A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.1/14219 |
Resumo: | Pyrroloquinoline quinone is a prominent redox cofactor in many prokaryotes, produced from a ribosomally synthesized and post-translationally modified peptide PqqA via a pathway comprising four conserved proteins PqqB?E. These four proteins are now fairly well-characterized and span radical SAM activity (PqqE), aided by a peptide chaperone (PqqD), a dual hydroxylase (PqqB), and an eight-electron, eight-proton oxidase (PqqC). A full description of this pathway has been hampered by a lack of information regarding a protease/peptidase required for the excision of an early, cross-linked di-amino acid precursor to pyrroloquinoline quinone. Herein, we isolated and characterized a two-component heterodimer protein from the ?-proteobacterium Methylobacterium (Methylorubrum) extorquens that can rapidly catalyze cleavage of PqqA into smaller peptides. Using pulldown assays, surface plasmon resonance, and isothermal calorimetry, we demonstrated the formation of a complex PqqF/PqqG, with a K-D of 300 nm. We created a molecular model of the heterodimer by comparison with the Sphingomonas sp. A1 M16B Sph2681/Sph2682 protease. Analysis of time-dependent patterns for the appearance of proteolysis products indicates high specificity of PqqF/PqqG for serine side chains. We hypothesize that PqqF/PqqG initially cleaves between the PqqE/PqqD-generated cross-linked form of PqqA, with nonspecific cellular proteases completing the release of a suitable substrate for the downstream enzyme PqqB. The finding of a protease that specifically targets serine side chains is rare, and we propose that this activity may be useful in proteomic analyses of the large family of proteins that have undergone post-translational phosphorylation at serine. |
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A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinoneGlucose-dehydrogenaseNucleotide-sequenceEscherichia-coliMammalian enzymePqqMethylobacteriumBiosynthesisBiogenesisSiteOxidationProteaseZincSerineEnzyme catalysisPeptidesHeterodimerM16B ProteasePQQPyrroloquinoline quinone is a prominent redox cofactor in many prokaryotes, produced from a ribosomally synthesized and post-translationally modified peptide PqqA via a pathway comprising four conserved proteins PqqB?E. These four proteins are now fairly well-characterized and span radical SAM activity (PqqE), aided by a peptide chaperone (PqqD), a dual hydroxylase (PqqB), and an eight-electron, eight-proton oxidase (PqqC). A full description of this pathway has been hampered by a lack of information regarding a protease/peptidase required for the excision of an early, cross-linked di-amino acid precursor to pyrroloquinoline quinone. Herein, we isolated and characterized a two-component heterodimer protein from the ?-proteobacterium Methylobacterium (Methylorubrum) extorquens that can rapidly catalyze cleavage of PqqA into smaller peptides. Using pulldown assays, surface plasmon resonance, and isothermal calorimetry, we demonstrated the formation of a complex PqqF/PqqG, with a K-D of 300 nm. We created a molecular model of the heterodimer by comparison with the Sphingomonas sp. A1 M16B Sph2681/Sph2682 protease. Analysis of time-dependent patterns for the appearance of proteolysis products indicates high specificity of PqqF/PqqG for serine side chains. We hypothesize that PqqF/PqqG initially cleaves between the PqqE/PqqD-generated cross-linked form of PqqA, with nonspecific cellular proteases completing the release of a suitable substrate for the downstream enzyme PqqB. The finding of a protease that specifically targets serine side chains is rare, and we propose that this activity may be useful in proteomic analyses of the large family of proteins that have undergone post-translational phosphorylation at serine.National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [GM118117, GM124002, 1S10OD020062-01]American Society for Biochemistry and Molecular BiologySapientiaMartins, Ana M.Latham, John A.Martel, PauloBarr, IanIavarone, Anthony T.Klinman, Judith P.2020-07-24T10:51:20Z2019-102019-10-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.1/14219eng0021-925810.1074/jbc.RA119.009684info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-24T10:26:28Zoai:sapientia.ualg.pt:10400.1/14219Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T20:05:15.697939Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone |
title |
A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone |
spellingShingle |
A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone Martins, Ana M. Glucose-dehydrogenase Nucleotide-sequence Escherichia-coli Mammalian enzyme Pqq Methylobacterium Biosynthesis Biogenesis Site Oxidation Protease Zinc Serine Enzyme catalysis Peptides Heterodimer M16B Protease PQQ |
title_short |
A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone |
title_full |
A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone |
title_fullStr |
A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone |
title_full_unstemmed |
A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone |
title_sort |
A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone |
author |
Martins, Ana M. |
author_facet |
Martins, Ana M. Latham, John A. Martel, Paulo Barr, Ian Iavarone, Anthony T. Klinman, Judith P. |
author_role |
author |
author2 |
Latham, John A. Martel, Paulo Barr, Ian Iavarone, Anthony T. Klinman, Judith P. |
author2_role |
author author author author author |
dc.contributor.none.fl_str_mv |
Sapientia |
dc.contributor.author.fl_str_mv |
Martins, Ana M. Latham, John A. Martel, Paulo Barr, Ian Iavarone, Anthony T. Klinman, Judith P. |
dc.subject.por.fl_str_mv |
Glucose-dehydrogenase Nucleotide-sequence Escherichia-coli Mammalian enzyme Pqq Methylobacterium Biosynthesis Biogenesis Site Oxidation Protease Zinc Serine Enzyme catalysis Peptides Heterodimer M16B Protease PQQ |
topic |
Glucose-dehydrogenase Nucleotide-sequence Escherichia-coli Mammalian enzyme Pqq Methylobacterium Biosynthesis Biogenesis Site Oxidation Protease Zinc Serine Enzyme catalysis Peptides Heterodimer M16B Protease PQQ |
description |
Pyrroloquinoline quinone is a prominent redox cofactor in many prokaryotes, produced from a ribosomally synthesized and post-translationally modified peptide PqqA via a pathway comprising four conserved proteins PqqB?E. These four proteins are now fairly well-characterized and span radical SAM activity (PqqE), aided by a peptide chaperone (PqqD), a dual hydroxylase (PqqB), and an eight-electron, eight-proton oxidase (PqqC). A full description of this pathway has been hampered by a lack of information regarding a protease/peptidase required for the excision of an early, cross-linked di-amino acid precursor to pyrroloquinoline quinone. Herein, we isolated and characterized a two-component heterodimer protein from the ?-proteobacterium Methylobacterium (Methylorubrum) extorquens that can rapidly catalyze cleavage of PqqA into smaller peptides. Using pulldown assays, surface plasmon resonance, and isothermal calorimetry, we demonstrated the formation of a complex PqqF/PqqG, with a K-D of 300 nm. We created a molecular model of the heterodimer by comparison with the Sphingomonas sp. A1 M16B Sph2681/Sph2682 protease. Analysis of time-dependent patterns for the appearance of proteolysis products indicates high specificity of PqqF/PqqG for serine side chains. We hypothesize that PqqF/PqqG initially cleaves between the PqqE/PqqD-generated cross-linked form of PqqA, with nonspecific cellular proteases completing the release of a suitable substrate for the downstream enzyme PqqB. The finding of a protease that specifically targets serine side chains is rare, and we propose that this activity may be useful in proteomic analyses of the large family of proteins that have undergone post-translational phosphorylation at serine. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-10 2019-10-01T00:00:00Z 2020-07-24T10:51:20Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10400.1/14219 |
url |
http://hdl.handle.net/10400.1/14219 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
0021-9258 10.1074/jbc.RA119.009684 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
American Society for Biochemistry and Molecular Biology |
publisher.none.fl_str_mv |
American Society for Biochemistry and Molecular Biology |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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1799133292634570752 |