Expression and biochemical characterisation of recombinant AceA, a bacterial α-mannosyltransferase

Biosynthesis of repeat-unit polysaccharides and N-linked glycans proceeds by sequential transfer of sugars from the appropriate sugar donor to an activated lipid carrier. The transfer of each sugar is catalysed by a specific glycosyltransferase. The molecular basis of the specificity of sugar additi...

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Autores principales:	Geremía, Roberto Alejandro, Ferreiro, Diego U., Ielpi, Luis
Publicado:	1999
Materias:	Acetan Acetobacter xylinum Bacterial polysaccharide Biosynthesis Glycosyltransferase bacterial enzyme cellobiose guanosine diphosphate mannose mannose mannosyltransferase recombinant enzyme acetobacter article controlled study enzyme activity escherichia coli expression vector gene overexpression molecular cloning nonhuman open reading frame priority journal structure activity relation Amino Acid Sequence Base Sequence Carbohydrate Sequence Cloning, Molecular DNA, Recombinant Mannosyltransferases Mutagenesis, Site-Directed Open Reading Frames Substrate Specificity Acetobacter Bacteria (microorganisms) Escherichia coli Gluconacetobacter xylinus
Acceso en línea:	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00268925_v261_n6_p933_Geremia http://hdl.handle.net/20.500.12110/paper_00268925_v261_n6_p933_Geremia
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	paper:paper_00268925_v261_n6_p933_Geremia
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spelling	paper:paper_00268925_v261_n6_p933_Geremia2023-06-08T14:53:54Z Expression and biochemical characterisation of recombinant AceA, a bacterial α-mannosyltransferase Geremía, Roberto Alejandro Ferreiro, Diego U. Ielpi, Luis Acetan Acetobacter xylinum Bacterial polysaccharide Biosynthesis Glycosyltransferase bacterial enzyme cellobiose guanosine diphosphate mannose mannose mannosyltransferase recombinant enzyme acetobacter article controlled study enzyme activity escherichia coli expression vector gene overexpression molecular cloning nonhuman open reading frame priority journal structure activity relation Amino Acid Sequence Base Sequence Carbohydrate Sequence Cloning, Molecular DNA, Recombinant Mannosyltransferases Mutagenesis, Site-Directed Open Reading Frames Substrate Specificity Acetobacter Bacteria (microorganisms) Escherichia coli Gluconacetobacter xylinus Biosynthesis of repeat-unit polysaccharides and N-linked glycans proceeds by sequential transfer of sugars from the appropriate sugar donor to an activated lipid carrier. The transfer of each sugar is catalysed by a specific glycosyltransferase. The molecular basis of the specificity of sugar addition is not yet well understood, mainly because of the difficulty of isolating these proteins. In this study, the aceA gene product expressed by Acetobacter xylinum, which is involved in the biosynthesis of the exopolysaccharide acetan, was overproduced in Escherichia coli and its function was characterised. The aceA ORF was subcloned into the expression vector pET29 in frame with the S·tag epitope. The recombinant protein was identified, and culture conditions were optimised for production of the soluble protein. The results of test reactions showed that AceA is able to transfer one α-mannose residue from GDP-mannose to cellobiose-P-P-lipid to produce α-mannose-cellobiose-P-P-lipid. AceA was not able to use free cellobiose as a substrate, indicating that the pyrophosphate-lipid moiety is needed for enzymatic activity. Fil:Geremia, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Ferreiro, D.U. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Ielpi, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 1999 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00268925_v261_n6_p933_Geremia http://hdl.handle.net/20.500.12110/paper_00268925_v261_n6_p933_Geremia
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	Acetan Acetobacter xylinum Bacterial polysaccharide Biosynthesis Glycosyltransferase bacterial enzyme cellobiose guanosine diphosphate mannose mannose mannosyltransferase recombinant enzyme acetobacter article controlled study enzyme activity escherichia coli expression vector gene overexpression molecular cloning nonhuman open reading frame priority journal structure activity relation Amino Acid Sequence Base Sequence Carbohydrate Sequence Cloning, Molecular DNA, Recombinant Mannosyltransferases Mutagenesis, Site-Directed Open Reading Frames Substrate Specificity Acetobacter Bacteria (microorganisms) Escherichia coli Gluconacetobacter xylinus
spellingShingle	Acetan Acetobacter xylinum Bacterial polysaccharide Biosynthesis Glycosyltransferase bacterial enzyme cellobiose guanosine diphosphate mannose mannose mannosyltransferase recombinant enzyme acetobacter article controlled study enzyme activity escherichia coli expression vector gene overexpression molecular cloning nonhuman open reading frame priority journal structure activity relation Amino Acid Sequence Base Sequence Carbohydrate Sequence Cloning, Molecular DNA, Recombinant Mannosyltransferases Mutagenesis, Site-Directed Open Reading Frames Substrate Specificity Acetobacter Bacteria (microorganisms) Escherichia coli Gluconacetobacter xylinus Geremía, Roberto Alejandro Ferreiro, Diego U. Ielpi, Luis Expression and biochemical characterisation of recombinant AceA, a bacterial α-mannosyltransferase
topic_facet	Acetan Acetobacter xylinum Bacterial polysaccharide Biosynthesis Glycosyltransferase bacterial enzyme cellobiose guanosine diphosphate mannose mannose mannosyltransferase recombinant enzyme acetobacter article controlled study enzyme activity escherichia coli expression vector gene overexpression molecular cloning nonhuman open reading frame priority journal structure activity relation Amino Acid Sequence Base Sequence Carbohydrate Sequence Cloning, Molecular DNA, Recombinant Mannosyltransferases Mutagenesis, Site-Directed Open Reading Frames Substrate Specificity Acetobacter Bacteria (microorganisms) Escherichia coli Gluconacetobacter xylinus
description	Biosynthesis of repeat-unit polysaccharides and N-linked glycans proceeds by sequential transfer of sugars from the appropriate sugar donor to an activated lipid carrier. The transfer of each sugar is catalysed by a specific glycosyltransferase. The molecular basis of the specificity of sugar addition is not yet well understood, mainly because of the difficulty of isolating these proteins. In this study, the aceA gene product expressed by Acetobacter xylinum, which is involved in the biosynthesis of the exopolysaccharide acetan, was overproduced in Escherichia coli and its function was characterised. The aceA ORF was subcloned into the expression vector pET29 in frame with the S·tag epitope. The recombinant protein was identified, and culture conditions were optimised for production of the soluble protein. The results of test reactions showed that AceA is able to transfer one α-mannose residue from GDP-mannose to cellobiose-P-P-lipid to produce α-mannose-cellobiose-P-P-lipid. AceA was not able to use free cellobiose as a substrate, indicating that the pyrophosphate-lipid moiety is needed for enzymatic activity.
author	Geremía, Roberto Alejandro Ferreiro, Diego U. Ielpi, Luis
author_facet	Geremía, Roberto Alejandro Ferreiro, Diego U. Ielpi, Luis
author_sort	Geremía, Roberto Alejandro
title	Expression and biochemical characterisation of recombinant AceA, a bacterial α-mannosyltransferase
title_short	Expression and biochemical characterisation of recombinant AceA, a bacterial α-mannosyltransferase
title_full	Expression and biochemical characterisation of recombinant AceA, a bacterial α-mannosyltransferase
title_fullStr	Expression and biochemical characterisation of recombinant AceA, a bacterial α-mannosyltransferase
title_full_unstemmed	Expression and biochemical characterisation of recombinant AceA, a bacterial α-mannosyltransferase
title_sort	expression and biochemical characterisation of recombinant acea, a bacterial α-mannosyltransferase
publishDate	1999
url	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00268925_v261_n6_p933_Geremia http://hdl.handle.net/20.500.12110/paper_00268925_v261_n6_p933_Geremia
work_keys_str_mv	AT geremiarobertoalejandro expressionandbiochemicalcharacterisationofrecombinantaceaabacterialamannosyltransferase AT ferreirodiegou expressionandbiochemicalcharacterisationofrecombinantaceaabacterialamannosyltransferase AT ielpiluis expressionandbiochemicalcharacterisationofrecombinantaceaabacterialamannosyltransferase
_version_	1768546432607846400

Expression and biochemical characterisation of recombinant AceA, a bacterial α-mannosyltransferase

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