Biochemical diversity among sulfur-dependent, hyperthermophilic microorganisms

Abstract: Hyperthermophiles are a recently discovered group of microorganisms that grow at and above 90°C. They currently comprise over 20 different genera, and except for two novel bacteria, all are classified as Archaea. The majority of these organisms are obligately anaerobic heterotrophs that reduce elemental sulfur (S°) to H₂S. The best studied from a biochemical perspective are the archaeon, Pyrococcus furiosus , and the bacterium, Thermotoga maritima , both of which are saccharolytic. P. furiosus is thought to contain a new type of Entner-Doudoroff pathway for the conversion of carbohydrates ultimately to acetate, H₂ and CO₂. The pathway is independent of nicotinamide nucleotides and involves novel types of ferredoxin-linked oxidoreductases, one of which has tungsten, a rarely used element, as a prosthetic group. The only site of energy conservation is at the level of acetyl CoA, which in the presence of ADP and phosphate is converted to acetate and ATP in a single step. In contrast, T. maritima utilizes a conventional Embden-Meyerhof pathway for sugar oxidation. P. furiosus also utilizes peptides as a sole carbon and energy source. Amino acid oxidation is thought to involve glutamate dehydrogenase together with at least three types of novel ferredoxin-linked oxidoreductases which catalyze the oxidation of 2-ketoglutarate, aryl pyruvates and formaldehyde. One of these enzymes also utilizes tungsten. In P. furiosus , virtually all of the reductant that is generated during the catabolism of both carbohydrates and peptides is channeled to a cytoplasmic hydrogenase. This enzyme is now termed sulhydrogenase, as it reduces both protons to H₂ and S°(or polysulfide) to H₂S. S° reduction appears to lead to the conservation of energy in P. furiosus but not in T. maritima , although the mechanism by which this occurs is not known.     

Gluconeogenesis from pyruvate in the hyperthermophilic archaeon Pyrococcus furiosus: involvement of reactions of the Embden-Meyerhof pathway

The hyperthermophilic archaeon Pyrococcus furiosus was grown on pyruvate as carbon and energy source. The enzymes involved in gluconeogenesis were investigated. The following findings indicate that glucose-6-phosphate formation from pyruvate involves phosphoenolpyruvate synthetase, enzymes of the Embden-Meyerhof pathway and fructose-1,6-bisphosphate phosphatase.Cell extracts of pyruvate-grown P.furiosus contained the following enzyme activities: phosphoenolpyruvate synthetase (0.025 U/mg, 50 °C), enolase (0.9 U/mg, 80 °C), phosphoglycerate mutase (0.13 U/mg, 55 °C), phosphoglycerate kinase (0.01 U/mg, 50 °C), glyceraldehyde-3-phosphate dehydrogenase reducing either NADP⁺ or NAD⁺ (NADP⁺: 0.019 U/mg, NAD⁺: 0.009 U/mg; 50 °C), triosephosphate isomerase (1.4 U/mg, 50 °C), fructose-1,6-bisphosphate aldolase (0.0045 U/mg, 55 °C), fructose-1,6-bisphosphate phosphatase (0.026 U/mg, 75 °C), and glucose-6-phosphate isomerase (0.22 U/mg, 50 °C). Kinetic properties (V _max values and apparent K _m values) of the enzymes indicate that they operate in the direction of sugar synthesis. The specific enzyme activities of phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase (NADP⁺-reducing) and fructose-1,6-bisphosphate phosphatase in pyruvate-grown P. furiosus were by a factor of 3, 10 and 4, respectively, higher as compared to maltose-grown cells suggesting that these enzymes are induced under conditions of gluconeogenesis. Furthermore, cell extracts contained ferredoxin: NADP⁺ oxidoreductase (0.023 U/mg, 60 °C); phosphoenolpyruvate carboxylase (0.018 U/mg, 50 °C) acts as an anaplerotic enzyme.Thus, in P. furiosus sugar formation from pyruvate involves reactions of the Embden-Meyerhof pathway, whereas sugar degradation to pyruvate proceeds via a modified non-phosphorylated Entner-Doudoroff pathway.     

Enhanced thermotolerance by hydrostatic pressure in the deep-sea hyperthermophile Pyrococcus strain ES4

Abstract: The combined effect of hydrostatic pressure and heat shock on thermotolerance was examined in the deep-sea hyperthermophilic archaeon Pyrococcus strain ES4. Pressure equivalent to the depth of isolation (22 MPa) enhanced ES4's survival at super-optimal temperatures (101–108°C) relative to low pressure (3 MPa). Pressure also raised the temperature at which a putative heat-shock protein (98 kDa) accumulated. ES4 grown at 95°C and 3 MPa displayed immediate enhanced thermotolerance to 105°C after being shifted to 22 MPa. Cultures grown at 95°C and 22 MPa and then heat shocked at 105°C and 3 MPa retained enhanced thermotolerance after decompression. These results suggest that this deep-sea hyperthermophile has developed pressure-induced responses that include increased survival to hyperthermal conditions.     

Structural Changes in Ribonuclease P RNA in the Hyperthermophilic Archaeon Pyrococcus horikoshii OT3 Induced on Interaction with Proteins

The activated structure of RNase P RNA (PhopRNA) in Pyrococcus horikoshii OT3 was characterized by circular dichroism (CD) and ultraviolet (UV) absorbance spectra. The results suggested that interaction of four RNase P proteins (PhoPop5, PhoRpp21, PhoRpp29, and PhoRpp30) with PhopRNA results in destabilization of base stacking in PhopRNA, whereas the addition of a fifth protein, PhoRpp38, increases base stacking in PhopRNA.     

New Deblocking Aminopeptidases from Pyrococcus horikoshii

It has been reported that one of the hyperthermostable aminopeptidases from Pyrococcus horikoshii exhibits hydrolytic activity toward short peptides and acyl-peptides (deblocking activity). In the genome database of P. horikoshii, two new open reading frames homologous to the hyperthermostable aminopeptidase of P. horikoshii were found. The two new genes for the proteins were cloned, expressed using E. coli, and characterized. The purified proteins gave a single band on SDS-PAGE corresponding to molecular masses of 42 kDa and 41 kDa respectively, and exhibited aminopeptidase activity, including deblocking activity. These enzymes are likely to exist as oligomeric structures at neutral pH. The optimum pHs of the two enzyme activities were in the range of 7.0 to 7.5, and the optimum temperatures for the activities were around 100 °C. The enzymes exhibited low hydrolytic activity for peptide substrates longer than 10 residues. They were activated by cobalt and zinc ions. Their substrate specificities and activation factors are different. It was confirmed that P. horikoshii has three similar aminopeptidases with deblocking activity and that these enzymes appear to play important roles in hydrolyzing small peptides in P. horikoshii cells.     

Insights into the molecular basis of thermal stability from the structure determination of Pyrococcus furiosus gluatamate dehydrogenase

Abstract: The structure determination of the glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus has been completed at 2.2 Å resolution. The structure has been compared with the glutamate dehydrogenases from the mesophiles Clostridium symbiosum, Escherichia coli and Neurospora crassa . This comparison has revealed that the hyperthermophilic enzyme contains a striking series of networks of ion-pairs which are formed by regions of the protein which contain a high density of charged residues. Such regions are not found in the mesophilic enzymes and the number and extent of ion-pair formation is much more limited. The ion-pair networks are clustered at both inter domain and inter subunit interfaces and may well represent a major stabilising feature associated with the adaptation of enzymes to extreme temperatures.     

Overexpression of an archaeal protein in yeast: secretion bottleneck at the ER

Archaeal enzymes have great potential for industrial use; however, expressing them in their natural hosts has proven challenging. Growth conditions for many archaea are beyond typical fermentation capabilities, and to compound the problem, archaea generally achieve much lower biomass yields than Escherichia coli or Saccharomyces cerevisiae. To determine whether a eukaryotic host, S. cerevisiae, would be a suitable alternative for archaeal protein production, we examined the expression of the tetrameric beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. We engineered the beta-glucosidase to facilitate secretion into the culture medium and have demonstrated the beta-glucosidase's secretion and activity. We determined the dependence of beta-glucosidase secretion on gene copy number and obtained a transformant capable of secreting approximately 10 mg/L in batch culture. All transformants retained large intracellular fractions of beta-glucosidase, indicative of an intracellular bottleneck. Cell fractionation by sucrose density centrifugation and immunofluorescence identified the endoplasmic reticulum as the secretion bottleneck. Preliminary evidence indicates that the cause of this bottleneck is misfolding of the monomeric beta-glucosidase, rather than tetrameric association. Expression at moderately elevated temperatures (between 30 and 40 degrees C) improved beta-glucosidase yields, suggesting that higher temperature expression may improve folding and secretion yields.     

Proteolysis in hyperthermophilic microorganisms

Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus, the crenarchaeote Sulfolobus solfataricus, and the bacterium Thermotoga maritima. An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putative proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.