Expression and biochemical characterisation of recombinant AceA, a bacterial alpha-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 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 alpha-mannose residue from GDP-mannose to cellobiose-P-P-lipid to produce alpha-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.
Geremia
R A
RA
Centre de Recherches sur les Macromolécules Végétales (CNRS), Université Joseph Fourier, Grenoble, France. geremia@cermav.cnrs.fr
Roux
M
M
Ferreiro
D U
DU
Dauphin-Dubois
R
R
Lellouch
A C
AC
Ielpi
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D008364:Mannosyltransferases / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D013379:Substrate Specificity
D004274:DNA, Recombinant; D008364:Mannosyltransferases