Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA

Autores
Abdian, Patricia Lorena; Lellouch, Annemarie C.; Gautier, Catherine; Ielpi, Luis; Geremia, Roberto A.
Año de publicación
2000
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The alpha-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues included Glu-287 and Glu-295, which comprise an EX(7)E motif and have been proposed to be involved in catalysis. Alanine replacements of each conserved residue were constructed by site-directed mutagenesis. The mannosyltransferase activity of each mutant was examined by both an in vitro transferase assay using recombinant mutant AceA expressed in Escherichia coli and by an in vivo rescue assay by expressing the mutant AceA in a Xanthomonas campestris gumH(-) strain. We found that only mutants K211A and E287A lost all detectable activity both in vitro and in vivo, whereas E295A retained residual activity in the more sensitive in vivo assay. H127A and S162A each retained reduced but significant activities both in vitro and in vivo. Secondary structure predictions of AceA and subsequent comparison with the crystal structures of the T4 beta-glucosyltransferase and MurG suggest that AceA Lys-211 and Glu-295 are involved in nucleotide sugar donor binding, leaving Glu-287 of the EX(7)E as a potential catalytic residue.
Fil: Abdian, Patricia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; 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: Lellouch, Annemarie C.. Centre National de la Recherche Scientifique; Francia
Fil: Gautier, Catherine. Centre National de la Recherche Scientifique; Francia
Fil: Ielpi, Luis. 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. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina
Fil: Geremia, Roberto A.. Centre National de la Recherche Scientifique; Francia
Materia
GLYCOSYLTRANSFERASE
SITE DIRECTED MUTAGENESIS
MECHANISM
ACETOBACTER XYLINUM
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:11336/47751
Acceder
id CONICETDig_ddf2cae694b749c5394e778e76018504
oai_identifier_str oai:ri.conicet.gov.ar:11336/47751
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceAAbdian, Patricia LorenaLellouch, Annemarie C.Gautier, CatherineIelpi, LuisGeremia, Roberto A.GLYCOSYLTRANSFERASESITE DIRECTED MUTAGENESISMECHANISMACETOBACTER XYLINUMhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The alpha-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues included Glu-287 and Glu-295, which comprise an EX(7)E motif and have been proposed to be involved in catalysis. Alanine replacements of each conserved residue were constructed by site-directed mutagenesis. The mannosyltransferase activity of each mutant was examined by both an in vitro transferase assay using recombinant mutant AceA expressed in Escherichia coli and by an in vivo rescue assay by expressing the mutant AceA in a Xanthomonas campestris gumH(-) strain. We found that only mutants K211A and E287A lost all detectable activity both in vitro and in vivo, whereas E295A retained residual activity in the more sensitive in vivo assay. H127A and S162A each retained reduced but significant activities both in vitro and in vivo. Secondary structure predictions of AceA and subsequent comparison with the crystal structures of the T4 beta-glucosyltransferase and MurG suggest that AceA Lys-211 and Glu-295 are involved in nucleotide sugar donor binding, leaving Glu-287 of the EX(7)E as a potential catalytic residue.Fil: Abdian, Patricia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; 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: Lellouch, Annemarie C.. Centre National de la Recherche Scientifique; FranciaFil: Gautier, Catherine. Centre National de la Recherche Scientifique; FranciaFil: Ielpi, Luis. 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. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Geremia, Roberto A.. Centre National de la Recherche Scientifique; FranciaAmerican Society for Biochemistry and Molecular Biology2000-12info: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/11336/47751Abdian, Patricia Lorena; Lellouch, Annemarie C.; Gautier, Catherine; Ielpi, Luis; Geremia, Roberto A.; Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 275; 51; 12-2000; 40568-405750021-92581083-351XCONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://www.jbc.org/content/275/51/40568.longinfo:eu-repo/semantics/altIdentifier/doi/10.1074/jbc.M007496200info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T12:13:53Zoai:ri.conicet.gov.ar:11336/47751instacron: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:34982025-10-22 12:13:53.653CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA
title Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA
spellingShingle Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA
Abdian, Patricia Lorena
GLYCOSYLTRANSFERASE
SITE DIRECTED MUTAGENESIS
MECHANISM
ACETOBACTER XYLINUM
title_short Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA
title_full Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA
title_fullStr Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA
title_full_unstemmed Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA
title_sort Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA
dc.creator.none.fl_str_mv Abdian, Patricia Lorena
Lellouch, Annemarie C.
Gautier, Catherine
Ielpi, Luis
Geremia, Roberto A.
author Abdian, Patricia Lorena
author_facet Abdian, Patricia Lorena
Lellouch, Annemarie C.
Gautier, Catherine
Ielpi, Luis
Geremia, Roberto A.
author_role author
author2 Lellouch, Annemarie C.
Gautier, Catherine
Ielpi, Luis
Geremia, Roberto A.
author2_role author
author
author
author
dc.subject.none.fl_str_mv GLYCOSYLTRANSFERASE
SITE DIRECTED MUTAGENESIS
MECHANISM
ACETOBACTER XYLINUM
topic GLYCOSYLTRANSFERASE
SITE DIRECTED MUTAGENESIS
MECHANISM
ACETOBACTER XYLINUM
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The alpha-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues included Glu-287 and Glu-295, which comprise an EX(7)E motif and have been proposed to be involved in catalysis. Alanine replacements of each conserved residue were constructed by site-directed mutagenesis. The mannosyltransferase activity of each mutant was examined by both an in vitro transferase assay using recombinant mutant AceA expressed in Escherichia coli and by an in vivo rescue assay by expressing the mutant AceA in a Xanthomonas campestris gumH(-) strain. We found that only mutants K211A and E287A lost all detectable activity both in vitro and in vivo, whereas E295A retained residual activity in the more sensitive in vivo assay. H127A and S162A each retained reduced but significant activities both in vitro and in vivo. Secondary structure predictions of AceA and subsequent comparison with the crystal structures of the T4 beta-glucosyltransferase and MurG suggest that AceA Lys-211 and Glu-295 are involved in nucleotide sugar donor binding, leaving Glu-287 of the EX(7)E as a potential catalytic residue.
Fil: Abdian, Patricia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; 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: Lellouch, Annemarie C.. Centre National de la Recherche Scientifique; Francia
Fil: Gautier, Catherine. Centre National de la Recherche Scientifique; Francia
Fil: Ielpi, Luis. 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. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina
Fil: Geremia, Roberto A.. Centre National de la Recherche Scientifique; Francia
description The alpha-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues included Glu-287 and Glu-295, which comprise an EX(7)E motif and have been proposed to be involved in catalysis. Alanine replacements of each conserved residue were constructed by site-directed mutagenesis. The mannosyltransferase activity of each mutant was examined by both an in vitro transferase assay using recombinant mutant AceA expressed in Escherichia coli and by an in vivo rescue assay by expressing the mutant AceA in a Xanthomonas campestris gumH(-) strain. We found that only mutants K211A and E287A lost all detectable activity both in vitro and in vivo, whereas E295A retained residual activity in the more sensitive in vivo assay. H127A and S162A each retained reduced but significant activities both in vitro and in vivo. Secondary structure predictions of AceA and subsequent comparison with the crystal structures of the T4 beta-glucosyltransferase and MurG suggest that AceA Lys-211 and Glu-295 are involved in nucleotide sugar donor binding, leaving Glu-287 of the EX(7)E as a potential catalytic residue.
publishDate 2000
dc.date.none.fl_str_mv 2000-12
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/11336/47751
Abdian, Patricia Lorena; Lellouch, Annemarie C.; Gautier, Catherine; Ielpi, Luis; Geremia, Roberto A.; Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 275; 51; 12-2000; 40568-40575
0021-9258
1083-351X
CONICET Digital
CONICET
url http://hdl.handle.net/11336/47751
identifier_str_mv Abdian, Patricia Lorena; Lellouch, Annemarie C.; Gautier, Catherine; Ielpi, Luis; Geremia, Roberto A.; Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 275; 51; 12-2000; 40568-40575
0021-9258
1083-351X
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://www.jbc.org/content/275/51/40568.long
info:eu-repo/semantics/altIdentifier/doi/10.1074/jbc.M007496200
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 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: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 12.982451

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