首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
A gene, treX, encoding a debranching enzyme previously cloned from the trehalose biosynthesis gene cluster of Sulfolobus solfataricus P2 was expressed in Escherichia coli as a His-tagged protein and the biochemical properties were studied. The specific activity of the S. solfataricus debranching enzyme (TreX) was highest at 75°C and pH 5.5. The enzyme exhibited hydrolysing activity toward α-1,6-glycosidic linkages of amylopectin, glycogen, pullulan, and other branched substrates, and glycogen was the preferred substrate. TreX has a high specificity for hydrolysis of maltohexaosyl α-1,6-β-cyclodextrin, indicating the high preference for side chains consisting of 6 glucose residues or more. The enzyme also exhibited 4-α-sulfoxide-glucan transferase activity, catalysing transfer of α-1,4-glucan oligosaccharides from one chain to another. Dimethyl sulfoxide (10%, v/v) increased the hydrolytic activity of TreX. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation revealed that the enzyme exists mostly as a dimer at pH 7.0, and as a mixture of dimers and tetramers at pH 5.5. Interestingly, TreX existed as a tetramer in the presence of DMSO at pH 5.5–6.5. The tetramer showed a 4-fold higher catalytic efficiency than the dimer. The enzyme catalysed not only intermolecular trans-glycosylation of malto-oligosaccharides (disproportionation) to produce linear α-1,4-glucans, but also intramolecular trans-glycosylation of glycogen. The results presented in this study indicated that TreX may be associated with glycogen metabolism by selective cleavage of the outer side chain.  相似文献   

2.
A gene encoding a putative glycogen-debranching enzyme in Sulfolobus shibatae (abbreviated as SSGDE) was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. The recombinant SSGDE was extremely thermostable, with an optimal temperature at 85 degrees C. The enzyme had an optimum pH of 5.5 and was highly stable from pH 4.5 to 6.5. The substrate specificity of SSGDE suggested that it possesses characteristics of both amylo-1,6-glucosidase and alpha-1,4-glucanotransferase. SSGDE clearly hydrolyzed pullulan to maltotrlose, and 6-O-alpha-maltosyl-beta-cyclodextrin (G2-beta-CD) to maltose and beta-cyclodextrin. At the same time, SSGDE transferred maltooligosyl residues to the maltooligosaccharides employed, and maltosyl residues to G2-beta-CD. The enzyme preferentially hydrolyzed amylopectin, followed in a decreasing order by glycogen, pullulan, and amylose. Therefore, the present results suggest that the glycogen-debranching enzyme from S. shibatae may have industrial application for the efficient debranching and modification of starch to dextrins at a high temperature.  相似文献   

3.
Homoserine dehydrogenase (HSD; 305 amino acid residues) catalyzes an NAD(P)-dependent reversible reaction between l-homoserine and aspartate 4-semialdehyde and is involved in the aspartate pathway. HSD from the hyperthermophilic archaeon Sulfolobus tokodaii was markedly activated (2.5-fold) by the addition of 0.8 mM dithiothreitol. The crystal structure of the homodimer indicated that the activation was caused by cleavage of the disulfide bond formed between two cysteine residues (C303) in the C-terminal regions of the two subunits.  相似文献   

4.
Organophosphates (OPs) constitute the largest class of insecticides used worldwide and certain of them are potent nerve agents. Consequently, enzymes degrading OPs are of paramount interest, as they could be used as bioscavengers and biodecontaminants. Looking for a stable OPs catalyst, able to support industrial process constraints, a hyperthermophilic phosphotriesterase (PTE) (SsoPox) was isolated from the archaeon Sulfolobus solfataricus and was found to be highly thermostable. The solved 3D structure revealed that SsoPox is a noncovalent dimer, with lactonase activity against “quorum sensing signals”, and therefore could represent also a potential weapon against certain pathogens. The structural basis of the high thermostability of SsoPox has been investigated by performing a careful comparison between its structure and that of two mesophilic PTEs from Pseudomonas diminuta and Agrobacterium radiobacter. In addition, the conformational stability of SsoPox against the denaturing action of temperature and GuHCl has been determined by means of circular dichroism and fluorescence measurements. The data suggest that the two fundamental differences between SsoPox and the mesophilic counterparts are: (a) a larger number of surface salt bridges, also involved in complex networks; (b) a tighter quaternary structure due to an optimization of the interactions at the interface between the two monomers. Pompea Del Vecchio, Mikael Elias and Luigia Merone were contributed equally to this paper.  相似文献   

5.
A treX in the trehalose biosynthesis gene cluster of Sulfolobus solfataricus ATCC 35092 has been reported to produce TreX, which hydrolyzes the alpha-1,6-branch portion of amylopectin and glycogen. TreX exhibited 4-alpha-D-glucan transferase activity, catalyzing the transfer of alpha-1,4-glucan oligosaccharides from one molecule to another in the case of linear maltooligosaccharides (G3-G7), and it produced cyclic glucans from amylopectin and amylose like 4-alpha-glucanotransferase. These results suggest that TreX is a novel isoamylase possessing the properties of 4-alpha-glucanotransferase.  相似文献   

6.
When soluble extracts of the extreme acidothermophilic archaeon Sulfolobus solfataricus were incubated with [gamma-(32)P]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of approximately 46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy. The best match to the partial sequence obtained was the potential polypeptide product of open reading frame sso0417, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs]. Open reading frame sso0417 was therefore cloned, and its protein product was expressed in Escherichia coli. Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity. PGA mutase activity was dependent upon the presence of divalent metal ions such as Co(2+) or Mn(2+). The recombinant protein underwent autophosphorylation when incubated with either [gamma-(32)P]ATP or [gamma-(32)P]GTP. The site of phosphorylation was identified as Ser(59), which corresponds to the catalytically essential serine residue in bacterial and eucaryal iPGMs. The phosphoenzyme intermediate behaved in a chemically and kinetically competent manner. Incubation of the (32)P-labeled phosphoenzyme with 3-PGA resulted in the disappearance of radioactive phosphate and the concomitant appearance of (32)P-labeled PGA at rates comparable to those measured in steady-state assays of PGA mutase activity.  相似文献   

7.
Examination of the sequence of a hypothetical gene with an unknown function included in the carotenogenic gene cluster in the genome of a thermoacidophilic archaeon Sulfolobus solfataricus led to the prediction that the gene encodes a novel-type lycopene beta-cyclase, whose N- and C-terminal halves are homologous to the subunits of the bacterial heterodimeric enzymes. The recombinant expression of the gene in lycopene-producing Escherichia coli resulted in the accumulation of beta-carotene in the cells, which verifies the function of the gene. Homologues of the archaeal lycopene beta-cyclase from various organisms such as bacteria, archaea, and fungi have been reported. Although their primary structures are clearly specific to the biological taxa, a phylogenetic analysis revealed the unexpected complicity of the evolutional route of these enzymes.  相似文献   

8.
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.  相似文献   

9.
In the three domains of life, the archaea, bacteria, and eukarya, there are two general lineages of DNA replication proteins: the bacterial and the eukaryal/archaeal lineages. The hyperthermophilic archaeon Sulfolobus solfataricus provides an attractive model for biochemical study of DNA replication. Its relative simplicity in both genomic and biochemical contexts, together with high protein thermostability, has already provided insight into the function of the more complex yet homologous molecules of the eukaryotic domain. Here, we provide an overview of recent insights into the functioning of the chromosome replication machinery of S. solfataricus, focusing on some of the relatively well characterized core components that act at the DNA replication fork.  相似文献   

10.
11.
Dihydroxy-acid dehydratase (DHAD) is one of the key enzymes involved in the biosynthetic pathway of the branched chain amino acids. Although the enzyme has been purified and characterized in various mesophiles, including bacteria and eukarya, the biochemical properties of DHAD from hyperthermophilic archaea have not yet been reported. In this study we cloned, expressed in Escherichia coli, and purified a DHAD homologue from the thermoacidophilic archaeon Sulfolobus solfataricus, which grows optimally at 80 degrees C and pH 3. The recombinant S. solfataricus DHAD (rSso_DHAD) showed the highest activity on 2,3-dihydroxyisovalerate among 17 aldonic acids tested. Interestingly, this enzyme also displayed high activity toward d-gluconate and some other pentonic and hexonic sugar acids. The k(cat)/K(m) values were 140.3 mM(-1) s(-1) for 2,3-dihydroxyisovalerate and 20.0 mM(-1) s(-1) for d-gluconate, respectively. A possible evolutionary explanation for substrate promiscuity was provided through amino acid sequence alignments of DHADs and 6-phosphogluconate dehydratases from archaea, bacteria and eukarya.  相似文献   

12.
The sliding clamp, PCNA, of the archaeon Sulfolobus solfataricus P2 is a heterotrimer of three distinct subunits (PCNA1, 2, and 3) that assembles in a defined manner. The PCNA heterotrimer, but not individual subunits, stimulates the activities of the DNA polymerase, DNA ligase I, and the flap endonuclease (FEN1) of S. solfataricus. Distinct PCNA subunits contact DNA polymerase, DNA ligase, or FEN1, imposing a defined architecture at the lagging strand fork and suggesting the existence of a preformed scanning complex at the fork. This provides a mechanism to tightly couple DNA synthesis and Okazaki fragment maturation. Additionally, unique subunit-specific interactions between components of the clamp loader, RFC, suggest a model for clamp loading of PCNA.  相似文献   

13.
Although the Archaea exhibit an intriguing combination of bacterial- and eukaryotic-like features, it is not known how these prokaryotic cells segregate their chromosomes before the process of cell division. In the course of our analysis of the third replication origin in the archaeon Sulfolobus solfataricus, we identify and characterise sister chromatid junctions in this prokaryote. This pairing appears to be mediated by hemicatenane-like structures, and we provide evidence that these junctions persist in both replicating and postreplicative cells. These data, in conjunction with fluorescent in situ hybridisation analyses, suggest that Sulfolobus chromosomes have a significant period of postreplicative sister chromatid synapsis, a situation that is more reminiscent of eukaryotic than bacterial chromosome segregation mechanisms.  相似文献   

14.
Di-O-α-maltosyl-β-cyclodextrin ((G2)2-β-CD) was synthesized from 6-O-α-maltosyl-β-cyclodextrin (G2-β-CD) via a transglycosylation reaction catalyzed by TreX, a debranching enzyme from Sulfolobus solfataricus P2. TreX showed no activity toward glucosyl-β-CD, but a transfer product (1) was detected when the enzyme was incubated with maltosyl-β-CD, indicating specificity for a branched glucosyl chain bigger than DP2. Analysis of the structure of the transfer product (1) using MALDI-TOF/MS and isoamylase or glucoamylase treatment revealed it to be dimaltosyl-β-CD, suggesting that TreX transferred the maltosyl residue of a G2-β-CD to another molecule of G2-β-CD by forming an α-1,6-glucosidic linkage. When [14C]-maltose and maltosyl-β-CD were reacted with the enzyme, the radiogram showed no labeled dimaltosyl-β-CD; no condensation product between the two substrates was detected, indicating that the synthesis of dimaltosyl-β-CD occurred exclusively via transglycosylation of an α-1,6-glucosidic linkage. Based on the HPLC elution profile, the transfer product (1) was identified to be isomers of 61,63- and 61,64-dimaltosyl-β-CD. Inhibition studies with β-CD on the transglycosylation activity revealed that β-CD was a mixed-type inhibitor, with a Ki value of 55.6 μmol/mL. Thus, dimaltosyl-β-CD can be more efficiently synthesized by a transglycosylation reaction with TreX in the absence of β-CD. Our findings suggest that the high yield of (G2)2-β-CD from G2-β-CD was based on both the transglycosylation action mode and elimination of the inhibitory effect of β-CD.  相似文献   

15.
The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-mediated virus defense represents an adaptive immune system in many bacteria and archaea. Small CRISPR RNAs cause cleavage of complementary invading nucleic acids in conjunction with an associated protein or a protein complex. Here, we show CRISPR-mediated cleavage of mRNA from an invading virus in the hyperthermophilic archaeon Sulfolobus solfataricus. More than 40% of the targeted mRNA could be cleaved, as demonstrated by quantitative polymerase chain reaction. Cleavage of the mRNA was visualized by northern analyses and cleavage sites were mapped. In vitro, the same substrates were cleaved by the purified CRISPR-associated CMR complex from Sulfolobus solfataricus. The in vivo system was also re-programmed to knock down mRNA of a selected chromosomal gene (β-galactosidase) using an artificial miniCRISPR locus. With a single complementary spacer, ∼50% reduction of the targeted mRNA and of corresponding intracellular protein activity was achieved. Our results demonstrate in vivo cleavage of mRNA in a prokaryote mediated by small RNAs (i.e. analogous to RNA interference in eukaryotes) and the re-programming of the system to silence specific genes of interest.  相似文献   

16.
17.
The eukaryotic exosome is a protein complex with essential functions in processing and degradation of RNA. Exosome-like complexes were recently found in Archaea. Here we characterize the exosome of Sulfolobus solfataricus. Two exosome fractions can be discriminated by density gradient centrifugation. We show that the Cdc48 protein is associated with the exosome from the 30S-50S fraction but not with the exosome of the 11.3S fraction. While only some complexes contain Cdc48, the archaeal DnaG-like protein was found to be a core exosome subunit in addition to Rrp4, Rrp41, Rrp42 and Csl4. Assays with depleted extracts revealed that the exosome is responsible for major ribonucleolytic activity in S. solfataricus. Various complexes consisting of the Rrp41-Rrp42 hexameric ring and Rrp4, Csl4 and DnaG were reconstituted. Dependent on their composition, different complexes showed variations in RNase activity indicating functional interdependence of the subunits. The catalytic activity of these complexes and of the native exosome can be ascribed to the Rrp41-Rrp42 ring, which degrades RNA phosphorolytically. Rrp4 and Csl4 do not exhibit any hydrolytic RNase activity, either when assayed alone or in context of the complex, but influence the activity of the archaeal exosome.  相似文献   

18.
19.
Flagellation in archaea is widespread and is involved in swimming motility. Here, we demonstrate that the structural flagellin gene from the crenarchaeaon Sulfolobus solfataricus is highly expressed in stationary-phase-grown cells and under unfavorable nutritional conditions. A mutant in a flagellar auxiliary gene, flaJ, was found to be nonmotile. Electron microscopic imaging of the flagellum indicates that the filaments are composed of right-handed helices.  相似文献   

20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号