Catalytic promiscuity in dihydroxy-acid dehydratase from the thermoacidophilic archaeon Sulfolobus solfataricus

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.     

Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector

Abstract The comparative chromosomal locations of polymeric β-fructosidase SUC genes have been determined by Southern blot hybridization with the SUC2 probe in 91 different strains of Saccharomyces cerevisiae . Most of the strains exhibited a single SUC2 gene, but in some strains two or three SUC genes were found. All Suc⁻ strains carried a silent suc2⁰ sequence. The accumulation of SUC genes was observed in populations derived from sources containing sucrose and seems to be absent in strains from sources promoting the MEL gene.     

Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

An NAD(P)-dependent glutamate dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme is a hexamer (subunit mass 45 kDa) which dissociates into lower states of association when submitted to gel filtration. Isoelectric focusing analysis of the purified enzyme showed a pI of 5.7 and occasionally revealed microheterogeneity. The enzyme is strictly specific for the natural substrates 2-oxoglutarate and L-glutamate, but is active with both NADH and NADPH. S. solfataricus glutamate dehydrogenase revealed a high degree of thermal stability (at 80 C the half-life was 15 h) which was strictly dependent on the protein concentration. Very high levels of glutamate dehydrogenase were found in this archaebacterium which suggests that the conversion of 2-oxoglutarate and ammonia to glutamate is of central importance to the nitrogen metabolism in this bacterium.     

A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 degrees C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 degrees C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C8) with Km and kcat values of 71 microM and 14,700 s(-1), respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.     

Allosteric properties of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermoacidophilic archaeon Sulfolobus solfataricus

The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when assayed at 60 degrees C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number kcat and raised the Km values for 5-phosphoribosyl-1-diphosphate (PRPP) and uracil by two- and >10-fold, respectively. The inhibition by CTP was complex as it depended on the presence of the reaction product UMP. Neither CTP nor UMP were strong inhibitors of the enzyme, but when present in combination their inhibition was extremely powerful. Ligand binding analyses showed that GTP and PRPP bind cooperatively to the enzyme and that the inhibitors CTP and UMP can be bound simultaneously (KD equal to 2 and 0.5 microm, respectively). The binding of each of the inhibitors was incompatible with binding of PRPP or GTP. The data indicate that UPRTase undergoes a transition from a weakly active or inactive T-state, favored by binding of UMP and CTP, to an active R-state, favored by binding of GTP and PRPP.     

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.     

Type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon Sulfolobus shibatae.

Although isopentenyl diphosphate-dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea. A novel type of the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains. In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. The gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. The thermostable archaeal enzyme is tetrameric, and requires NAD(P)H and Mg2+ for activity, similar to its bacterial homologues. Using its apoenzyme, we were able to confirm that the archaeal enzyme is strictly dependent on FMN. Moreover, we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH.     

Purification and characterization of aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus, a thermoacidophilic organism isolated from an acidic hot spring (optimal growth conditions: 87 degrees C, pH 3.5) was purified to homogeneity. The enzyme is a dimer (Mr subunit = 53,000) showing microheterogeneity when submitted to chromatofocusing and/or isoelectric focusing analysis (two main bands having pI = 6.8 and 6.3 were observed). The N-terminal sequence (22 residues) does not show any homology with any stretch of known sequence of aspartate aminotransferases from animal and bacterial sources. The apoenzyme can be reconstituted with pyridoxamine 5'-phosphate and/or pyridoxal 5'-phosphate, each subunit binding 1 mol of coenzyme. The absorption maxima of the pyridoxamine and pyridoxal form are centered at 325 and 335 nm, respectively; the shape of the pyridoxal form band does not change with pH. The enzyme has an optimum temperature higher than 95 degrees C, and at 100 degrees C shows a half-inactivation time of 2 h. The above properties seem to be unique even for enzymes from extreme thermophiles (Daniel, R. M. (1986) in Protein Structure, Folding, and Design (Oxender, D. L., ed) pp. 291-296, Alan R. Liss, Inc., New York) and lead to the conclusion that aspartate aminotransferase from S. solfataricus is one of the most thermophilic and thermostable enzymes so far known.     

Phosphoprotein with phosphoglycerate mutase activity from the archaeon Sulfolobus solfataricus

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.     

Gelrite plate technique for culturing Sulfolobus solfataricus,a thermoacidophilic archaebacterium

Summary A rapid procedure for plating the thermo-acidophilic archaebacterium Sulfolobus solfataricus using Gerlite as a gelling agent is reported. The technique uses a clear, single layer in which the colonies are grown.     

A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7

We have characterized an esterase expressed from the putative esterase gene (ST0071) selected from the total genome analysis from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as a fusion protein in Escherichia coli. The protein was purified with heat treatment, affinity column chromatography, and size exclusion filtration. The optimum activity for ester cleavage against p-nitrophenyl esters was observed at around 70 degrees C and pH 7.5-8.0. The enzyme exhibited high thermostability and also showed activity in a mixture of a buffer and water-miscible organic solvents, such as acetonitrile and dimethyl sulfoxide. From the kinetic analysis, p-nitrophenyl butyrate was found to be a better substrate than caproate and caprylate.     

Crystal structure of a thermophilic cytochrome P450 from the archaeon Sulfolobus solfataricus

The structure of the first P450 identified in Archaea, CYP119 from Sulfolobus solfataricus, has been solved in two different crystal forms that differ by the ligand (imidazole or 4-phenylimidazole) coordinated to the heme iron. A comparison of the two structures reveals an unprecedented rearrangement of the active site to adapt to the different size and shape of ligands bound to the heme iron. These changes involve unraveling of the F helix C-terminal segment to extend a loop structure connecting the F and G helices, allowing the longer loop to dip down into the active site and interact with the smaller imidazole ligand. A comparison of CYP119 with P450cam and P450eryF indicates an extensive clustering of aromatic residues may provide the structural basis for the enhanced thermal stability of CYP119. An additional feature of the 4-phenylimidazole-bound structure is a zinc ion tetrahedrally bound by symmetry-related His and Glu residues.     

Comparative analysis of the catechol 2,3-dioxygenase gene locus in thermoacidophilic archaeon Sulfolobus solfataricus strain 98/2

A catechol 2,3-dioxygenase (C23O) gene was found from Sulfolobus solfataricus strain 98/2. Heterologous thermophilic C23O expressed in Escherichia coli showed the highest activity against catechol and 4-chlorocatechol, and at neutral pH. The C23O gene located with a putative multicomponent monooxygenase (MM) gene cluster that exactly matched with the homologous region of S. solfataricus strain P2. Primary sequence comparison identified an insertion sequence (IS) element inserted into a putative MM protein A N-terminal fragment gene in strain 98/2. Both ends of the transposase gene in the IS element, ISC1234, were flanked by 19 bp inverted repeat and 4 bp direct repeat sequences which are typical features of mobile elements. Our analysis and the two geographically distant origins of strains 98/2 and P2 (USA and Italy, respectively) suggest that the two strains have evolved from a common ancestor.     

Sister chromatid junctions in the hyperthermophilic archaeon Sulfolobus solfataricus

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.     

A heterotrimeric PCNA in the hyperthermophilic archaeon Sulfolobus solfataricus

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.     

The chromosome replication machinery of the archaeon Sulfolobus solfataricus

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.     

Identification of a thermostable and enantioselective amidase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7

We have characterized an amidase expressed from the putative amidase gene (ST0478) selected from the total genome analysis from the thermoacidophilic archaeon, Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as an insoluble aggregated 6 x His-tagged fusion protein in Escherichia coli. The protein was purified with denaturing, refolding on affinity column chromatography, size exclusion filtration, and heat treatment. The enzyme exhibited high thermostability and the optimum activity for amide cleavage against benzamide was observed at around 75 degrees C and pH 7.0-8.0. It also showed enantioselectivity for (R,S)-2-phenylpropionamide and preferentially hydrolyzed the S-enantiomer. This novel enzyme is the second characterized archaeal amidase.     

Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus

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.