Expression and study of recombinant ExoM, a beta 1-4 glucosyltransferase involved in succinoglycan biosynthesis in Sinorhizobium meliloti

Here we report on the overexpression and in vitro characterization of a rec ombinant form of ExoM, a putative beta 1-4 glucosyltransferase involved in the assembly of the octasaccharide repeating subunit of succinoglycan from Sinorhizobium meliloti. The open reading frame exoM was isolated by PCR and subcloned into the expression vector pET29b, allowing inducible expression under the control of the T7 promoter. Escherichia coli BL21(DE3)/pLysS con taining exoM expressed a novel 38-kDa protein corresponding to ExoM in N-te rminal fusion with the S-tag peptide. Cell fractionation studies showed tha t the protein is expressed in E. coli as a membrane-bound protein in agreem ent with the presence of a predicted C-terminal transmembrane region. E. co li membrane preparations containing ExoM were shown to be capable of transf erring glucose from UDP-glucose to glycolipid extracts from an S. meliloti mutant strain which accumulates the ExoM substrate (Glc beta 1-4Glc beta 1- 3Gal-pyrophosphate-polyprenol). Thin-layer chromatography of the glycosidic portion of the ExoM product showed that the oligosaccharide formed comigra tes with an authentic standard. The oligosaccharide produced by the recombi nant ExoM, but not the starting substrate, was sensitive to cleavage with a specific cellobiohydrolase, consistent with the formation of a beta 1-4 gl ucosidic linkage. No evidence for the transfer of multiple glucose residues to the glycolipid substrate was observed. It was also found that ExoM does not transfer glucose to an acceptor substrate that has been hydrolyzed fro m the polyprenol anchor. Furthermore, neither glucose, cellobiose, nor the trisaccharide Glc beta 1-4Glc beta 1-3Glc inhibited the transferase activit y, suggesting that some feature of the lipid anchor is necessary for activi ty.