ABCC8 p.Asp96Glu
| 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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[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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No. Sentence Comment
131 However, the two mutants N45A and D96E were not detected on Coomassie Blue-stained SDS-PAGE gels (Fig. 3).
X
ABCC8 p.Asp96Glu 10908566:131:34
status: NEW144 Unfortunately, the mutant protein containing the conservative D96E mutation was not stably expressed in E. coli (Fig. 3) but, as will be presented below, was stably expressed in S. meliloti.
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ABCC8 p.Asp96Glu 10908566:144:62
status: NEW163 However, of the conservative mutations D44E, D96E, and D187E, only D187E was completely incapable of restoring succinoglycan production.
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ABCC8 p.Asp96Glu 10908566:163:45
status: NEW164 S. meliloti, harboring ExoM D44E and ExoM D96E, was able to produce 77 and 41% of the wild type succinoglycan level, respectively, demonstrating that these conservative mutations do not completely inactivate the protein.
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ABCC8 p.Asp96Glu 10908566:164:42
status: NEW175 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.
X
ABCC8 p.Asp96Glu 10908566:175:191
status: NEW