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J Bacteriol. May 1996; 178(9): 2605–2612.
PMCID: PMC177986

Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus.

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adams MW. Enzymes and proteins from organisms that grow near and above 100 degrees C. Annu Rev Microbiol. 1993;47:627–658. [PubMed]
  • Adams MW, Kelly RM. Thermostability and thermoactivity of enzymes from hyperthermophilic Archaea. Bioorg Med Chem. 1994 Jul;2(7):659–667. [PubMed]
  • Blumentals II, Robinson AS, Kelly RM. Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus. Appl Environ Microbiol. 1990 Jul;56(7):1992–1998. [PMC free article] [PubMed]
  • Borchardt SA, Babwah AV, Jayaswal RK. Sequence analysis of the region downstream from a peptidoglycan hydrolase-encoding gene from Staphylococcus aureus NCTC8325. Gene. 1993 Dec 31;137(2):253–258. [PubMed]
  • Burlini N, Magnani P, Villa A, Macchi F, Tortora P, Guerritore A. A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus. Biochim Biophys Acta. 1992 Aug 21;1122(3):283–292. [PubMed]
  • Cowan DA, Smolenski KA, Daniel RM, Morgan HW. An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88 degrees C. Biochem J. 1987 Oct 1;247(1):121–133. [PMC free article] [PubMed]
  • Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. [PMC free article] [PubMed]
  • Eggen RI, Geerling AC, Waldkötter K, Antranikian G, de Vos WM. The glutamate dehydrogenase-encoding gene of the hyperthermophilic archaeon Pyrococcus furiosus: sequence, transcription and analysis of the deduced amino acid sequence. Gene. 1993 Sep 30;132(1):143–148. [PubMed]
  • Fusek M, Lin XL, Tang J. Enzymic properties of thermopsin. J Biol Chem. 1990 Jan 25;265(3):1496–1501. [PubMed]
  • Goldberg AL, Rock KL. Proteolysis, proteasomes and antigen presentation. Nature. 1992 Jun 4;357(6377):375–379. [PubMed]
  • Hain J, Reiter WD, Hüdepohl U, Zillig W. Elements of an archaeal promoter defined by mutational analysis. Nucleic Acids Res. 1992 Oct 25;20(20):5423–5428. [PMC free article] [PubMed]
  • Hanner M, Redl B, Stöffler G. Isolation and characterization of an intracellular aminopeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus. Biochim Biophys Acta. 1990 Feb 26;1033(2):148–153. [PubMed]
  • Heltzel A, Smith ET, Zhou ZH, Blamey JM, Adams MW. Cloning, expression, and molecular characterization of the gene encoding an extremely thermostable [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol. 1994 Aug;176(15):4790–4793. [PMC free article] [PubMed]
  • Kelly RM, Adams MW. Metabolism in hyperthermophilic microorganisms. Antonie Van Leeuwenhoek. 1994;66(1-3):247–270. [PubMed]
  • Kengen SW, de Bok FA, van Loo ND, Dijkema C, Stams AJ, de Vos WM. Evidence for the operation of a novel Embden-Meyerhof pathway that involves ADP-dependent kinases during sugar fermentation by Pyrococcus furiosus. J Biol Chem. 1994 Jul 1;269(26):17537–17541. [PubMed]
  • Klingeberg M, Galunsky B, Sjoholm C, Kasche V, Antranikian G. Purification and Properties of a Highly Thermostable, Sodium Dodecyl Sulfate-Resistant and Stereospecific Proteinase from the Extremely Thermophilic Archaeon Thermococcus stetteri. Appl Environ Microbiol. 1995 Aug;61(8):3098–3104. [PMC free article] [PubMed]
  • Laderman KA, Asada K, Uemori T, Mukai H, Taguchi Y, Kato I, Anfinsen CB. Alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus. Cloning and sequencing of the gene and expression in Escherichia coli. J Biol Chem. 1993 Nov 15;268(32):24402–24407. [PubMed]
  • Lin X, Tang J. Purification, characterization, and gene cloning of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius. J Biol Chem. 1990 Jan 25;265(3):1490–1495. [PubMed]
  • Löwe J, Stock D, Jap B, Zwickl P, Baumeister W, Huber R. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science. 1995 Apr 28;268(5210):533–539. [PubMed]
  • Ma K, Schicho RN, Kelly RM, Adams MW. Hydrogenase of the hyperthermophile Pyrococcus furiosus is an elemental sulfur reductase or sulfhydrogenase: evidence for a sulfur-reducing hydrogenase ancestor. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5341–5344. [PMC free article] [PubMed]
  • Maurizi MR, Clark WP, Katayama Y, Rudikoff S, Pumphrey J, Bowers B, Gottesman S. Sequence and structure of Clp P, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli. J Biol Chem. 1990 Jul 25;265(21):12536–12545. [PubMed]
  • McDonald OB, Merrill BM, Bland MM, Taylor LC, Sahyoun N. Site and consequences of the autophosphorylation of Ca2+/calmodulin-dependent protein kinase type "Gr". J Biol Chem. 1993 May 15;268(14):10054–10059. [PubMed]
  • Morikawa M, Izawa Y, Rashid N, Hoaki T, Imanaka T. Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp. Appl Environ Microbiol. 1994 Dec;60(12):4559–4566. [PMC free article] [PubMed]
  • Mukund S, Adams MW. The novel tungsten-iron-sulfur protein of the hyperthermophilic archaebacterium, Pyrococcus furiosus, is an aldehyde ferredoxin oxidoreductase. Evidence for its participation in a unique glycolytic pathway. J Biol Chem. 1991 Aug 5;266(22):14208–14216. [PubMed]
  • Mukund S, Adams MW. Glyceraldehyde-3-phosphate ferredoxin oxidoreductase, a novel tungsten-containing enzyme with a potential glycolytic role in the hyperthermophilic archaeon Pyrococcus furiosus. J Biol Chem. 1995 Apr 14;270(15):8389–8392. [PubMed]
  • Orlowski M. The multicatalytic proteinase complex (proteasome) and intracellular protein degradation: diverse functions of an intracellular particle. J Lab Clin Med. 1993 Feb;121(2):187–189. [PubMed]
  • Peters J, Nitsch M, Kühlmorgen B, Golbik R, Lupas A, Kellermann J, Engelhardt H, Pfander JP, Müller S, Goldie K, et al. Tetrabrachion: a filamentous archaebacterial surface protein assembly of unusual structure and extreme stability. J Mol Biol. 1995 Jan 27;245(4):385–401. [PubMed]
  • Reiter WD, Hüdepohl U, Zillig W. Mutational analysis of an archaebacterial promoter: essential role of a TATA box for transcription efficiency and start-site selection in vitro. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9509–9513. [PMC free article] [PubMed]
  • Robinson KA, Bartley DA, Robb FT, Schreier HJ. A gene from the hyperthermophile Pyrococcus furiosus whose deduced product is homologous to members of the prolyl oligopeptidase family of proteases. Gene. 1995 Jan 11;152(1):103–106. [PubMed]
  • Rowlands T, Baumann P, Jackson SP. The TATA-binding protein: a general transcription factor in eukaryotes and archaebacteria. Science. 1994 May 27;264(5163):1326–1329. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Schicho RN, Ma K, Adams MW, Kelly RM. Bioenergetics of sulfur reduction in the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol. 1993 Mar;175(6):1823–1830. [PMC free article] [PubMed]
  • Segerer AH, Burggraf S, Fiala G, Huber G, Huber R, Pley U, Stetter KO. Life in hot springs and hydrothermal vents. Orig Life Evol Biosph. 1993 Feb;23(1):77–90. [PubMed]
  • Snowden LJ, Blumentals II, Kelly RM. Regulation of Proteolytic Activity in the Hyperthermophile Pyrococcus furiosus. Appl Environ Microbiol. 1992 Apr;58(4):1134–1141. [PMC free article] [PubMed]
  • Thompson MW, Maurizi MR. Activity and specificity of Escherichia coli ClpAP protease in cleaving model peptide substrates. J Biol Chem. 1994 Jul 8;269(27):18201–18208. [PubMed]
  • Tiboni O, Cammarano P, Sanangelantoni AM. Cloning and sequencing of the gene encoding glutamine synthetase I from the archaeum Pyrococcus woesei: anomalous phylogenies inferred from analysis of archaeal and bacterial glutamine synthetase I sequences. J Bacteriol. 1993 May;175(10):2961–2969. [PMC free article] [PubMed]
  • Uemori T, Ishino Y, Toh H, Asada K, Kato I. Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus. Nucleic Acids Res. 1993 Jan 25;21(2):259–265. [PMC free article] [PubMed]
  • Völkl P, Markiewicz P, Stetter KO, Miller JH. The sequence of a subtilisin-type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability. Protein Sci. 1994 Aug;3(8):1329–1340. [PMC free article] [PubMed]
  • Waxman L, Goldberg AL. Protease La, the lon gene product, cleaves specific fluorogenic peptides in an ATP-dependent reaction. J Biol Chem. 1985 Oct 5;260(22):12022–12028. [PubMed]
  • Wesson L, Eisenberg D. Atomic solvation parameters applied to molecular dynamics of proteins in solution. Protein Sci. 1992 Feb;1(2):227–235. [PMC free article] [PubMed]
  • Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4576–4579. [PMC free article] [PubMed]
  • Zwickl P, Fabry S, Bogedain C, Haas A, Hensel R. Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli. J Bacteriol. 1990 Aug;172(8):4329–4338. [PMC free article] [PubMed]
  • Zwickl P, Grziwa A, Pühler G, Dahlmann B, Lottspeich F, Baumeister W. Primary structure of the Thermoplasma proteasome and its implications for the structure, function, and evolution of the multicatalytic proteinase. Biochemistry. 1992 Feb 4;31(4):964–972. [PubMed]
  • Zwickl P, Lottspeich F, Baumeister W. Expression of functional Thermoplasma acidophilum proteasomes in Escherichia coli. FEBS Lett. 1992 Nov 9;312(2-3):157–160. [PubMed]

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