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.     

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.     

The structural basis for substrate promiscuity in 2-keto-3-deoxygluconate aldolase from the Entner-Doudoroff pathway in Sulfolobus solfataricus

The hyperthermophilic Archaea Sulfolobus solfataricus grows optimally above 80 degrees C and metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to D-2-keto-3-deoxygluconate (KDG). KDG aldolase (KDGA) then catalyzes the cleavage of KDG to D-glyceraldehyde and pyruvate. It has recently been shown that all the enzymes of this pathway exhibit a catalytic promiscuity that also enables them to be used for the metabolism of galactose. This phenomenon, known as metabolic pathway promiscuity, depends crucially on the ability of KDGA to cleave KDG and D-2-keto-3-deoxygalactonate (KDGal), in both cases producing pyruvate and D-glyceraldehyde. In turn, the aldolase exhibits a remarkable lack of stereoselectivity in the condensation reaction of pyruvate and D-glyceraldehyde, forming a mixture of KDG and KDGal. We now report the structure of KDGA, determined by multiwavelength anomalous diffraction phasing, and confirm that it is a member of the tetrameric N-acetylneuraminate lyase superfamily of Schiff base-forming aldolases. Furthermore, by soaking crystals of the aldolase at more than 80 degrees C below its temperature activity optimum, we have been able to trap Schiff base complexes of the natural substrates pyruvate, KDG, KDGal, and pyruvate plus D-glyceraldehyde, which have allowed rationalization of the structural basis of promiscuous substrate recognition and catalysis. It is proposed that the active site of the enzyme is rigid to keep its thermostability but incorporates extra functionality to be promiscuous.     

Peptidyl-tRNA hydrolase from Sulfolobus solfataricus

An enzyme capable of liberating functional tRNA^Lys from Escherichia coli diacetyl-lysyl-tRNA^Lys was purified from the archae Sulfolobus solfataricus. Contrasting with the specificity of peptidyl- tRNA hydrolase (PTH) from E.coli, the S.solfataricus enzyme readily accepts E.coli formyl-methionyl-tRNA^fMet as a substrate. N-terminal sequencing of this enzyme identifies a gene that has homologs in the whole archaeal kingdom. Involvement of this gene (SS00175) in the recycling of peptidyl-tRNA is supported by its capacity to complement an E.coli strain lacking PTH activity. The archaeal gene, the product of which appears markedly different from bacterial PTHs, also has homologs in all the available eukaryal genomes. Since most of the eukaryotes already display a bacterial-like PTH gene, this observation suggests the occurrence in many eukaryotes of two distinct PTH activities, either of a bacterial or of an archaeal type. Indeed, the bacterial- and archaeal-like genes encoding the two full-length PTHs of Saccharomyces cerevisiae, YHR189w and YBL057c, respectively, can each rescue the growth of an E.coli strain lacking endogeneous PTH. In vitro assays confirm that the two enzymes ensure the recycling of tRNA^Lys from diacetyl-lysyl-tRNA^Lys. Finally, the growth of yeast cells in which either YHR189w or YBL057c has been disrupted was compared under various culture conditions. Evidence is presented that YHR189w, the gene encoding a bacterial-like PTH, should be involved in mitochondrial function.     

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.     

Glucose dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus.

Glucose dehydrogenase has been purified to homogeneity from cell extracts of the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme utilizes both NAD+ and NADP+ as coenzyme and catalyses the oxidation of several monosaccharides to the corresponding glyconic acid. Substrate specificity and oxidation rate depend on the coenzyme present; when NAD+ is used, the enzyme binds and oxidizes specifically sugars presenting equatorial orientation of hydroxy groups at C-2, C-3 and C-4. The Mr of the native enzyme is 124,000 and decreases to about 60,000 in the presence of 6 M-guanidinium chloride and to about 30,000 in the presence of 5% (w/v) SDS. The enzyme shows maximal activity at pH 9, 77 degrees C and 20 mM-Mg2+, -Mn2+ or -Ca2+ and is fairly stable in the presence of chaotropic agents and water-miscible organic solvents such as methanol or acetone.     

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.     

Characterization of ribonuclease P from the archaebacterium Sulfolobus solfataricus

Ribonuclease P is the endonuclease that removes the leader fragments from the 5'-ends of precursor tRNAs. The enzyme isolated from eubacteria contains a catalytic RNA subunit. RNAs also copurify with eukaryotic RNase P, although catalysis by those RNAs has not been demonstrated. This paper reports the isolation and characterization of ribonuclease P from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Archaebacteria are a primary evolutionary lineage, distinct from both eukaryotes and eubacteria. Ribonuclease P of S. solfataricus has reaction component requirements and a Km for substrate tRNA (2.5 X 10(-7) M) that are roughly similar to those reported for eubacterial and eukaryotic ribonuclease P. The temperature optimum for the reaction is 77 degrees C, reflecting the thermophilic character of the organism. The enzyme activity is not affected by treatment with micrococcal nuclease, suggesting that there is no RNA subunit or that it is protected from nuclease action. The density of the enzyme in cesium sulfate equilibrium density gradients is 1.27 g/ml, which is similar to that of protein. However, several RNAs between 200 and 400 nucleotides in size copurify with the enzyme activity on the density gradients, and one of them remains after micrococcal nuclease treatment. These properties of the S. solfataricus enzyme are compared with those of ribonuclease P from eukaryotes and eubacteria.     

A thioredoxin from the extreme thermophilic Archaeon Sulfolobus solfataricus

• 1.1. A purification procedure for a thioredoxin from the extremophilic archaeon Sulfolobus solfataricus is described.
• 2.2. The thioredoxin is active in the dithiothreitol-dependent reduction of insulin disulfide bonds.
• 3.3. The thioredoxin is a monomer of 24,800 Da; it is an acidic protein with a pi of 4.5.
• 4.4. The protein is stable to heating for 3 hr at 90°C.
• 5.5. The amino acid composition of S. solfataricus thioredoxin is reported.

     

An exosome-like complex in Sulfolobus solfataricus

We present the first experimental evidence for the existence of an exosome-like protein complex in Archaea. In Eukarya, the exosome is essential for many pathways of RNA processing and degradation. Co-immunoprecipitation with antibodies directed against the previously predicted Sulfolobus solfataricus orthologue of the exosome subunit ribosomal-RNA-processing protein 41 (Rrp41) led to the purification of a 250-kDa protein complex from S. solfataricus. Approximately half of the complex cosediments with ribosomal subunits. It comprises four previously predicted orthologues of the core exosome subunits from yeast (Rrp41, Rrp42, Rrp4 and Csl4 (cep1 synthetic lethality 4; an RNA-binding protein and exosome subunit)), whereas other predicted subunits were not found. Surprisingly, the archaeal homologue of the bacterial DNA primase DnaG was tightly associated with the complex. This suggests an RNA-related function for the archaeal DnaG-like proteins. Comparison of experimental data from different organisms shows that the minimal core of the exosome consists of at least one phosphate-dependent ribonuclease PH homologue, and of Rrp4 and Csl4. Such a protein complex was probably present in the last common ancestor of Archaea and Eukarya.     

Regulatory Properties of Glutamate Dehydrogenase from Sulfolobus solfataricus

The purified glutamate dehydrogenase (GDH) from Sulfolobus solfataricus showed remarkable thermostability and retained 90–95% of the initial activity after incubation at –20°C, 4°C, and 25°C for up to 6 months. Unlike mammalian GDHs, the activity of GDH from Sulfolobus solfataricus was not significantly affected by the presence of various allosteric effectors such as ADP, GTP, and leucine. Incubation of GDH with increasing concentration of o-phthalaldehyde resulted in a progressive decrease in enzyme activity, suggesting that the o-phthalaldehyde-modified lysine or cysteine is directly involved in catalysis. The inhibition was competitive with respect to both 2-oxoglutarate (K_i = 30 M) and NADH (K_i = 100 M), further supporting a possibility that the o-phthalaldehyde-modified residues may be directly involved at the catalytic site. The modification of GDH by the arginine-specific dicarbonyl reagent phenylglyoxal was also examined with the view that arginine residues might play a general role in the binding of coenzyme throughout the family of pyridine nucleotide-dependent dehydro-genases. The purified GDH was inactivated in a dose-dependent manner by phenylglyoxal. Either NADH or 2-oxoglutarate did not gave any protection against the inactivation caused by a phenylglyoxal. This result indicates that GDH saturated with NADH or 2-oxoglutarate is still open to attack by phenylglyoxal. Phenylglyoxal was an uncompetitive inhibitor (K_i = 5 M) with respect to 2-oxoglutarate and a noncompetitive inhibitor (K_i = 6 M) with respect to NADH. The above results suggests that the phenylglyoxal-modified arginine residues are not located at the catalytic site and the inactivation of GDH by phenylglyoxal might be due to a steric hindrance or a conformational change affected by the interaction of the enzyme with its inhibitor.     

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.     

A distinctive single-strand DNA-binding protein from the Archaeon Sulfolobus solfataricus

Single-stranded DNA binding proteins (SSBs) have been identified in all three domains of life. Here, we report the identification of a novel crenarchaeal SSB protein that is distinctly different from its euryarchaeal counterparts. Rather than comprising four DNA-binding domains and a zinc-finger motif within a single polypeptide of 645 amino acids, as for Methanococcus jannaschii, the Sulfolobus solfataricus SSB protein (SsoSSB) has a single DNA-binding domain in a polypeptide of just 148 amino acids with a eubacterial-like acidic C-terminus. SsoSSB protein was purified to homogeneity and found to form tetramers in solution, suggesting a quaternary structure analogous to that of E. coli SSB protein,despite possessing DNA-binding domains more similar to those of eukaryotic Replication Protein A (RPA). We demonstrate distributive binding of SsoSSB to ssDNA at high temperature with an apparent site size of approximately five nucleotides (nt)per monomer. Additionally, the protein is functional both in vitro and in vivo, stimulating RecA protein-mediated DNA strand-exchange and rescuing the ssb-1 lethal mutation of E. coli respectively. We discuss possible evolutionary relationships amongst the various members of the SSB/RPA family.     

Non-autonomous mobile elements in the crenarchaeon Sulfolobus solfataricus

The genome of the archaeon Sulfolobus solfataricus P2 contains at least four types of short sequence elements lacking open reading frames which are similar to eukaryal non-autonomous mobile elements. The most- conserved elements SM1 (79-80 bp) and SM2 (183-186 bp), with 95 % sequence identity, are present in 40 and 25 copies, respectively. The less-conserved elements SM3 (127-139 bp) and SM4 (160-168 bp), with 75-97 % identity, occur in 44 and 34 copies, respectively. In total, the 143 SM elements constitute about 0.6 % of the genome. The wide distribution of each class of conserved element throughout the genome, and their precise locations, indicate that they are mobile. Direct evidence arises from the presence of SM1 and SM2 in only a fraction of genomic copies of a given class of insertion element, and within copies of open reading frames that are conserved in sequence. SM1 to SM4 are likely to be mobilized by transposases encoded by insertion elements ISC1048, ISC1217, ISC1058 and ISC1173, respectively. Furthermore, the occurrence of clusters of interwoven SM and insertion elements, in potentially mobile units, suggests a mechanism for the transfer of SM elements to other organisms.     

Fusion-type lycopene beta-cyclase from a thermoacidophilic archaeon Sulfolobus solfataricus

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.     

Highly UV-absorbing complex in selenomethionine-substituted alcohol dehydrogenase from Sulfolobus solfataricus

The aim of this work was to explain the previously discovered effect of significant decrease in intrinsic fluorescence intensity of SsADH caused by replacement of S atoms of methionine residues to Se (Giordano, A.; Raia, C. A. J. Fluorescence 2003, 13, 17-24) on the basis of the analysis of its 3D structure. It was found that all selenium atoms are located far from both Trp95 and Trp117 and could not cause their fluorescence quenching. At the same time, it was determined that substitution of S by Se causes enhanced protein absorption in the UV-region. This effect was explained by the formation of Se complex with some groups of protein. It was revealed that this complex does not participate in fluorescence and does not transfer excitation energy to tryptophan or tyrosine residues.     

Metabolism of Pentose Sugars in the Hyperthermophilic Archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.     

Applications in Biocatalysis of Glycosyl Hydrolases from the Hyperthermophilic Archaeon Sulfolobus solfataricus

Abstract

Carbohydrates serve as structural components and energy sources of cells. More interestingly, however, these biomolecules are involved in a variety of molecular recognition processes in intercellular communication and signal transduction such as cell adhesion, differentiation, development and regulation. For these reasons, great interest has arisen in carbohydrate-based pharmaceuticals and on the development of techniques for the analysis and synthesis of oligosaccharides. In this respect, enzymes involved in carbohydrates hydrolysis and modification are increasingly being utilised for the bioconversion of sugars, for the synthesis of oligosaccharides with potential application, and for the characterisation of carbohydrate compounds of unknown structure.

In this review, the enzymology and the applications of three glycosyl hydrolases from the archaeon Sulfolobus solfataricus are described. In particular, we focus on the enzymological properties of β-glycosidase, an α-xylosidase, and an α-fucosidase; their exploitation in oligosaccharides synthesis will also be discussed.