ABCMdb

PMID: 10908566

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., [PubMed]

Sentences

No. Mutations Sentence Comment

40 ABCC8 p.Asp96Ala 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 131 ABCC8 p.Asp96Glu However, the two mutants N45A and D96E were not detected on Coomassie Blue-stained SDS-PAGE gels (Fig. 3). Login to comment 143 ABCC8 p.Asp96Ala Only the mutation of the conserved Asp, D96A, resulted in complete loss of activity. Login to comment 144 ABCC8 p.Asp96Glu 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. Login to comment 162 ABCC8 p.Asp96Ala 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 163 ABCC8 p.Asp96Glu However, of the conservative mutations D44E, D96E, and D187E, only D187E was completely incapable of restoring succinoglycan production. Login to comment 164 ABCC8 p.Asp96Glu 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. Login to comment 175 ABCC8 p.Asp96Ala ABCC8 p.Asp96Glu 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