Identification of essential amino acid residues in the Sinorhizobium meliloti glucosyltransferase ExoM

ExoM is a beta(1-4)-glucosyltransferase involved in the assembly of the rep eat unit of the exopolysaccharide succinoglycan from Sinorhizobium meliloti , By comparing the sequence of ExoM to those of other members of the Pfam G lyco Domain 2 family, most notably SpsA (Bacillus subtilis) for whom the th ree-dimensional structure has been resolved, three potentially important as partic acid residues of ExoM were identified, Single substitutions of each of the Asp amino acids at positions 44, 96, and 187 with Ala resulted in th e loss of mutant recombinant protein activity in vitro as well as the loss of succinoglycan production in an in vivo rescue assay. Mutants harboring G lu instead of Asp 44 or Asp-96 possessed no in vitro activity but could res tore succinoglycan production in vivo. However, replacement of Asp-187 with Glu completely inactivated ExoM as judged by both the in vitro and in vivo assays. These results indicate that Asp-44, Asp-96, and Asp-187 are essent ial for the activity of ExoM. Furthermore, these data are consistent with t he functions proposed for each of the analogous aspartic acids of SpsA base d on the SpsA-UDP structure, namely, that Asp-44 and Asp-96 are involved in UDP substrate binding and that Asp-187 is the catalytic base in the glycos yltransferase reaction.