Characterization of pyrolysin, a hyperthermoactive serine protease from the archaebacterium Pyrococcus furiosus

Eggen, Rik I.L.; Geerling, A.C.M.; Watts, J. E.; Vos, Willem M. de

doi:10.1111/j.1574-6968.1990.tb03791.x

Cited by 91 publications

(48 citation statements)

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“…Little is known of the novel biochemistry that must be required to sustain life above 100 "C. However, the quest for biomolecules displaying unusual and interesting physicochemical properties has recently led to the discovery and isolation of specific enzymes from these hyperthermophilic organisms. Among those that have been purified so far are proteases (Eggen et al, 1990;Cowan et al, 1987), redox proteins (Aono et al, 1989;Blake et al, 1991), hydrogenases Juszczak et al, 1991;Pihl & Maier, 1991), dehydrogenases (Robb et al, 1992), ferredoxin-dependent oxidoreductases (Mukund & Adams, 1990), DNA polymerases (Lundberg et al, 1991), and enzymes involved in carbohydrate metabolism (Koch & Zablowski, 1990;Constantino et al, 1990). These enzymes are composed of the regular 20 amino acids and display a thermal stability unknown to their mesophilic counterparts.…”

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Response of Rubredoxin from Pyrococcus furiosus to Environmental Changes: Implications for the Origin of Hyperthermostability

Cavagnero¹,

Zhou²,

Adams³

et al. 1995

Biochemistry

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The bases of the hyperthennostability of rubredoxin from Py~ococcus furiosus (RdPf) have been probed by structural perturbations induced by solution pH and ionic strength changes. Comparison of the solution behavior at pH 7 and pH 2, as probed by far-and near-UV circular dichroism, Trp fluorescence emission, 1 -anilinonaphthalene-8-sulfonate (ANS) binding, and NMR spectroscopy, reveals the presence of only minimal structural variations at room temperature. At pH 2, the protein displays a surprising nearly native-like behavior at high ionic strength while, at low ionic strength, it is capable of strongly binding the hydrophobic probe ANS. All the secondary and tertiary structural features, including the environment of the hydrophobic core, appear to be intact regardless of pH and ionic strength. The apparent "melting" or denaturation temperature at pH 2, however, is 42 "C lower than at pH 7. This is attributed to the perturbation of many electrostatic interactions, including the disruption of all the ion pairs, which is complete at pH 2, as indicated by electrometric pH titrations. The implications of these findings for the origins of the hyperthennostability of rubredoxin are discussed.The existence of life forms that live at elevated temperature and pressure is an open manifestation of nature's ingenuity in managing to support life even under the most adverse conditions. A variety of so-called hyperthermophilic microorganisms have been isolated in recent years (Stetter et al., 1990;Adams, 1990;Stetter, 1986) in the vicinity of both terrestrial and submarine hydrothermal vents. The majority of them are classified as Archaea (Woese et al., 1990) and thrive at temperatures up to 110 "C, the highest temperature known to be compatible with life. They are also the most slowly evolving or "primitive" of known organisms. Little is known of the novel biochemistry that must be required to sustain life above 100 "C. However, the quest for biomolecules displaying unusual and interesting physicochemical properties has recently led to the discovery and isolation of specific enzymes from these hyperthermophilic organisms. Among those that have been purified so far are proteases (Eggen et al., 1990;Cowan et al., 1987), redox proteins (Aono et al., 1989;Blake et al., 1991), hydrogenases Juszczak et al., 1991;Pihl & Maier, 1991), dehydrogenases (Robb et al., 1992), ferredoxin-dependent oxidoreductases (Mukund & Adams, 1990), DNA polymerases (Lundberg et al., 1991), and enzymes involved in carbohydrate metabolism (Koch & Zablowski, 1990;Constantino et al., 1990). These enzymes are composed of the regular 20 amino acids and display a thermal stability unknown to their mesophilic counterparts. Yet, sequence comparisons reveal no apparent striking differences. At this juncture, the origin of protein hyperthermostability (Flam, * To whom correspondence should be addressed.

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Response of Rubredoxin from Pyrococcus furiosus to Environmental Changes: Implications for the Origin of Hyperthermostability

Cavagnero¹,

Zhou²,

Adams³

et al. 1995

Biochemistry

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“…Several of these organisms have been shown to contain high protease (9,21,33), glutamate dehydrogenase (16,19,40,54) and aromatic amino acid transaminase (3,4) activities, and these enzymes have been purified from one or more species. In addition, three distinct types of ferredoxin-and coenzyme A (CoA)-dependent 2-ketoacid oxidoreductases from these organisms have been characterized.…”

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Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea

1996

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Cell extracts of the proteolytic and hyperthermophilic archaea Thermococcus litoralis, Thermococcus sp. strain ES-1, Pyrococcus furiosus, and Pyrococcus sp. strain ES-4 contain an enzyme which catalyzes the coenzyme A-dependent oxidation of branched-chain 2-ketoacids coupled to the reduction of viologen dyes or ferredoxin. This enzyme, termed VOR (for keto-valine-ferredoxin oxidoreductase), has been purified from all four organisms. All four VORs comprise four different subunits and show amino-terminal sequence homology. T. litoralis VOR has an M r of ca. 230,000, with subunit M r values of 47,000 (␣), 34,000 (␤), 23,000 (␥), and 13,000 (␦). It contains about 11 iron and 12 acid-labile sulfide atoms and 13 cysteine residues per heterotetramer (␣␤␥␦), but thiamine pyrophosphate, which is required for catalytic activity, was lost during purification. The most efficient substrates (k cat /K m > 1.0 M ؊1 s ؊1; K m < 100 M) for the enzyme were the 2-ketoacid derivatives of valine, leucine, isoleucine, and methionine, while pyruvate and aryl pyruvates were very poor substrates (k cat /K m < 0.2 M ؊1 s ؊1) and 2-ketoglutarate was not utilized. T. litoralis VOR also functioned as a 2-ketoisovalerate synthase at 85؇C, producing 2-ketoisovalerate and coenzyme A from isobutyryl-coenzyme A (apparent K m , 250 M) and CO 2 (apparent K m , 48 mM) with reduced viologen as the electron donor. The rate of 2-ketoisovalerate synthesis was about 5% of the rate of 2-ketoisovalerate oxidation. The optimum pH for both reactions was 7.0. A mechanism for 2-ketoisovalerate oxidation based on data from substrate-induced electron paramagnetic resonance spectra is proposed, and the physiological role of VOR is discussed.In the last decade, a remarkable group of microorganisms capable of growing at temperatures around 100ЊC have been isolated from geothermally heated habitats (59,60). Virtually all of these so-called hyperthermophiles are classified as archaea (formerly archaebacteria [68]). Most of them are obligately anaerobic organisms and obtain energy for growth by either fermentation, sulfate reduction, or methanogenesis (1, 2, 28). The fermentative hyperthermophiles typically require elemental sulfur (S 0 ), which is reduced to H 2 S, for growth, although some, such as species of Pyrococcus and Thermococcus, grow well in the absence of S 0 (22,38,48). A characteristic of these organisms is their ability to ferment peptides, and some can also utilize carbohydrates (28,59,60). The pathways of carbohydrate fermentation have recently been shown to resemble the well-known bacterial pathways but with some unique additions and possibly a combination of different pathways (29,46,56).Only limited information is available on the degradation of amino acids by hyperthermophilic archaea (2, 28). Several of these organisms have been shown to contain high protease (9, 21, 33), glutamate dehydrogenase (16,19,40,54) and aromatic amino acid transaminase (3, 4) activities, and these enzymes have been purified from one or more species. In addition, three ...

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“…It was thermostable up to a temperature of 115°C and had a half-life of more than 96 hours at 80°C and 4 h at 100°C. It was found to have the highest homology to the subgroup of the subtilisin-like serine proteases (Eggen et al, 1990;Voorhorst et al, 1996).…”

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confidence: 99%

Enhanced production of subtilisin of Pyrococcus furiosus expressed in Escherichia coli using auto-inducing medium

Ikram¹,

Naz²,

Rajoka³

et al. 2009

Afr. J. Biotechnol.

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A subtilisin gene identified in the reported genome sequence of Pyrococcus furiosus was amplified and inserted in pET-22b(+) vector to produce the recombinant plasmid pET-SB. Escherichia coli BL-21 (DE3) CodonPlus was transformed with this plasmid and the enzyme was expressed up to 30% of the total cell protein on induction with IPTG. The expressed protein appeared at a position corresponding to ~20 kDa on SDS-PAGE as compared to theoretical molecular mass of 17.6 kDa. This aberrant electrophoresis mobility could be due to specific amino acid composition of the protein. Auto-induction with lactose also produced a similar level of expression but the total amount of the enzyme produced was 2.4 fold greater than that when produced with IPTG induction. This was due to a higher cell density obtainable in the auto-inducing medium. The enzyme expressed in the insoluble state could be partially refolded after denaturation with urea at high pH. This study reports for the first time high-level expression of subtilisin of P. furiosus in E. coli using an auto-inducing medium.

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Characterization of pyrolysin, a hyperthermoactive serine protease from the archaebacterium Pyrococcus furiosus

Cited by 91 publications

References 14 publications

Response of Rubredoxin from Pyrococcus furiosus to Environmental Changes: Implications for the Origin of Hyperthermostability

Response of Rubredoxin from Pyrococcus furiosus to Environmental Changes: Implications for the Origin of Hyperthermostability

Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea

Enhanced production of subtilisin of Pyrococcus furiosus expressed in Escherichia coli using auto-inducing medium

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