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

Biosynthesis of repeat-unit polysaccharides and N-linked glycans proceeds b y sequential transfer of sugars from the appropriate sugar donor to an acti vated lipid carrier. The transfer of each sugar is catalysed by a specific glycosyltransferase. The molecular basis of the specificity of sugar additi on is not yet well understood, mainly because of the difficulty of isolatin g these proteins. In this study, the aceA gene product expressed by Acetoba cter xylinum, which is involved in the biosynthesis of the exopolysaccharid e acetan, was overproduced in Escherichia coli and its function was charact erised. The aceA ORF was subcloned into the expression vector pET29 in fram e with the Stag epitope. The recombinant protein was identified, and cultur e conditions were optimised for production of the soluble protein. The resu lts of test reactions showed that AceA is able to transfer one alpha-mannos e 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 substra te, indicating that the pyrophosphate-lipid moiety is needed for enzymatic activity.