Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campes...

Autores
Salinas, Silvina Rosa; Bianco, Maria Isabel; Barreras, Maximo; Ielpi, Luis
Año de publicación
2011
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
We describe the first biochemical characterization of the gumI gene product, an essential protein for xanthan polysaccharide synthesis. Cellular fractionation experiments reveal the presence of a protein associated with the membrane fraction, even in the absence of the other proteins responsible for the synthesis of glycolipid intermediates and the proteins involved in the polymerization and transport of the xanthan chains. By alkaline buffer extraction and detergent phase partitioning, GumI was categorized as a monotopic membrane protein. GumI was overexpressed in Escherichia coli, solubilized and purified in an active and stable form using a simple and reproducible two-step procedure. The purified recombinant GumI is a nonprocessive β-mannosyltransferase that uses GDP-Man as a donor substrate and glucuronic acid-β-1,2-mannose-α-1,3-glucose-β-1,4-glucose-PP-polyisoprenyl as an acceptor. We also established the optimal biochemical conditions for GumI enzymatic activity. Sequence analysis revealed the presence of a conserved domain for glycosyltransferases (GTs) of the GT-B superfamily and homologous proteins in several prokaryote organisms. On the basis of this biochemical characterization, GumI may represent the founding member of a new GT family in the Carbohydrate-Active EnZymes classification.
Fil: Salinas, Silvina Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina
Fil: Bianco, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina
Fil: Barreras, Maximo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina
Fil: Ielpi, Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina
Materia
Glycosyltransferase
Mannosyltransferase
Membrane-Associated Proteins
Xanthan
Xanthomonas Campestris
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:11336/12803
Acceder
id CONICETDig_0e78738ecde40772699cb132a492bf64
oai_identifier_str oai:ri.conicet.gov.ar:11336/12803
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestrisSalinas, Silvina RosaBianco, Maria IsabelBarreras, MaximoIelpi, LuisGlycosyltransferaseMannosyltransferaseMembrane-Associated ProteinsXanthanXanthomonas Campestrishttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1We describe the first biochemical characterization of the gumI gene product, an essential protein for xanthan polysaccharide synthesis. Cellular fractionation experiments reveal the presence of a protein associated with the membrane fraction, even in the absence of the other proteins responsible for the synthesis of glycolipid intermediates and the proteins involved in the polymerization and transport of the xanthan chains. By alkaline buffer extraction and detergent phase partitioning, GumI was categorized as a monotopic membrane protein. GumI was overexpressed in Escherichia coli, solubilized and purified in an active and stable form using a simple and reproducible two-step procedure. The purified recombinant GumI is a nonprocessive β-mannosyltransferase that uses GDP-Man as a donor substrate and glucuronic acid-β-1,2-mannose-α-1,3-glucose-β-1,4-glucose-PP-polyisoprenyl as an acceptor. We also established the optimal biochemical conditions for GumI enzymatic activity. Sequence analysis revealed the presence of a conserved domain for glycosyltransferases (GTs) of the GT-B superfamily and homologous proteins in several prokaryote organisms. On the basis of this biochemical characterization, GumI may represent the founding member of a new GT family in the Carbohydrate-Active EnZymes classification.Fil: Salinas, Silvina Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Bianco, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Barreras, Maximo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Ielpi, Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaOxford Univ Press Inc2011-07info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/12803Salinas, Silvina Rosa; Bianco, Maria Isabel; Barreras, Maximo; Ielpi, Luis; Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestris; Oxford Univ Press Inc; Glycobiology; 21; 7; 7-2011; 903-9130959-6658enginfo:eu-repo/semantics/altIdentifier/url/https://academic.oup.com/glycob/article-lookup/doi/10.1093/glycob/cwr022info:eu-repo/semantics/altIdentifier/doi/10.1093/glycob/cwr022info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-17T11:17:43Zoai:ri.conicet.gov.ar:11336/12803instacron: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:34982025-09-17 11:17:43.585CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestris
title Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestris
spellingShingle Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestris
Salinas, Silvina Rosa
Glycosyltransferase
Mannosyltransferase
Membrane-Associated Proteins
Xanthan
Xanthomonas Campestris
title_short Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestris
title_full Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestris
title_fullStr Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestris
title_full_unstemmed Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestris
title_sort Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestris
dc.creator.none.fl_str_mv Salinas, Silvina Rosa
Bianco, Maria Isabel
Barreras, Maximo
Ielpi, Luis
author Salinas, Silvina Rosa
author_facet Salinas, Silvina Rosa
Bianco, Maria Isabel
Barreras, Maximo
Ielpi, Luis
author_role author
author2 Bianco, Maria Isabel
Barreras, Maximo
Ielpi, Luis
author2_role author
author
author
dc.subject.none.fl_str_mv Glycosyltransferase
Mannosyltransferase
Membrane-Associated Proteins
Xanthan
Xanthomonas Campestris
topic Glycosyltransferase
Mannosyltransferase
Membrane-Associated Proteins
Xanthan
Xanthomonas Campestris
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv We describe the first biochemical characterization of the gumI gene product, an essential protein for xanthan polysaccharide synthesis. Cellular fractionation experiments reveal the presence of a protein associated with the membrane fraction, even in the absence of the other proteins responsible for the synthesis of glycolipid intermediates and the proteins involved in the polymerization and transport of the xanthan chains. By alkaline buffer extraction and detergent phase partitioning, GumI was categorized as a monotopic membrane protein. GumI was overexpressed in Escherichia coli, solubilized and purified in an active and stable form using a simple and reproducible two-step procedure. The purified recombinant GumI is a nonprocessive β-mannosyltransferase that uses GDP-Man as a donor substrate and glucuronic acid-β-1,2-mannose-α-1,3-glucose-β-1,4-glucose-PP-polyisoprenyl as an acceptor. We also established the optimal biochemical conditions for GumI enzymatic activity. Sequence analysis revealed the presence of a conserved domain for glycosyltransferases (GTs) of the GT-B superfamily and homologous proteins in several prokaryote organisms. On the basis of this biochemical characterization, GumI may represent the founding member of a new GT family in the Carbohydrate-Active EnZymes classification.
Fil: Salinas, Silvina Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina
Fil: Bianco, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina
Fil: Barreras, Maximo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina
Fil: Ielpi, Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina
description We describe the first biochemical characterization of the gumI gene product, an essential protein for xanthan polysaccharide synthesis. Cellular fractionation experiments reveal the presence of a protein associated with the membrane fraction, even in the absence of the other proteins responsible for the synthesis of glycolipid intermediates and the proteins involved in the polymerization and transport of the xanthan chains. By alkaline buffer extraction and detergent phase partitioning, GumI was categorized as a monotopic membrane protein. GumI was overexpressed in Escherichia coli, solubilized and purified in an active and stable form using a simple and reproducible two-step procedure. The purified recombinant GumI is a nonprocessive β-mannosyltransferase that uses GDP-Man as a donor substrate and glucuronic acid-β-1,2-mannose-α-1,3-glucose-β-1,4-glucose-PP-polyisoprenyl as an acceptor. We also established the optimal biochemical conditions for GumI enzymatic activity. Sequence analysis revealed the presence of a conserved domain for glycosyltransferases (GTs) of the GT-B superfamily and homologous proteins in several prokaryote organisms. On the basis of this biochemical characterization, GumI may represent the founding member of a new GT family in the Carbohydrate-Active EnZymes classification.
publishDate 2011
dc.date.none.fl_str_mv 2011-07
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/11336/12803
Salinas, Silvina Rosa; Bianco, Maria Isabel; Barreras, Maximo; Ielpi, Luis; Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestris; Oxford Univ Press Inc; Glycobiology; 21; 7; 7-2011; 903-913
0959-6658
url http://hdl.handle.net/11336/12803
identifier_str_mv Salinas, Silvina Rosa; Bianco, Maria Isabel; Barreras, Maximo; Ielpi, Luis; Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestris; Oxford Univ Press Inc; Glycobiology; 21; 7; 7-2011; 903-913
0959-6658
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://academic.oup.com/glycob/article-lookup/doi/10.1093/glycob/cwr022
info:eu-repo/semantics/altIdentifier/doi/10.1093/glycob/cwr022
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
dc.publisher.none.fl_str_mv Oxford Univ Press Inc
publisher.none.fl_str_mv Oxford Univ Press Inc
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 13.001348

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