Proteases and glycosyl hydrolases from hyperthermophilic microorganisms.
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Archaea↗
Biomedical subjects
R M Kelly
Publications and source records attributed to R M Kelly.
Growth Physiology of the Hyperthermophilic Archaeon Thermococcus litoralis: Development of a Sulfur-Free Defined Medium, Characterization of an Exopolysaccharide, and Evidence of Biofilm Formation.
Nutritional characteristics of the hyperthermophilic archaeon Thermococcus litoralis have been investigated with emphasis on the development of a sulfur-free, defined growth medium, analysis of an exocellular polysaccharide, and formation of a biofilm. An artificial-seawater-based medium, containing 16 amino acids, adenine, uracil, vitamins, and trace elements, allowed T. litoralis to attain growth rates and cell densities similar to those found with complex media. Four amino acids (alanine, asparagine, glutamine, and glutamate) were not included due to their lack of effect on growth rates and cell yields. In this medium, cultures reached densities of 10(sup8) cells per ml, with doubling times of 55 min (without maltose) or 43 min (with maltose). Neither the addition of elemental sulfur nor the presence of H(inf2) significantly affected cell growth. A sparingly soluble exopolysaccharide was produced by T. litoralis grown in either defined or complex media. Analysis of the acid-hydrolyzed exopolysaccharide yielded mannose as the only monosaccharidic constituent. This exopolysaccharide is apparently involved in the formation of a biofilm on polycarbonate filters and glass slides, which is inhabited by high levels of T. litoralis. Biofilm formation by hyperthermophilic microorganisms in geothermal environments has not been examined to any extent, but further work in this area may provide information related to the interactions among high-temperature organisms.
Journal Article↗
Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus.
A previously identified intracellular proteolytic activity in the hyperthermophilic archaeon Pyrococcus furiosus (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1992-1998, 1990) was found to be a homomultimer consisting of 18.8-kDa subunits. Dissociation of this native P. furiosus protease I (PfpI) into a single subunit was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but only after trichloroacetic acid precipitation; heating to 95 degrees C in the presence of 2% SDS and 80 mM dithiothreitol did not dissociate the protein. The gene (pfpI) coding for this protease was located in genomic digests by Southern blotting with probes derived from the N-terminal amino acid sequence. pfpI was cloned, sequenced, and expressed in active form in Escherichia coli as a fusion protein with a histidine tag. The recombinant protease from E. coli showed maximum proteolytic activity at 95 degrees C, and its half-life was 19 min at this temperature. This level of stability was significantly below that previously reported for the enzyme purified by electroelution of a 66-kDa band from SDS-PAGE after extended incubation of cell extracts at 98 degrees C in 1% SDS (>30 h). The pfpI gene codes for a polypeptide of 166 amino acid residues lacking any conserved protease motifs; no protease activity was detected for the 18.8-kDa PfpI subunit (native or recombinant) by substrate gel assay. Although an immunological relationship of this protease to the eukaryotic proteasome has been seen previously, searches of the available databases identified only two similar amino acid sequences: an open reading frame of unknown function from Staphylococcus aureus NCTC 8325 (171 amino acid residues, 18.6 kDa, 41% identity) and an open reading frame also of unknown function in E. coli (172 amino acid residues, 18.8 kDa, 47% identity). Primer extension experiments with P. furiosus total RNA defined the 5' end of the transcript. There are only 10 nucleotides upstream of the start of translation; therefore, it is unlikely that there are any pre- or pro-regions associated with PfpI which could have been used for targeting or assembly of this protease. Although PfpI activity appears to be the dominant proteolytic activity in P. furiosus cell extracts, the physiological function of PfpI is unclear.
Amino Acid Sequence↗
Behavioral and neurobiological alterations induced by the immunotoxin 192-IgG-saporin: cholinergic and non-cholinergic effects following i.c.v. injection.
192-IgG-Saporin is an anti-neuronal immunotoxin that combines the 192 monoclonal antibody to the p75 neurotrophin receptor found on terminals and cell bodies of neurons in the cholinergic basal forebrain with the ribosome-inactivating protein saporin. Bilateral intraventricular injection of the 192-saporin produced a variety of dose-related behavioral, neurochemical, and histological alterations in adult male rats. While both the 2 micrograms and 4 micrograms dose produced comparable cholinergic hypofunction only the high dose produced behavioral changes. Behavioral deficits induced by the 4 micrograms dose of 192-saporin induced alterations in rotorod performance and reactivity on the hot-plate which recovered over 8 weeks. In addition, the 4 micrograms dose produced a persistent impairment in the acquisition and performance of standard Morris water maze task as well as a cued version of the task. The neurobiological alterations induced by 192-saporin involved both cholinergic and non-cholinergic systems. Both doses of 192-saporin produced a 60-80% decrease in high affinity choline transport in the hippocampus and cortex without altering this parameter in the striatum. In addition, there was a significant dose-related decrease of norepinephrine in the hippocampus in the high dose group. 192-saporin did not alter the content of dopamine, serotonin, or their metabolites in any region examined. 192-saporin also produced a loss of Purkinje cells in the cerebellum. This cell type also expresses the p75 receptor and appears to be a target for intraventricular 192-saporin. This complex interplay of factors makes the i.c.v. model of 192-saporin very problematic for studying the functional properties of the cholinergic basal forebrain. However, recent data suggest that injection of 192-saporin directly into components of the cholinergic basal forebrain can be used to further elaborate the function of this brain system and to model disorders of cholinergic hypofunction such as Alzheimer's disease.
Animals↗
Extremozymes: expanding the limits of biocatalysis.
The study of enzymes isolated from organisms inhabiting unconventional ecosystems has led to the realization that biocatalysis need not be constrained to mild conditions and can be considered at pH's, temperatures, pressures, ionic and solvent environments long thought to be destructive to biomolecules. Parallel to this, it has been demonstrated that even conventional enzymes will catalyze reactions in solvents other than water. However, the intrinsic basis for biological function under extreme conditions is only starting to be addressed, as are associated applications. This was the focus of a recent NSF/NIST-sponsored workshop on extremozymes. Given the information acquired from the study of extremozymes, modification of enzymes to improve their ranges of stability and activity remains a possibility. Ultimately, by expanding the range of conditions suitable for enzyme function, new opportunities to use biocatalysis will be created.
Catalysis↗
xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana.
The xylA gene coding for xylose isomerase from the hyperthermophile Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 444 residues with a calculated molecular weight of 50,892. The native enzyme was a homotetramer with a molecular weight of 200,000. This xylose isomerase was a member of the family II enzymes (these differ from family I isomerases by the presence of approximately 50 additional residues at the amino terminus). The enzyme was extremely thermostable, with optimal activity above 95 degrees C. The xylose isomerase showed maximum activity at pH 7.1, but it had high relative activity over a broad pH range. The catalytic efficiency (kcat/Km) of the enzyme was essentially constant between 60 and 90 degrees C, and the catalytic efficiency decreased between 90 and 98 degrees C primarily because of a large increase in Km. The T. neapolitana xylose isomerase had a higher turnover number and a lower Km for glucose than other family II xylose isomerases. Comparisons with other xylose isomerases showed that the catalytic and cation binding regions were well conserved. Comparison of different xylose isomerase sequences showed that numbers of asparagine and glutamine residues decreased with increasing enzyme thermostability, presumably as a thermophilic strategy for diminishing the potential for chemical denaturation through deamidation at elevated temperatures.
Aldose-Ketose Isomerases↗
Bioenergetic Response of the Extreme Thermoacidophile Metallosphaera sedula to Thermal and Nutritional Stresses.
The bioenergetic response of the extremely thermoacidophilic archaeon Metallosphaera sedula to thermal and nutritional stresses was examined. Continuous cultures (pH 2.0, 70(deg)C, and dilution rate of 0.05 h(sup-1)) in which the levels of Casamino Acids and ferrous iron in growth media were reduced by a step change of 25 to 50% resulted in higher levels of several proteins, including a 62-kDa protein immunologically related to the molecular chaperone designated thermophilic factor 55 in Sulfolobus shibatae (J. D. Trent, J. Osipiuk, and T. Pinkau, J. Bacteriol. 172:1478-1484, 1990), on sodium dodecyl sulfate-polyacrylamide gels. The 62-kDa protein was also noted at elevated levels in cells that had been shifted from 70 to either 80 or 85(deg)C. The proton motive force ((Delta)p), transmembrane pH ((Delta)pH), and membrane potential ((Delta)(psi)) were determined for samples obtained from continuous cultures (pH 2.0, 70(deg)C, and dilution rate of 0.05 h(sup-1)) and incubated under nutritionally and/or thermally stressed and unstressed conditions. At 70(deg)C under optimal growth conditions, M. sedula was typically found to have a (Delta)p of approximately -190 to -200 mV, the result of an intracellular pH of 5.4 (extracellular pH, 2.0) and a (Delta)(psi) of +40 to +50 mV (positive inside). After cells had been shifted to either 80 or 85(deg)C, (Delta)(psi) decreased to nearly 0 mV and internal pH approached 4.0 within 4 h of the shift; respiratory activity, as evidenced by iron speciation in parallel temperature-shifted cultures on iron pyrite, had ceased by this point. If cultures shifted from 70 to 80(deg)C were shifted back to 70(deg)C after 4 h, cells were able to regain pyrite oxidation capacity and internal pH increased to nearly normal levels after 13 h. However, (Delta)(psi) remained close to 0 mV, possibly the result of enhanced ionic exchange with media upon thermal damage to cell membranes. Further, when M. sedula was subjected to an intermediate temperature shift from 73 to 79(deg)C, an increase in pyrite dissolution (ferric iron levels doubled) over that of the unshifted control at 73(deg)C was noted. The improvement in leaching was attributed to the synergistic effect of chemical and biological factors. As such, periodic exposure to higher temperatures, followed by a suitable recovery period, may provide a basis for improving bioleaching rates of acidophilic chemolithotrophs.
Journal Article↗
Extremely thermophilic microorganisms. Metabolic strategies, genetic characteristics, and biotechnological potential.
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Aerobiosis↗
Chloride acts as a novel negative heterotropic effector of hemoglobin Rothschild (beta 37 Trp-->Arg) in solution.
The effects of chloride ion concentration on the rate constants for association of carbon monoxide with human hemoglobin A and a synthetic form of the mutant hemoglobin Rothschild (beta 37 Trp-->Arg) have been investigated by stopped-flow techniques. Previous studies of the structure [Kavanaugh et al. (1992) Biochemistry 31, 4111] and functional properties [Rivetti et al. (1993) Biochemistry 32, 2888] of hemoglobin Rothschild crystallized in the T state have demonstrated that the mutant arginine residues create new chloride ion binding sites and that chloride ions act to lower the oxygen affinity of hemoglobin Rothschild in these crystals. The studies reported here demonstrate a parallel effect of chloride ions on the rate of CO association with deoxygenated hemoglobin Rothschild in solution. Although the kinetics of CO binding to this hemoglobin in solution exhibit a Bohr effect, the chloride effect is independent of pH. In addition, we find that other halide ions have similar effects on the rate constants for the association of CO with this hemoglobin variant.
Binding Sites↗
Metabolism in hyperthermophilic microorganisms.
Hyperthermophilic microorganisms grow at temperatures of 90 degrees C and above and are a recent discovery in the microbial world. They are considered to be the most ancient of all extant life forms, and have been isolated mainly from near shallow and deep sea hydrothermal vents. All but two of the nearly twenty known genera are classified as Archaea (formerly archaebacteria). Virtually all of them are strict anaerobes. The majority are obligate heterotrophs that utilize proteinaceous materials as carbon and energy sources, although a few species are also saccharolytic. Most also depend on the reduction of elemental sulfur to hydrogen sulfide (H2S) for significant growth. Peptide fermentation involves transaminases and glutamate dehydrogenase, together with several unusual ferredoxin-linked oxidoreductases not found in mesophilic organisms. Similarly, a novel pathway based on a partially non-phosphorylated Entner-Doudoroff scheme has been postulated to convert carbohydrates to acetate, H2 and CO2, although a more conventional Embden-Meyerhof pathway has also been identified in one saccharolytic species. The few hypethermophiles known that can assimilate CO2 do so via a reductive citric acid cycle. Two S(o)-reducing enzymes termed sulfhydrogenase and sulfide dehydrogenase have been purified from the cytoplasm of a hyperthermophile that is able to grow either with or without S(o). A scheme for electron flow during the oxidation of carbohydrates and peptides and the reduction of S(o) has been proposed. However, the mechanisms by which S(o) reduction is coupled to energy conservation in this organism and in obligate S(o)-reducing hyperthermophiles is not known.
Anaerobiosis↗
Thermostability and thermoactivity of enzymes from hyperthermophilic Archaea.
Enzymes from hyperthermophilic microorganisms are characteristically thermostable and thermoactive at extremely high temperatures. Information about the basis for the structure and function of these novel proteins is beginning to emerge. However, there are very few generalizations that can be drawn at this point that can be derived from the limited number of studies that have focused on biocatalysis and thermostability at extremely high temperatures.
Archaea↗
Strategies to limit brain injury and promote recovery of function.
Neurotoxicology integrates the best aspects of pharmacology, neuroscience, and toxicology and has established unique approaches, questions, and model systems. It has contributed to (i) isolating the multitude of environmental factors that damage the nervous system, (ii) characterizing the nature of the insult in both developing and mature organisms, and (iii) identifying the populations at risk. These efforts have symbolized the scope of modern neurotoxicology but there is now a growing appreciation that neurotoxicology, neurology, and biological psychiatry share common interests that are based upon a set of unified conceptual questions. The unifying goals for all of these disciplines are to discern the biology of neural insult, its functional consequences, and the extent and underlying mechanisms of recovery of function. While research is beginning to unravel the biology of neurological disorders there is a widening gap between our understanding of the substrates of these diseases and our ability to prevent or treat them. A concerted effort must be mounted to develop new and appropriate treatments for neurodegenerative disorders. The vulnerability of the hippocampus to a broad spectrum of insults (cerebrovascular insufficiency, excitotoxins, age-related neurodegenerative disorders, drugs of abuse) and its involvement in cognitive function makes it a logical focal point for the study of the behavioral and neurobiological correlates of recovery of function. This article discusses models of neurodegeneration and different therapeutic strategies that might limit injury or promote recovery of function.
Alzheimer Disease↗
Regulation of ribosomal RNA transcription by growth rate of the hyperthermophilic Archaeon, Pyrococcus furiosus.
We have studied the single rRNA gene cluster from the Archaeon, Pyrococcus furiosus. This isolate grows optimally at 100 degrees C and is thus a hyperthermophile. In P. furiosus, transcription of 16S rRNA is subject to regulation over a 7.5-fold range in response to a 20-fold increase in growth rate. The single cluster encoding the 16S and 23S rRNA genes of P. furiosus was cloned and the 1.9 kb region upstream of the 16S rRNA gene was sequenced.
Archaea↗
Hydrogenase of the hyperthermophile Pyrococcus furiosus is an elemental sulfur reductase or sulfhydrogenase: evidence for a sulfur-reducing hydrogenase ancestor.
Microorganisms growing near and above 100 degrees C have recently been discovered near shallow and deep sea hydrothermal vents. Most are obligately dependent upon the reduction of elemental sulfur (S0) to hydrogen sulfide (H2S) for optimal growth, even though S0 reduction readily occurs abiotically at their growth temperatures. The sulfur reductase activity of the anaerobic archaeon Pyrococcus furiosus, which grows optimally at 100 degrees C by a metabolism that produces H2S if S0 is present, was found in the cytoplasm. It was purified anaerobically and was shown to be identical to the hydrogenase that had been previously purified from this organism. Both S0 and polysulfide served as substrates for H2S production, and the S0 reduction activity but not the H2-oxidation activity was enhanced by the redox protein rubredoxin. The H2-oxidizing and S0-reduction activities of the enzyme also showed different responses to pH, temperature, and inhibitors. This bifunctional "sulfhydrogenase" enzyme can, therefore, dispose of the excess reductant generated during fermentation using either protons or polysulfides as the electron acceptor. In addition, purified hydrogenases from both hyperthermophilic and mesophilic representatives of the archaeal and bacterial domains were shown to reduce S0 to H2S. It is suggested that the function of some form of ancestral hydrogenase was S0 reduction rather than, or in addition to, the reduction of protons.
Archaea↗
Enzymes from high-temperature microorganisms.
Enzymes derived from microorganisms growing at extreme temperatures are of biotechnological use as highly thermostable biocatalysts and should provide insight into the intrinsic basis of protein stability. So far, only DNA polymerases from these organisms have been put to commercial use, although the application of other classes of highly thermostable enzymes is being considered. Problems in the cultivation of high-temperature microorganisms and in the production of their enzymes still hampers progress in this field.
Archaea↗
Characterization of Amylolytic Enzymes, Having Both alpha-1,4 and alpha-1,6 Hydrolytic Activity, from the Thermophilic Archaea Pyrococcus furiosus and Thermococcus litoralis.
Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90 degrees C) and Pyrococcus furiosus (optimal growth temperature, 98 degrees C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140 degrees C. The addition of Ca also affected substrate binding, as evidenced by a decrease in K(m) for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the alpha-1,6 linkages in pullulan, alpha-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller alpha-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.
Journal Article↗
Bioenergetics of sulfur reduction in the hyperthermophilic archaeon Pyrococcus furiosus.
The bioenergetic role of the reduction of elemental sulfur (S0) in the hyperthermophilic archaeon (formerly archaebacterium) Pyrococcus furiosus was investigated with chemostat cultures with maltose as the limiting carbon source. The maximal yield coefficient was 99.8 g (dry weight) of cells (cdw) per mol of maltose in the presence of S0 but only 51.3 g (cdw) per mol of maltose if S0 was omitted. However, the corresponding maintenance coefficients were not found to be significantly different. The primary fermentation products detected were H2, CO2, and acetate, together with H2S, when S0 was also added to the growth medium. If H2S was summed with H2 to represent total reducing equivalents released during fermentation, the presence of S0 had no significant effect on the pattern of fermentation products. In addition, the presence of S0 did not significantly affect the specific activities in cell extracts of hydrogenase, sulfur reductase, alpha-glucosidase, or protease. These results suggest either that S0 reduction is an energy-conserving reaction, i.e., S0 respiration, or that S0 has a stimulatory effect on or helps overcome a process that is yield limiting. A modification of the Entner-Doudoroff glycolytic pathway has been proposed as the primary route of glucose catabolism in P. furiosus (S. Mukund and M. W. W. Adams, J. Biol. Chem. 266:14208-14216, 1991). Operation of this pathway should yield 4 mol of ATP per mol of maltose oxidized, from which one can calculate a value of 12.9 g (cdw) per mol of ATP for non-S0 growth. Comparison of this value to the yield data for growth in the presence of S0 reduction is equivalent to an ATP yield of 0.5 mol of ATP per mol of S0 reduced. Possible mechanism to account for this apparent energy conservation are discussed.
Archaea↗
Regulation of Proteolytic Activity in the Hyperthermophile Pyrococcus furiosus.
Pyrococcus furiosus was shown to grow on casein or peptides as the sole carbon, energy, and nitrogen sources, while maltose could be used as a carbon and energy source only if peptides were present in the medium. A mixture of all 20 single amino acids could not replace the peptide requirement. Specific intracellular proteolytic activity was induced under low casein or tryptone levels and was decreased by the addition of maltose to both peptide-limiting and peptide-rich media in batch and continuous cultures. In a peptide-limited chemostat, activity towards azocasein and MeO-Suc-Arg-Pro-Tyr-p-nitroanilide reached a maximum at a dilution rate of 0.28 h, while activity toward l-lysine-p-nitroanilide reached a maximum at 0.50 h. Under peptide-limiting conditions, levels of the 66-kDa protease (S66) were enhanced relative to those of other cell proteins. Preliminary evidence suggests that this protease is immunologically related to the eukaryotic multicatalytic proteinase complex (proteosome).
Journal Article↗