ABCMdb

ABCC8 p.Asp96Ala

Predicted by SNAP2:	A: N (72%), C: N (72%), E: N (72%), F: D (71%), G: N (66%), H: N (61%), I: D (71%), K: D (59%), L: N (53%), M: D (71%), N: N (87%), P: N (53%), Q: N (61%), R: D (53%), S: N (72%), T: N (66%), V: N (61%), W: D (75%), Y: D (53%),
Predicted by PROVEAN:	A: D, C: D, E: N, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: N, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Identification of essential amino acid residues in... J Biol Chem. 2000 Oct 6;275(40):31407-13. Garinot-Schneider C, Lellouch AC, Geremia RA
Identification of essential amino acid residues in the Sinorhizobium meliloti glucosyltransferase ExoM.
J Biol Chem. 2000 Oct 6;275(40):31407-13., [PMID:10908566]

Abstract [show]
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.

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Sentences [show]
No. Sentence Comment
40 We found that replacement of each of the three positions by alanine (D44A, D96A, and the newly identified D187A) abolished glucosyltransferase activity in vitro and resulted in the loss of the ability to restore succinoglycan production in an in vivo rescue assay. Login to comment
143 Only the mutation of the conserved Asp, D96A, resulted in complete loss of activity. Login to comment
162 The substitutions of the conserved aspartic acids, D44A, D96A, and D187A, which were expressed in E. coli but inactive in vitro, were expressed in S. meliloti and were also not able to restore succinoglycan production in vivo. Login to comment
175 In vitro activity Succinoglycan production (%wt) %wt ExoM 100 100 Site I V42I 100 69 A43D 3 Ͼ3 D44Aa 0 Ͼ3 D44E 7 77 N45A NEb NE D46A 75 NE Site II F94V 69 77 L95A 30 114 D96A 0 7 D96E NE 41 D97A 15 115 D98A 26 139 E99A 100 84 Site III D187A 0 3 D187E 0 4 a The mutations involving conserved amino acid residues are in italics. b NE, not expressed. Login to comment