2.4.1.B62: small GTPase glucosyltransferase
This is an abbreviated version!
For detailed information about small GTPase glucosyltransferase, go to the full flat file.
Reaction
Synonyms
Clostridium sordellii lethal toxin, cytotoxin B, cytotoxin L, glucosyltransferase TcdA, glucosyltransferase TcdB, haemorrhagic toxin, hemorrhagic toxin, letal toxin, lethal toxin, lethal-toxin, TcdA, TcdB, TcsH, TcsL, toxA, ToxB, toxin, toxin A, toxin B
ECTree
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Engineering
Engineering on EC 2.4.1.B62 - small GTPase glucosyltransferase
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C698A
mutant is able to glucosylate Rac protein but does not display cytotoxicity. Mutant does not show autocatalytical activity
D587N
mutant is able to glucosylate Rac protein but does not display cytotoxicity. Mutant does not show autocatalytical activity
H653A
mutant is able to glucosylate Rac protein but does not display cytotoxicity. Mutant does not show autocatalytical activity
I383S
mutation in toxin B, 67% of wild-type catalytic efficiency with substrate UDP-alpha-D-glucose
I383S/Q385A
mutation in toxin B, 23% of wild-type catalytic efficiency with substrate UDP-alpha-D-glucose. Mutation largely increases the acceptance of UDP-Nacetylglucosamine as a sugar donor for modification of RhoA
Q385A
mutation in toxin B, 58% of wild-type catalytic efficiency with substrate UDP-alpha-D-glucose
I383S
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mutation in toxin B, 67% of wild-type catalytic efficiency with substrate UDP-alpha-D-glucose
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I383S/Q385A
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mutation in toxin B, 23% of wild-type catalytic efficiency with substrate UDP-alpha-D-glucose. Mutation largely increases the acceptance of UDP-Nacetylglucosamine as a sugar donor for modification of RhoA
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Q385A
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mutation in toxin B, 58% of wild-type catalytic efficiency with substrate UDP-alpha-D-glucose
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S385/A387Q
C698A
site-directed mutagenesis of the autoprocessing domain, mutant TcsL C698A is able to quickly glucosylate Rac1, similar to wild-type TcsL, but is attenuated in its ability to glucosylate Ras GTPases. The introduction of the autoprocessing mutation does not impact the glucosylation of Rac1 or H-Ras in an in vitro assay
F17K
mutation strongly decreases binding to brain phosphatidylserine
F17N/R18A
site-directed mutagenesis in the GTD membrane localization domain, on the surface of the membrane localization domain (MLD), the mutant shows a defect in membrane association in a liposome binding assay
F17N/R18A/C698A
site-directed mutagenesis in the GTD membrane localization domain, on the surface of the membrane localization domain (MLD), the mutant shows a defect in membrane association in a liposome binding assay. The triple mutant is also inhibited in both Rac1 and Ras glucosylation
I383S/Q385A
mutation allow modification of Ras in the presence of UDP-N-acetyl-glucosamine and reduces the acceptance of UDP-glucose as a donor for glycosylation
K11I
mutation moderately decreases binding to brain phosphatidylserine
K16I
mutation moderately decreases binding to brain phosphatidylserine
Q10A
mutation does not significantly decrease binding to brain phosphatidylserine
Q20A
mutation moderately decreases binding to brain phosphatidylserine
R18P
mutation strongly decreases binding to brain phosphatidylserine
R68A
mutation strongly decreases binding to brain phosphatidylserine
S38A
mutation does not significantly decrease binding to brain phosphatidylserine
V15S
mutation strongly decreases binding to brain phosphatidylserine
Y14A
mutation strongly decreases binding to brain phosphatidylserine
Y78A
mutation does not significantly decrease binding to brain phosphatidylserine
I383S/Q385A
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mutation allow modification of Ras in the presence of UDP-N-acetyl-glucosamine and reduces the acceptance of UDP-glucose as a donor for glycosylation
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C698A
Paeniclostridium sordellii JGS6382
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site-directed mutagenesis of the autoprocessing domain, mutant TcsL C698A is able to quickly glucosylate Rac1, similar to wild-type TcsL, but is attenuated in its ability to glucosylate Ras GTPases. The introduction of the autoprocessing mutation does not impact the glucosylation of Rac1 or H-Ras in an in vitro assay
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F17N/R18A
Paeniclostridium sordellii JGS6382
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site-directed mutagenesis in the GTD membrane localization domain, on the surface of the membrane localization domain (MLD), the mutant shows a defect in membrane association in a liposome binding assay
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F17N/R18A/C698A
Paeniclostridium sordellii JGS6382
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site-directed mutagenesis in the GTD membrane localization domain, on the surface of the membrane localization domain (MLD), the mutant shows a defect in membrane association in a liposome binding assay. The triple mutant is also inhibited in both Rac1 and Ras glucosylation
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additional information
mutation reverses the donor specificity of alpha-toxin from UDP-N-acetylglucosamine to UDP-glucose
S385/A387Q
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mutation reverses the donor specificity of alpha-toxin from UDP-N-acetylglucosamine to UDP-glucose
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a nontoxigenic peptide corresponding to amino acids 2394 to 2706 of toxin A acts a a mucosal adjuvant. The adjuvant effect following oral, but not intranasal, immunization is dose dependent. Splenocytes of immunized mice challenged in vitro with keyhole limpet hemocyanin indicate the induction of a mixed Th1/Th2-type immune response, with prevalence of the Th1 branch
additional information
construction of truncation mutants. The enzymatic domain for binding UDP-glucose, for catalytically transferring glucose to Rho A and for recognizing the interaction interface of Rho A resides in the first N-terminal 659 amino acids of toxin A
additional information
expression of a fragment of toxin B, covering residues 1955. Wild-type fragment of toxin B is always recovered as a cleaved product with fragments of 63 and 47 kDa, whereas the mutants with changes C698A or H653A are expressed in full length
additional information
expression of a fragment of toxin B, covering residues 1955. Wild-type fragment of toxin B is always recovered as a cleaved product with fragments of 63 and 47 kDa, whereas the mutants with changes C698A or H653A are expressed in full length
additional information
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modification of toxin A with diethyl dicarbonate leads to concentration dependent labelling of histidine residues and abolishes both its cytotoxic activity and the ability of the toxin to bind Zn-Sepharose gel. Histidine modification has no effect on the glucosyl transferase enzyme activity of toxin A. However, modification abolishes the binding of toxin to bovine thyroglobulin in an ELISA and reduces ligand binding activity in a rabbit erythrocyte haemagglutination assay. The data suggest that the histidine residues may be crucial to the receptor-binding activity of toxin A
additional information
the Dxd motif, i.e. residues D286-D288, is required at least for activation of the UDP-glucose hydrolysis activity by Mn2+
additional information
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the Dxd motif, i.e. residues D286-D288, is required at least for activation of the UDP-glucose hydrolysis activity by Mn2+
additional information
25AA (1756-1780) are deleted to construct the deletion mutant TcdBD1756-1780. The deletion mutant TcdBD1827-1851, with deletion of a different 25AA, 1827-1851, located in the C-terminus of D97, serves as the control. The deletion mutants are expressed with His6-tags at the C-terminal in a Bacillus megaterium expression system. Wild-type and mutant toxins are introduced into mouse CT26 host cells. Both TcdBD1756-1780 and TcdBfl successfully induce autocleavage, releasing a 63kD fragment containing GTD, in the presence of InsP6. TcdBD1756-1780 undergoes autocleavage after incubation with a series of concentrations of InsP6 for several hours. The TcdBD1756-1780 mutant efficiently induces Rac1 glucosylation using CT26 cell lysate as the substrate, but fails to glucosylate Rac1 in intact CT26 cells. Thus, the deletion of amino acids 1756-1780 does not change the structure and function of the cysteine protease and glucosytransferase domains, but may result in an inability to deliver GTD into the host cytosol. The deletion of region 1756-1780 might lead to locking of the TcdB in endosomes, resulting in a failure to deliver GTD
additional information
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a nontoxigenic peptide corresponding to amino acids 2394 to 2706 of toxin A acts a a mucosal adjuvant. The adjuvant effect following oral, but not intranasal, immunization is dose dependent. Splenocytes of immunized mice challenged in vitro with keyhole limpet hemocyanin indicate the induction of a mixed Th1/Th2-type immune response, with prevalence of the Th1 branch
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additional information
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modification of toxin A with diethyl dicarbonate leads to concentration dependent labelling of histidine residues and abolishes both its cytotoxic activity and the ability of the toxin to bind Zn-Sepharose gel. Histidine modification has no effect on the glucosyl transferase enzyme activity of toxin A. However, modification abolishes the binding of toxin to bovine thyroglobulin in an ELISA and reduces ligand binding activity in a rabbit erythrocyte haemagglutination assay. The data suggest that the histidine residues may be crucial to the receptor-binding activity of toxin A
-
additional information
the Dxd motif, i.e. residues D286-D288, is required at least for activation of the UDP-glucose hydrolysis activity by Mn2+
additional information
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the Dxd motif, i.e. residues D286-D288, is required at least for activation of the UDP-glucose hydrolysis activity by Mn2+
additional information
when glucosyltransferase-deficient TcsL mutant, TcsL DxD, is used to intoxicate cells, the loss of the glucosyltransferase activity renders the mutant unable to glucosylate both Rac1 and Ras
additional information
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when glucosyltransferase-deficient TcsL mutant, TcsL DxD, is used to intoxicate cells, the loss of the glucosyltransferase activity renders the mutant unable to glucosylate both Rac1 and Ras
additional information
Paeniclostridium sordellii JGS6382
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when glucosyltransferase-deficient TcsL mutant, TcsL DxD, is used to intoxicate cells, the loss of the glucosyltransferase activity renders the mutant unable to glucosylate both Rac1 and Ras
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