Isolation and Characterization of the Hyperthermostable Serine Protease, Pyrolysin, and Its Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus*

The hyperthermostable serine protease pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus was purified from membrane fractions. Two proteolytically active fractions were obtained, designated high (HMW) and low (LMW) molecular weight pyrolysin, that showed immunological cross-reaction and identical NH2-terminal sequences in which the third residue could be glycosylated. The HMW pyrolysin showed a subunit mass of 150 kDa after acid denaturation. Incubation of HMW pyrolysin at 95°C resulted in the formation of LMW pyrolysin, probably as a consequence of COOH-terminal autoproteolysis. The 4194-base pair pls gene encoding pyrolysin was isolated and characterized, and its transcription initiation site was identified. The deduced pyrolysin sequence indicated a prepro-enzyme organization, with a 1249-residue mature protein composed of an NH2-terminal catalytic domain with considerable homology to subtilisin-like serine proteases and a COOH-terminal domain that contained most of the 32 possible N-glycosylation sites. The archaeal pyrolysin showed highest homology with eucaryal tripeptidyl peptidases II on the amino acid level but a different cleavage specificity as shown by its endopeptidase activity toward caseins, casein fragments including αS1-casein and synthetic peptides.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  W. D. de Vos,et al.  Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli , 1995, Journal of bacteriology.

[3]  G. Antranikian,et al.  Purification and Properties of a Highly Thermostable, Sodium Dodecyl Sulfate-Resistant and Stereospecific Proteinase from the Extremely Thermophilic Archaeon Thermococcus stetteri , 1995, Applied and environmental microbiology.

[4]  M. Adams,et al.  Glyceraldehyde-3-phosphate Ferredoxin Oxidoreductase, a Novel Tungsten-containing Enzyme with a Potential Glycolytic Role in the Hyperthermophilic Archaeon Pyrococcus furiosus(*) , 1995, The Journal of Biological Chemistry.

[5]  A. Lupas,et al.  Tetrabrachion: a filamentous archaebacterial surface protein assembly of unusual structure and extreme stability. , 1995, Journal of molecular biology.

[6]  T. Imanaka,et al.  Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp , 1994, Applied and environmental microbiology.

[7]  O. Zetterqvist,et al.  Use of a dehydroalanine-containing peptide as an efficient inhibitor of tripeptidyl peptidase II. , 1994, Archives of biochemistry and biophysics.

[8]  Jeffrey H. Miller,et al.  The sequence of a subtilisin‐type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability , 1994, Protein science : a publication of the Protein Society.

[9]  W. D. de Vos,et al.  Evidence for the operation of a novel Embden-Meyerhof pathway that involves ADP-dependent kinases during sugar fermentation by Pyrococcus furiosus. , 1994, The Journal of biological chemistry.

[10]  P. Baumann,et al.  The TATA-binding protein: a general transcription factor in eukaryotes and archaebacteria. , 1994, Science.

[11]  M. Inouye,et al.  Intramolecular chaperones and protein folding. , 1993, Trends in biochemical sciences.

[12]  W. D. de Vos,et al.  Engineering of the substrate-binding region of the subtilisin-like, cell-envelope proteinase of Lactococcus lactis. , 1993, Protein engineering.

[13]  M. Inouye,et al.  Folding pathway mediated by an intramolecular chaperone. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[14]  R. Leduc,et al.  Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage. , 1992, The Journal of biological chemistry.

[15]  R. Kelly,et al.  Regulation of Proteolytic Activity in the Hyperthermophile Pyrococcus furiosus , 1992, Applied and environmental microbiology.

[16]  T. Vernet,et al.  The pro‐region of the Kex2 endoprotease of Saccharomyces cerevisiae is removed by self‐processing , 1992, FEBS letters.

[17]  J. Leunissen,et al.  Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases. , 1991, Protein engineering.

[18]  H. Connaris,et al.  Heterogeneity of proteinases from the hyperthermophilic archaeobacterium Pyrococcus furiosus , 1991 .

[19]  W. D. de Vos,et al.  Cloning, expression, and sequence analysis of the genes for carbon monoxide dehydrogenase of Methanothrix soehngenii. , 1991, The Journal of biological chemistry.

[20]  W. M. Vos,et al.  Characterization of pyrolysin, a hyperthermoactive serine protease from the archaebacterium Pyrococcus furiosus. , 1990 .

[21]  R. Kelly,et al.  Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus , 1990, Applied and environmental microbiology.

[22]  O. Kandler,et al.  Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[23]  X. Lin,et al.  Purification, characterization, and gene cloning of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius. , 1990, The Journal of biological chemistry.

[24]  X. Lin,et al.  Enzymic properties of thermopsin. , 1990, The Journal of biological chemistry.

[25]  S. Höglund,et al.  Supramolecular structure of tripeptidyl peptidase II from human erythrocytes as studied by electron microscopy, and its correlation to enzyme activity. , 1987, The Biochemical journal.

[26]  U. Hellman,et al.  Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[27]  D. Cowan,et al.  An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88 degrees C. , 1987, The Biochemical journal.

[28]  S. Power,et al.  Secretion and autoproteolytic maturation of subtilisin. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[29]  O. Zetterqvist,et al.  Purification, substrate specificity, and classification of tripeptidyl peptidase II. , 1986, The Journal of biological chemistry.

[30]  G. von Heijne,et al.  Signal sequences: The limits of variation , 1985 .

[31]  J. Devereux,et al.  A comprehensive set of sequence analysis programs for the VAX , 1984, Nucleic Acids Res..

[32]  M. Ihara,et al.  Initiator tRNAs from archaebacteria show common unique sequence characteristics , 1982, Nature.

[33]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[34]  J. Woodlock,et al.  Glycoprotein staining following electrophoresis on acrylamide gels. , 1969, Analytical biochemistry.

[35]  A. Berger,et al.  On the size of the active site in proteases. I. Papain. , 1967, Biochemical and biophysical research communications.

[36]  A. Stams,et al.  An extremely thermostable β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus : a comparison with other glycosidases , 1994 .

[37]  M. Kates,et al.  The Biochemistry of archaea (archaebacteria) , 1993 .

[38]  J. Dalgaard,et al.  Chapter 17 Archaeal hyperthermophile genes , 1993 .

[39]  M. Adams.  Enzymes and proteins from organisms that grow near and above 100 degrees C. , 1993, Annual review of microbiology.

[40]  A. Alting,et al.  Specificity of two genetically related cell-envelope proteinases of Lactococcus lactis subsp. cremoris towards alpha s1-casein-(1-23)-fragment. , 1991, The Biochemical journal.

[41]  G. Klein.  Studies on the Epstein-Barr Virus genome and the EBV-determined nuclear antigen in human malignant disease. , 1975, Cold Spring Harbor symposia on quantitative biology.