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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todo:paper_00268925_v261_n6_p933_Geremia2023-10-03T14:37:10Z Expression and biochemical characterisation of recombinant AceA, a bacterial α-mannosyltransferase Geremia, R.A. Roux, M. Ferreiro, D.U. Dauphin-Dubois, R. Lellouch, A.C. Ielpi, L. 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. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar 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 Geremia, R.A. Roux, M. Ferreiro, D.U. Dauphin-Dubois, R. Lellouch, A.C. Ielpi, L. 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. |
format |
JOUR |
author |
Geremia, R.A. Roux, M. Ferreiro, D.U. Dauphin-Dubois, R. Lellouch, A.C. Ielpi, L. |
author_facet |
Geremia, R.A. Roux, M. Ferreiro, D.U. Dauphin-Dubois, R. Lellouch, A.C. Ielpi, L. |
author_sort |
Geremia, R.A. |
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 |
url |
http://hdl.handle.net/20.500.12110/paper_00268925_v261_n6_p933_Geremia |
work_keys_str_mv |
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