Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from <i>Araujia angustifolia</i> latex

Autores
Obregón, Walter David; Lufrano, Daniela; Liggieri, Constanza Silvina; Trejo, Sebastián Alejandro; Vairo Cavalli, Sandra Elizabeth; Avilés, Francesc X.; Priolo de Lufrano, Nora Silvia
Año de publicación
2011
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Araujiain aII, the protease with highest specific activity purified from latex of Araujia angustifolia (Apocynaceae), shows optimum proteolytic activity at alkaline pH, and it is completely inhibited by the irreversible inhibitor of cysteine proteases trans-epoxysucciny-l-leucyl-amido(4-guanidino) butane. It exhibits esterolytic activity on several N-α-Cbz-amino acid p-nitrophenyl esters with a preference for Gln, Ala, and Gly derivatives. Kinetic enzymatic assays were performed with the thiol proteinase substrate p-Glu-Phe-Leu-p-nitroanilide (Kₘ = 0.18 ± 0.03 mM, kcat = 1.078 ± 0.055 s⁻¹, kcat/Kₘ = 5.99 ± 0.57 s⁻¹ mM⁻¹). The enzyme has a pI value above 9.3 and a molecular mass of 23.528 kDa determined by mass spectrometry. cDNA of the peptidase was obtained by reverse transcription-PCR starting from total RNA isolated from latex. The deduced amino acid sequence was confirmed by peptide mass fingerprinting analysis. The N-terminus of the mature protein was determined by automated sequencing using Edman’s degradation and compared with the sequence deduced from cDNA. The full araujiain aII sequence was thus obtained with a total of 213 amino acid residues. The peptidase, as well as other Apocynaceae latex peptidases, is a member of the subfamily C1A of cysteine proteases. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22–Cys63 and Cys56–Cys95) in the alpha domain, and another one (Cys150–Cys201) in the beta domain, as was suggested by molecular modeling.
Facultad de Ciencias Exactas
Centro de Investigación de Proteínas Vegetales
Materia
Ciencias Exactas
Biología
Araujia angustifolia
Cysteine protease
Latex peptidase
Papain-like protease
Araujiain
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by/4.0/
Repositorio
SEDICI (UNLP)
Institución
Universidad Nacional de La Plata
OAI Identificador
oai:sedici.unlp.edu.ar:10915/133393

id SEDICI_507bad693bed76352620e89c4c27fe2a
oai_identifier_str oai:sedici.unlp.edu.ar:10915/133393
network_acronym_str SEDICI
repository_id_str 1329
network_name_str SEDICI (UNLP)
spelling Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from <i>Araujia angustifolia</i> latexObregón, Walter DavidLufrano, DanielaLiggieri, Constanza SilvinaTrejo, Sebastián AlejandroVairo Cavalli, Sandra ElizabethAvilés, Francesc X.Priolo de Lufrano, Nora SilviaCiencias ExactasBiologíaAraujia angustifoliaCysteine proteaseLatex peptidasePapain-like proteaseAraujiainAraujiain aII, the protease with highest specific activity purified from latex of <i>Araujia angustifolia</i> (Apocynaceae), shows optimum proteolytic activity at alkaline pH, and it is completely inhibited by the irreversible inhibitor of cysteine proteases <i>trans</i>-epoxysucciny-l-leucyl-amido(4-guanidino) butane. It exhibits esterolytic activity on several N-α-Cbz-amino acid p-nitrophenyl esters with a preference for Gln, Ala, and Gly derivatives. Kinetic enzymatic assays were performed with the thiol proteinase substrate p-Glu-Phe-Leu-p-nitroanilide (Kₘ = 0.18 ± 0.03 mM, k<sub>cat</sub> = 1.078 ± 0.055 s⁻¹, k<sub>cat</sub>/Kₘ = 5.99 ± 0.57 s⁻¹ mM⁻¹). The enzyme has a pI value above 9.3 and a molecular mass of 23.528 kDa determined by mass spectrometry. cDNA of the peptidase was obtained by reverse transcription-PCR starting from total RNA isolated from latex. The deduced amino acid sequence was confirmed by peptide mass fingerprinting analysis. The N-terminus of the mature protein was determined by automated sequencing using Edman’s degradation and compared with the sequence deduced from cDNA. The full araujiain aII sequence was thus obtained with a total of 213 amino acid residues. The peptidase, as well as other Apocynaceae latex peptidases, is a member of the subfamily C1A of cysteine proteases. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22–Cys63 and Cys56–Cys95) in the alpha domain, and another one (Cys150–Cys201) in the beta domain, as was suggested by molecular modeling.Facultad de Ciencias ExactasCentro de Investigación de Proteínas Vegetales2011-08info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionArticulohttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdf293-304http://sedici.unlp.edu.ar/handle/10915/133393enginfo:eu-repo/semantics/altIdentifier/issn/1432-2048info:eu-repo/semantics/altIdentifier/issn/0032-0935info:eu-repo/semantics/altIdentifier/doi/10.1007/s00425-011-1399-7info:eu-repo/semantics/altIdentifier/pmid/21424535info:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International (CC BY 4.0)reponame:SEDICI (UNLP)instname:Universidad Nacional de La Platainstacron:UNLP2025-09-29T11:31:52Zoai:sedici.unlp.edu.ar:10915/133393Institucionalhttp://sedici.unlp.edu.ar/Universidad públicaNo correspondehttp://sedici.unlp.edu.ar/oai/snrdalira@sedici.unlp.edu.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:13292025-09-29 11:31:53.161SEDICI (UNLP) - Universidad Nacional de La Platafalse
dc.title.none.fl_str_mv Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from <i>Araujia angustifolia</i> latex
title Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from <i>Araujia angustifolia</i> latex
spellingShingle Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from <i>Araujia angustifolia</i> latex
Obregón, Walter David
Ciencias Exactas
Biología
Araujia angustifolia
Cysteine protease
Latex peptidase
Papain-like protease
Araujiain
title_short Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from <i>Araujia angustifolia</i> latex
title_full Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from <i>Araujia angustifolia</i> latex
title_fullStr Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from <i>Araujia angustifolia</i> latex
title_full_unstemmed Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from <i>Araujia angustifolia</i> latex
title_sort Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from <i>Araujia angustifolia</i> latex
dc.creator.none.fl_str_mv Obregón, Walter David
Lufrano, Daniela
Liggieri, Constanza Silvina
Trejo, Sebastián Alejandro
Vairo Cavalli, Sandra Elizabeth
Avilés, Francesc X.
Priolo de Lufrano, Nora Silvia
author Obregón, Walter David
author_facet Obregón, Walter David
Lufrano, Daniela
Liggieri, Constanza Silvina
Trejo, Sebastián Alejandro
Vairo Cavalli, Sandra Elizabeth
Avilés, Francesc X.
Priolo de Lufrano, Nora Silvia
author_role author
author2 Lufrano, Daniela
Liggieri, Constanza Silvina
Trejo, Sebastián Alejandro
Vairo Cavalli, Sandra Elizabeth
Avilés, Francesc X.
Priolo de Lufrano, Nora Silvia
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv Ciencias Exactas
Biología
Araujia angustifolia
Cysteine protease
Latex peptidase
Papain-like protease
Araujiain
topic Ciencias Exactas
Biología
Araujia angustifolia
Cysteine protease
Latex peptidase
Papain-like protease
Araujiain
dc.description.none.fl_txt_mv Araujiain aII, the protease with highest specific activity purified from latex of <i>Araujia angustifolia</i> (Apocynaceae), shows optimum proteolytic activity at alkaline pH, and it is completely inhibited by the irreversible inhibitor of cysteine proteases <i>trans</i>-epoxysucciny-l-leucyl-amido(4-guanidino) butane. It exhibits esterolytic activity on several N-α-Cbz-amino acid p-nitrophenyl esters with a preference for Gln, Ala, and Gly derivatives. Kinetic enzymatic assays were performed with the thiol proteinase substrate p-Glu-Phe-Leu-p-nitroanilide (Kₘ = 0.18 ± 0.03 mM, k<sub>cat</sub> = 1.078 ± 0.055 s⁻¹, k<sub>cat</sub>/Kₘ = 5.99 ± 0.57 s⁻¹ mM⁻¹). The enzyme has a pI value above 9.3 and a molecular mass of 23.528 kDa determined by mass spectrometry. cDNA of the peptidase was obtained by reverse transcription-PCR starting from total RNA isolated from latex. The deduced amino acid sequence was confirmed by peptide mass fingerprinting analysis. The N-terminus of the mature protein was determined by automated sequencing using Edman’s degradation and compared with the sequence deduced from cDNA. The full araujiain aII sequence was thus obtained with a total of 213 amino acid residues. The peptidase, as well as other Apocynaceae latex peptidases, is a member of the subfamily C1A of cysteine proteases. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22–Cys63 and Cys56–Cys95) in the alpha domain, and another one (Cys150–Cys201) in the beta domain, as was suggested by molecular modeling.
Facultad de Ciencias Exactas
Centro de Investigación de Proteínas Vegetales
description Araujiain aII, the protease with highest specific activity purified from latex of <i>Araujia angustifolia</i> (Apocynaceae), shows optimum proteolytic activity at alkaline pH, and it is completely inhibited by the irreversible inhibitor of cysteine proteases <i>trans</i>-epoxysucciny-l-leucyl-amido(4-guanidino) butane. It exhibits esterolytic activity on several N-α-Cbz-amino acid p-nitrophenyl esters with a preference for Gln, Ala, and Gly derivatives. Kinetic enzymatic assays were performed with the thiol proteinase substrate p-Glu-Phe-Leu-p-nitroanilide (Kₘ = 0.18 ± 0.03 mM, k<sub>cat</sub> = 1.078 ± 0.055 s⁻¹, k<sub>cat</sub>/Kₘ = 5.99 ± 0.57 s⁻¹ mM⁻¹). The enzyme has a pI value above 9.3 and a molecular mass of 23.528 kDa determined by mass spectrometry. cDNA of the peptidase was obtained by reverse transcription-PCR starting from total RNA isolated from latex. The deduced amino acid sequence was confirmed by peptide mass fingerprinting analysis. The N-terminus of the mature protein was determined by automated sequencing using Edman’s degradation and compared with the sequence deduced from cDNA. The full araujiain aII sequence was thus obtained with a total of 213 amino acid residues. The peptidase, as well as other Apocynaceae latex peptidases, is a member of the subfamily C1A of cysteine proteases. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22–Cys63 and Cys56–Cys95) in the alpha domain, and another one (Cys150–Cys201) in the beta domain, as was suggested by molecular modeling.
publishDate 2011
dc.date.none.fl_str_mv 2011-08
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
Articulo
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://sedici.unlp.edu.ar/handle/10915/133393
url http://sedici.unlp.edu.ar/handle/10915/133393
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/issn/1432-2048
info:eu-repo/semantics/altIdentifier/issn/0032-0935
info:eu-repo/semantics/altIdentifier/doi/10.1007/s00425-011-1399-7
info:eu-repo/semantics/altIdentifier/pmid/21424535
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by/4.0/
Creative Commons Attribution 4.0 International (CC BY 4.0)
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by/4.0/
Creative Commons Attribution 4.0 International (CC BY 4.0)
dc.format.none.fl_str_mv application/pdf
293-304
dc.source.none.fl_str_mv reponame:SEDICI (UNLP)
instname:Universidad Nacional de La Plata
instacron:UNLP
reponame_str SEDICI (UNLP)
collection SEDICI (UNLP)
instname_str Universidad Nacional de La Plata
instacron_str UNLP
institution UNLP
repository.name.fl_str_mv SEDICI (UNLP) - Universidad Nacional de La Plata
repository.mail.fl_str_mv alira@sedici.unlp.edu.ar
_version_ 1844616198365380608
score 13.070432