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Originally published In Press as doi:10.1074/jbc.M004524200 on July 24, 2000

J. Biol. Chem., Vol. 275, Issue 40, 31407-31413, October 6, 2000
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Identification of Essential Amino Acid Residues in the Sinorhizobium meliloti Glucosyltransferase ExoM*

Carole Garinot-SchneiderDagger , Annemarie C. Lellouch, and Roberto A. Geremia§

From the Centre de Recherches sur les Macromolécules Végétales, CNRS, Affiliated with the Joseph Fourier University, BP 53X, Grenoble 38041, Cedex 9, France

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.

* This work was supported in part by the program "Physique et chimie du vivant" (CNRS).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a postdoctoral fellowship from the "Société de secours des Amis des Sciences."

§ To whom correspondence should be addressed: CERMAV/CNRS BP 53. Grenoble cedex 9, France. Tel.: 33-04-76-03-76-47; Fax: 33-04-76-54-72-03; E-mail: roberto.geremia@cermav.cnrs.fr.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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