A sialidase mutant displaying trans-sialidase activity

Autores
Paris, Gastón; Ratier, Laura; Amaya, María Fernanda; Nguyen, Tong; Alzari, Pedro M.; Frasch, Alberto Carlos C.
Año de publicación
2005
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Trypanosoma cruzi, the agent of Chagas disease, expresses a modified sialidase, the trans-sialidase, which transfers sialic acid from host glycoconjugates to β-galactose present in parasite mucins. Another American trypanosome, Trypanosoma rangeli, expresses a homologous protein that has sialidase activity but is devoid of transglycosidase activity. Based on the recently determined structures of T. rangeli sialidase (TrSA) and T. cruzi trans-sialidase (TcTS), we have now constructed mutants of TrSA with the aim of studing the relevant residues in transfer activity. Five mutations, Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr and Gln284-Pro, were enough to obtain a sialidase mutant (TrSA 5mut) with trans-sialidase activity; and a sixth mutation increased the activity to about 10% that of wild-type TcTS. The crystal structure of TrSA 5mut revealed the formation of a trans-sialidase-like binding site for the acceptor galactose, primarily defined by the phenol group of Tyr120 and the indole ring of Trp313, which adopts a new conformation, similar to that in TcTS, induced by the Gln284-Pro mutation. The transition state analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which inhibits sialidases but is a poor inhibitor of trans-sialidase, was used to probe the active site conformation of mutant enzymes. The results show that the presence of a sugar acceptor binding-site, the fine-tuning of protein-substrate interactions and the flexibility of crucial active site residues are all important to achieve transglycosidase activity from the TrSA sialidase scaffold.
Fil: Paris, Gastón. Universidad Nacional de San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Ratier, Laura. Universidad Nacional de San Martín; Argentina
Fil: Amaya, María Fernanda. Instituto Pasteur; Francia
Fil: Nguyen, Tong. Instituto Pasteur; Francia
Fil: Alzari, Pedro M.. Instituto Pasteur; Francia
Fil: Frasch, Alberto Carlos C.. Universidad Nacional de San Martín; Argentina
Materia
Protein Engineering
Sialidase
Trans-Sialidase
Trypanosoma Cruzi
Trypanosoma Rangeli
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/39546
Acceder
id CONICETDig_5f6c7ea836c76ff9084b4546cf47f374
oai_identifier_str oai:ri.conicet.gov.ar:11336/39546
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling A sialidase mutant displaying trans-sialidase activityParis, GastónRatier, LauraAmaya, María FernandaNguyen, TongAlzari, Pedro M.Frasch, Alberto Carlos C.Protein EngineeringSialidaseTrans-SialidaseTrypanosoma CruziTrypanosoma Rangelihttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Trypanosoma cruzi, the agent of Chagas disease, expresses a modified sialidase, the trans-sialidase, which transfers sialic acid from host glycoconjugates to β-galactose present in parasite mucins. Another American trypanosome, Trypanosoma rangeli, expresses a homologous protein that has sialidase activity but is devoid of transglycosidase activity. Based on the recently determined structures of T. rangeli sialidase (TrSA) and T. cruzi trans-sialidase (TcTS), we have now constructed mutants of TrSA with the aim of studing the relevant residues in transfer activity. Five mutations, Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr and Gln284-Pro, were enough to obtain a sialidase mutant (TrSA 5mut) with trans-sialidase activity; and a sixth mutation increased the activity to about 10% that of wild-type TcTS. The crystal structure of TrSA 5mut revealed the formation of a trans-sialidase-like binding site for the acceptor galactose, primarily defined by the phenol group of Tyr120 and the indole ring of Trp313, which adopts a new conformation, similar to that in TcTS, induced by the Gln284-Pro mutation. The transition state analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which inhibits sialidases but is a poor inhibitor of trans-sialidase, was used to probe the active site conformation of mutant enzymes. The results show that the presence of a sugar acceptor binding-site, the fine-tuning of protein-substrate interactions and the flexibility of crucial active site residues are all important to achieve transglycosidase activity from the TrSA sialidase scaffold.Fil: Paris, Gastón. Universidad Nacional de San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Ratier, Laura. Universidad Nacional de San Martín; ArgentinaFil: Amaya, María Fernanda. Instituto Pasteur; FranciaFil: Nguyen, Tong. Instituto Pasteur; FranciaFil: Alzari, Pedro M.. Instituto Pasteur; FranciaFil: Frasch, Alberto Carlos C.. Universidad Nacional de San Martín; ArgentinaElsevier2005-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/39546Paris, Gastón; Ratier, Laura; Amaya, María Fernanda; Nguyen, Tong; Alzari, Pedro M.; et al.; A sialidase mutant displaying trans-sialidase activity; Elsevier; Journal Of Molecular Biology; 345; 4; 1-2005; 923-9340022-28361089-8638CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S002228360401174Xinfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.jmb.2004.09.031info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:07:33Zoai:ri.conicet.gov.ar:11336/39546instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 10:07:33.754CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv A sialidase mutant displaying trans-sialidase activity
title A sialidase mutant displaying trans-sialidase activity
spellingShingle A sialidase mutant displaying trans-sialidase activity
Paris, Gastón
Protein Engineering
Sialidase
Trans-Sialidase
Trypanosoma Cruzi
Trypanosoma Rangeli
title_short A sialidase mutant displaying trans-sialidase activity
title_full A sialidase mutant displaying trans-sialidase activity
title_fullStr A sialidase mutant displaying trans-sialidase activity
title_full_unstemmed A sialidase mutant displaying trans-sialidase activity
title_sort A sialidase mutant displaying trans-sialidase activity
dc.creator.none.fl_str_mv Paris, Gastón
Ratier, Laura
Amaya, María Fernanda
Nguyen, Tong
Alzari, Pedro M.
Frasch, Alberto Carlos C.
author Paris, Gastón
author_facet Paris, Gastón
Ratier, Laura
Amaya, María Fernanda
Nguyen, Tong
Alzari, Pedro M.
Frasch, Alberto Carlos C.
author_role author
author2 Ratier, Laura
Amaya, María Fernanda
Nguyen, Tong
Alzari, Pedro M.
Frasch, Alberto Carlos C.
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv Protein Engineering
Sialidase
Trans-Sialidase
Trypanosoma Cruzi
Trypanosoma Rangeli
topic Protein Engineering
Sialidase
Trans-Sialidase
Trypanosoma Cruzi
Trypanosoma Rangeli
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Trypanosoma cruzi, the agent of Chagas disease, expresses a modified sialidase, the trans-sialidase, which transfers sialic acid from host glycoconjugates to β-galactose present in parasite mucins. Another American trypanosome, Trypanosoma rangeli, expresses a homologous protein that has sialidase activity but is devoid of transglycosidase activity. Based on the recently determined structures of T. rangeli sialidase (TrSA) and T. cruzi trans-sialidase (TcTS), we have now constructed mutants of TrSA with the aim of studing the relevant residues in transfer activity. Five mutations, Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr and Gln284-Pro, were enough to obtain a sialidase mutant (TrSA 5mut) with trans-sialidase activity; and a sixth mutation increased the activity to about 10% that of wild-type TcTS. The crystal structure of TrSA 5mut revealed the formation of a trans-sialidase-like binding site for the acceptor galactose, primarily defined by the phenol group of Tyr120 and the indole ring of Trp313, which adopts a new conformation, similar to that in TcTS, induced by the Gln284-Pro mutation. The transition state analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which inhibits sialidases but is a poor inhibitor of trans-sialidase, was used to probe the active site conformation of mutant enzymes. The results show that the presence of a sugar acceptor binding-site, the fine-tuning of protein-substrate interactions and the flexibility of crucial active site residues are all important to achieve transglycosidase activity from the TrSA sialidase scaffold.
Fil: Paris, Gastón. Universidad Nacional de San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Ratier, Laura. Universidad Nacional de San Martín; Argentina
Fil: Amaya, María Fernanda. Instituto Pasteur; Francia
Fil: Nguyen, Tong. Instituto Pasteur; Francia
Fil: Alzari, Pedro M.. Instituto Pasteur; Francia
Fil: Frasch, Alberto Carlos C.. Universidad Nacional de San Martín; Argentina
description Trypanosoma cruzi, the agent of Chagas disease, expresses a modified sialidase, the trans-sialidase, which transfers sialic acid from host glycoconjugates to β-galactose present in parasite mucins. Another American trypanosome, Trypanosoma rangeli, expresses a homologous protein that has sialidase activity but is devoid of transglycosidase activity. Based on the recently determined structures of T. rangeli sialidase (TrSA) and T. cruzi trans-sialidase (TcTS), we have now constructed mutants of TrSA with the aim of studing the relevant residues in transfer activity. Five mutations, Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr and Gln284-Pro, were enough to obtain a sialidase mutant (TrSA 5mut) with trans-sialidase activity; and a sixth mutation increased the activity to about 10% that of wild-type TcTS. The crystal structure of TrSA 5mut revealed the formation of a trans-sialidase-like binding site for the acceptor galactose, primarily defined by the phenol group of Tyr120 and the indole ring of Trp313, which adopts a new conformation, similar to that in TcTS, induced by the Gln284-Pro mutation. The transition state analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which inhibits sialidases but is a poor inhibitor of trans-sialidase, was used to probe the active site conformation of mutant enzymes. The results show that the presence of a sugar acceptor binding-site, the fine-tuning of protein-substrate interactions and the flexibility of crucial active site residues are all important to achieve transglycosidase activity from the TrSA sialidase scaffold.
publishDate 2005
dc.date.none.fl_str_mv 2005-01
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
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://hdl.handle.net/11336/39546
Paris, Gastón; Ratier, Laura; Amaya, María Fernanda; Nguyen, Tong; Alzari, Pedro M.; et al.; A sialidase mutant displaying trans-sialidase activity; Elsevier; Journal Of Molecular Biology; 345; 4; 1-2005; 923-934
0022-2836
1089-8638
CONICET Digital
CONICET
url http://hdl.handle.net/11336/39546
identifier_str_mv Paris, Gastón; Ratier, Laura; Amaya, María Fernanda; Nguyen, Tong; Alzari, Pedro M.; et al.; A sialidase mutant displaying trans-sialidase activity; Elsevier; Journal Of Molecular Biology; 345; 4; 1-2005; 923-934
0022-2836
1089-8638
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S002228360401174X
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jmb.2004.09.031
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.13397

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