobus solfataricus.

A Sulfolobus solfataricus genomic library cloned in the EMBL3 phage was screened using as probes synthetic oligonucleotides designed from the known amino acid sequence of a peptide obtained from the purified glyceraldehyde-3-phosphate dehydrogenase (aGAPD) protein. The screening led to the isolation of six recombinant phages (lambda G1-lambda G6) and one of them (lambda G4) contained the entire GAPD gene. The deduced amino acid sequence accounts for a protein made of 341 amino acids and the initial methionine is encoded by a GTG triplet. Alignment of the S. solfataricus aGAPD sequence versus GAPD from archaea, eukarya, and bacteria showed that aGAPD is very similar to other archaebacterial but not to eukaryotic or eubacterial GAPD. For known archaebacterial GAPD sequences, the rate of nucleotide substitutions per site per year showed that these sequences are homologous not only at the amino acid but also at the nucleotide level. The evolutionary rates are nearly similar to those reported for other eukaryotic genes.

Amino Acid Sequence↗
PubMed1998

Characterization, crystallization and preliminary X-ray investigation of glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus.

Recombinant Sulfolobus solfataricus glyceraldehyde-3-phosphate dehydrogenase has been purified and found to be a tetramer of 148 kDa. The enzyme shows dual cofactor specificity and uses NADP+ in preference to NAD+. The sequence has been compared with other GAPDH proteins including those from other archaeal sources. The purified protein has been crystallized from ammonium sulfate to produce crystals that diffract to 2.4 A with a space group of P43212 or P41212. A native data set has been collected to 2.4 A using synchrotron radiation and cryocooling.

Amino Acid Sequence↗
PubMed1996

Identification of two glutamic acid residues essential for catalysis in the beta-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus.

The Sulfolobus solfataricus, strain MT4, beta-glycosidase (Ss beta-gly) is a thermophilic member of glycohydrolase family 1. To identify active-site residues, glutamic acids 206 and 387 have been changed to isosteric glutamine by site-directed mutagenesis. Mutant proteins have been purified to homogeneity using the Schistosoma japonicum glutathione S-transferase (GST) fusion system. The proteolytic cleavage of the chimeric protein with thrombin was only obtainable after the introduction of a molecular spacer between the GST and the Ss beta-gly domains. The Glu387-->Gln mutant showed no detectable activity, as expected for the residue acting as the nucleophile of the reaction. The Glu206-->Gln mutant showed 10- and 60-fold reduced activities on aryl-galacto and aryl-glucosides, respectively, when compared with the wild type. Moreover, a significant Km decrease with p/o-nitrophenyl-beta-D-glucoside was observed. The residual activity of the Glu206-->Gln mutant lost the typical pH dependence shown by the wild type. These data suggest that Glu206 acts as the general acid/base catalyst in the hydrolysis reaction.

Amino Acid Sequence↗
PubMed1986

1H and 13C NMR assignment of benzothiophenquinones from the sulfur-oxidizing archaebacterium Sulfolobus solfataricus.

From Sulfolobus solfataricus, a sulfur-oxidizing thermophilic member of archaebacteria, three unusual benzothiophenquinones were isolated. Detailed NMR studies on these quinones, including multipulse mono-dimensional and two-dimensional techniques, were performed to obtain carbon and proton assignments, one-bond, geminal and vicinal coupling constants and T1 relaxation times. This report extends the known quinone composition of thermophilic archaebacteria and further supports the concept that these biomolecules can serve as a useful chemotaxonomic tool.

Archaea↗
PubMed2004

The role of cis-acting sequences governing catabolite repression control of lacS expression in the archaeon Sulfolobus solfataricus.

The archaeon Sulfolobus solfataricus uses a catabolite repression-like system to control production of several glycoside hydrolases. To better understand this regulatory system, studies of the regulation of expression of the beta-glycosidase gene (lacS) were conducted. Expression of lacS varies in response to medium composition and to mutations at an unlinked gene called car. Despite gene overlap, expression of the lacS promoter proximal gene, SSO3017, exhibited coregulation but not cotranscription with lacS. Measurements of mRNA half-life excluded differential stability as a factor in lacS regulation. Chromosomal repositioning by homologous recombination of a lacS deletion series clarified critical cis-acting sequences required for lacS regulation. lacS repositioned at amyA exhibited increased lacS expression and compromised the response to medium composition independently of lacS 5' flanking sequence composition. In contrast, regulation of lacS by the car mutation was dependent on sequences upstream of the archaeal TATA box. Expression of a promoter fusion between lacS and the car-independent malA promoter integrated either at amyA or at the natural lacS locus was insensitive to the allelic state of car. In contrast, the promoter fusion retained a response to medium composition only at the lacS locus. These results indicate that car acts at the lacS promoter and that the response to medium composition involves locus-specific sequences exclusive of those present 5' to lacS or within the lacS transcription unit.

Archaeal Proteins↗
PubMed1994

Solution structure and DNA-binding properties of a thermostable protein from the archaeon Sulfolobus solfataricus.

The archaeon Sulfolobus solfataricus expresses large amounts of a small basic protein, Sso7d, which was previously identified as a DNA-binding protein possibly involved in compaction of DNA. We have determined the solution structure of Sso7d. The protein consists of a triple-stranded anti-parallel beta-sheet onto which an orthogonal double-stranded beta-sheet is packed. This topology is very similar to that found in eukaryotic Src homology-3 (SH3) domains. Sso7d binds strongly (Kd < 10 microM) to double-stranded DNA and protects it from thermal denaturation. In addition, we note that epsilon-mono-methylation of lysine side chains of Sso7d is governed by cell growth temperatures, suggesting that methylation is related to the heat-shock response.

Amino Acid Sequence↗
PubMed2000

The active site of the thermophilic CYP119 from Sulfolobus solfataricus.

CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85 degrees C. UV-visible and resonance Raman show the enzyme is in the low spin state and only modestly shifts to the high spin state at higher temperatures. Styrene only causes a small spin state shift, but T(1) NMR studies confirm that styrene is bound in the active site. CYP119 catalyzes the H(2)O(2)-dependent epoxidation of styrene, cis-beta-methylstyrene, and cis-stilbene with retention of stereochemistry. This catalytic activity is stable to preincubation at 80 degrees C for 90 min. Site-specific mutagenesis shows that Thr-213 is catalytically important and Thr-214 helps to control the iron spin state. Topological analysis by reaction with aryldiazenes shows that Thr-213 lies above pyrrole rings A and B and is close to the iron atom, whereas Thr-214 is some distance away. CYP119 is very slowly reduced by putidaredoxin and putidaredoxin reductase, but these proteins support catalytic turnover of the Thr-214 mutants. Protein melting curves indicate that the thermal stability of CYP119 does not depend on the iron spin state or the active site architecture defined by the threonine residues. Independence of thermal stability from active site structural factors should facilitate the engineering of novel thermostable catalysts.

Amino Acid Sequence↗
PubMed1991

The active site of Sulfolobus solfataricus aspartate aminotransferase.

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus binds pyridoxal 5' phosphate, via an aldimine bond, with Lys-241. This residue has been identified by reducing the enzyme in the pyridoxal form with sodium cyanoboro[3H]hydride and sequencing the specifically labeled peptic peptides. The amino acid sequence centered around the coenzyme binding site is highly conserved between thermophilic aspartate aminotransferases and differs from that found in mesophilic isoenzymes. An alignment of aspartate aminotransferase from Sulfolobus solfataricus with mesophilic isoenzymes, attempted in spite of the low degree of similarity, was confirmed by the correspondence between pyridoxal 5' phosphate binding residues. Using this alignment it was possible to insert the archaebacterial aspartate aminotransferase into a subclass, subclass I, of pyridoxal 5' phosphate binding enzymes comprising mesophilic aspartate aminotransferases, tyrosine aminotransferases and histidinol phosphate aminotransferases. These enzymes share 12 invariant amino acids most of which interact with the coenzyme or with the substrates. Some enzymes of subclass I and in particular aspartate aminotransferase from Sulfolobus solfataricus, lack a positively charged residue, corresponding to Arg-292, which in pig cytosolic aspartate aminotransferase interacts with the distal carboxylate of the substrates (and determines the specificity towards dicarboxylic acids). It was confirmed that aspartate aminotransferase from Sulfolobus solfataricus does not possess any arginine residue exposed to chemical modifications responsible for the binding of omega-carboxylate of the substrates. Furthermore, it has been found that aspartate aminotransferase from Sulfolobus solfataricus is fairly active when alanine is used as substrate and that this activity is not affected by the presence of formate. The KM value of the thermophilic aspartate aminotransferase towards alanine is at least one order of magnitude lower than that of the mesophilic analogue enzymes.

Amino Acid Sequence↗
PubMed2006

Regulation of expression of the arabinose and glucose transporter genes in the thermophilic archaeon Sulfolobus solfataricus.

Sugar uptake in Sulfolobus solfataricus, a thermoacidophilic archaeon, occurs through high-affinity binding of protein-dependent ABC transporters. We have investigated the expression patterns of two sugar transport operons, that is, the glucose and arabinose transporters. Analysis of the araS promoter activity, and the mRNA and protein levels in S. solfataricus cells grown on different carbon sources showed that expression of the arabinose transporter gene cluster is highly regulated and dependent on the presence of arabinose in the medium. Glucose in the growth medium repressed the expression of the arabinose transport genes. By means of primer extension, the transcriptional start site for the arabinose operon was mapped. Interestingly, expression of the arabinose transporter is down-regulated by addition of a selective set of amino acids to the medium. Expression of the glucose transporter genes appeared constitutive. These data confirm the earlier observation of a catabolite repression-like system in S. solfataricus.

Amino Acids↗
PubMed2005

Promiscuity in the part-phosphorylative Entner-Doudoroff pathway of the archaeon Sulfolobus solfataricus.

The hyperthermophilic archaeon Sulfolobus solfataricus metabolises glucose and galactose by a 'promiscuous' non-phosphorylative variant of the Entner-Doudoroff pathway, in which a series of enzymes have sufficient substrate promiscuity to permit the metabolism of both sugars. Recently, it has been proposed that the part-phosphorylative Entner-Doudoroff pathway occurs in parallel in S. solfataricus as an alternative route for glucose metabolism. In this report we demonstrate, by in vitro kinetic studies of D-2-keto-3-deoxygluconate (KDG) kinase and KDG aldolase, that the part-phosphorylative pathway in S. solfataricus is also promiscuous for the metabolism of both glucose and galactose.

Adenosine Triphosphate↗
PubMed2005

A highly acid-stable and thermostable endo-beta-glucanase from the thermoacidophilic archaeon Sulfolobus solfataricus.

The thermoacidophilic archaeon Sulfolobus solfataricus P2 encodes three hypothetic endo-beta-glucanases, SSO1354, SSO1949 and SSO2534. We cloned and expressed the gene sso1949 encoding the 334 amino acids containing protein SSO1949, which can be classified as a member of glycoside hydrolase family 12. The purified recombinant enzyme hydrolyses carboxymethylcellulose as well as cello-oligomers, with cellobiose and cellotriose as main reaction products. By following the hydrolysis of a fluorescently labelled cellohexaoside under a wide variety of conditions, we show that SSO1949 is a unique extremophilic enzyme. This archaeal enzyme has a pH optimum of approx. pH 1.8 and a temperature optimum of approx. 80 degrees C. Furthermore, the enzyme is thermostable, with a half-life of approx. 8 h at 80 degrees C and pH 1.8. The thermostability is strongly pH-dependent. At neutral pH, the thermal inactivation rate is nearly two orders of magnitude higher than at pH 1.8. Homology modelling suggests that the catalytic domain of SSO1949 has a similar fold to other mesophilic, acidophilic and neutral cellulases. The presence of a signal peptide indicates that SSO1949 is a secreted protein, which enables S. solfataricus to use cellulose as an external carbon source. It appears that SSO1949 is perfectly adapted to the extreme environment in solfataric pools. A cellulolytic enzyme with such a combination of stability and activity at high temperatures and low pH has not been described so far and could be a valuable tool for the large-scale hydrolysis of cellulose under acidic conditions.

Amino Acid Sequence↗
PubMed2004

Preliminary crystallographic studies of glucose dehydrogenase from the promiscuous Entner-Doudoroff pathway in the hyperthermophilic archaeon Sulfolobus solfataricus.

The hyperthermophilic archaeon Sulfolobus solfataricus grows optimally above 353 K and can metabolize glucose and its C4 epimer galactose via a non-phosphorylative variant of the Entner-Doudoroff pathway involving catalytically promiscuous enzymes that can operate with both sugars. The initial oxidation step is catalysed by glucose dehydrogenase (SsGDH), which can utilize both NAD and NADP as cofactors. The enzyme operates with glucose and galactose at similar catalytic efficiency, while its substrate profile also includes a range of other five- and six-carbon sugars. Crystals of the 164 kDa SsGDH homotetramer have been grown under a variety of conditions. The best crystals to date diffract to 1.8 A on a synchrotron source, have orthorhombic symmetry and belong to space group P2(1)2(1)2. Attempts are being made to solve the structure by MAD and MR.

Archaeal Proteins↗
PubMed2006

The structural basis of substrate promiscuity in glucose dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus.

The hyperthermophilic archaeon Sulfolobus solfataricus grows optimally above 80 degrees C and utilizes an unusual, promiscuous, non-phosphorylative Entner-Doudoroff pathway to metabolize both glucose and galactose. The first enzyme in this pathway, glucose dehydrogenase, catalyzes the oxidation of glucose to gluconate, but has been shown to have activity with a broad range of sugar substrates, including glucose, galactose, xylose, and L-arabinose, with a requirement for the glucose stereo configuration at the C2 and C3 positions. Here we report the crystal structure of the apo form of glucose dehydrogenase to a resolution of 1.8 A and a complex with its required cofactor, NADP+, to a resolution of 2.3 A. A T41A mutation was engineered to enable the trapping of substrate in the crystal. Complexes of the enzyme with D-glucose and D-xylose are presented to resolutions of 1.6 and 1.5 A, respectively, that provide evidence of selectivity for the beta-anomeric, pyranose form of the substrate, and indicate that this is the productive substrate form. The nature of the promiscuity of glucose dehydrogenase is also elucidated, and a physiological role for this enzyme in xylose metabolism is suggested. Finally, the structure suggests that the mechanism of sugar oxidation by this enzyme may be similar to that described for human sorbitol dehydrogenase.

Arabinose↗