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 repeat unit of the exopolysaccharide succinoglycan from Sinorhizobium meliloti. By comparing the sequence of ExoM to those of other members of the Pfam Glyco Domain 2 family, most notably SpsA (Bacillus subtilis) for whom the three-dimensional structure has been resolved, three potentially important aspartic 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 the loss of mutant recombinant protein activity in vitro as well as the loss of succinoglycan production in an in vivo rescue assay. Mutants harboring Glu instead of Asp-44 or Asp-96 possessed no in vitro activity but could restore 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 essential for the activity of ExoM. Furthermore, these data are consistent with the functions proposed for each of the analogous aspartic acids of SpsA based 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 glycosyltransferase reaction.
Garinot-Schneider
C
C
Centre de Recherches sur les Macromolécules Végétales, CNRS, Affiliated with the Joseph Fourier University, BP 53X, Grenoble 38041, Cedex 9, France.
Lellouch
A C
AC
Geremia
R A
RA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D001216:Asparagine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D015153:Blotting, Western; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics; D005973:Glutamine / Q000737:chemistry; D016695:Glycosyltransferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016962:Sinorhizobium meliloti / Q000201:enzymology*
D000596:Amino Acids; D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; C110344:SpsA protein, Streptococcus pneumoniae; D005973:Glutamine; D001216:Asparagine; C056779:succinoglycan; D016695:Glycosyltransferases; C117828:ExoM protein, Sinorhizobium meliloti; D005964:Glucosyltransferases