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 a...
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| Veröffentlicht in: | The Journal of biological chemistry 1996-08, Vol.271 (34), p.20426-20431 |
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| container_title | The Journal of biological chemistry |
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| creator | Voorhorst, Wilfried G.B. Eggen, Rik I.L. Geerling, Ans C.M. Platteeuw, Christ Siezen, Roland J. de Vos, Willem M. |
| description | 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. |
| doi_str_mv | 10.1074/jbc.271.34.20426 |
| format | Article |
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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.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.271.34.20426</identifier><identifier>PMID: 8702780</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Archaea - enzymology ; Archaeal Proteins ; Base Sequence ; Cloning, Molecular ; DNA Primers - chemistry ; Enzyme Precursors - genetics ; Enzyme Precursors - isolation & purification ; Enzyme Precursors - metabolism ; Genes, Bacterial ; Hot Temperature ; Molecular Sequence Data ; Molecular Weight ; Protein Precursors ; Pyrococcus furiosus ; Sequence Alignment ; Sequence Homology, Amino Acid ; Serine Endopeptidases - genetics ; Serine Endopeptidases - isolation & purification ; Serine Endopeptidases - metabolism ; Substrate Specificity ; Subtilisins - chemistry ; Transcription, Genetic</subject><ispartof>The Journal of biological chemistry, 1996-08, Vol.271 (34), p.20426-20431</ispartof><rights>1996 © 1996 ASBMB. 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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. 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Eggen, Rik I.L. ; Geerling, Ans C.M. ; Platteeuw, Christ ; Siezen, Roland J. ; de Vos, Willem M.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c513t-c5b1eab457390d537de88eae1880149a149dd39b3fbe66ea3c48bca4c382e95c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1996</creationdate><topic>Amino Acid Sequence</topic><topic>Archaea - enzymology</topic><topic>Archaeal Proteins</topic><topic>Base Sequence</topic><topic>Cloning, Molecular</topic><topic>DNA Primers - chemistry</topic><topic>Enzyme Precursors - genetics</topic><topic>Enzyme Precursors - isolation & purification</topic><topic>Enzyme Precursors - metabolism</topic><topic>Genes, Bacterial</topic><topic>Hot Temperature</topic><topic>Molecular Sequence Data</topic><topic>Molecular Weight</topic><topic>Protein Precursors</topic><topic>Pyrococcus furiosus</topic><topic>Sequence Alignment</topic><topic>Sequence Homology, Amino Acid</topic><topic>Serine Endopeptidases - genetics</topic><topic>Serine Endopeptidases - isolation & purification</topic><topic>Serine Endopeptidases - metabolism</topic><topic>Substrate Specificity</topic><topic>Subtilisins - chemistry</topic><topic>Transcription, Genetic</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Voorhorst, Wilfried G.B.</creatorcontrib><creatorcontrib>Eggen, Rik I.L.</creatorcontrib><creatorcontrib>Geerling, Ans C.M.</creatorcontrib><creatorcontrib>Platteeuw, Christ</creatorcontrib><creatorcontrib>Siezen, Roland J.</creatorcontrib><creatorcontrib>de Vos, Willem M.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Voorhorst, Wilfried G.B.</au><au>Eggen, Rik I.L.</au><au>Geerling, Ans C.M.</au><au>Platteeuw, Christ</au><au>Siezen, Roland J.</au><au>de Vos, Willem M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Isolation and Characterization of the Hyperthermostable Serine Protease, Pyrolysin, and Its Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1996-08-23</date><risdate>1996</risdate><volume>271</volume><issue>34</issue><spage>20426</spage><epage>20431</epage><pages>20426-20431</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>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.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>8702780</pmid><doi>10.1074/jbc.271.34.20426</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | The Journal of biological chemistry, 1996-08, Vol.271 (34), p.20426-20431 |
| issn | 0021-9258 1083-351X |
| language | eng |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Amino Acid Sequence Archaea - enzymology Archaeal Proteins Base Sequence Cloning, Molecular DNA Primers - chemistry Enzyme Precursors - genetics Enzyme Precursors - isolation & purification Enzyme Precursors - metabolism Genes, Bacterial Hot Temperature Molecular Sequence Data Molecular Weight Protein Precursors Pyrococcus furiosus Sequence Alignment Sequence Homology, Amino Acid Serine Endopeptidases - genetics Serine Endopeptidases - isolation & purification Serine Endopeptidases - metabolism Substrate Specificity Subtilisins - chemistry Transcription, Genetic |
| title | Isolation and Characterization of the Hyperthermostable Serine Protease, Pyrolysin, and Its Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus |
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