Difference in Protein Substrate Specificity between Hemorrhagic Toxin and Lethal Toxin fromClostridium sordellii

ABSTRACT Clostridium sordellii lethal toxin is a member of the family of large clostridial cytotoxins that glucosylate small GTPases. In contrast to Clostridium difficile toxins A and B, which exclusively modify Rho subfamily proteins, C. sordelliilethal toxin also glucosylates Ras subfamily proteins. By deletion analysis and construction of chimeric fusion proteins of C. sordellii lethal toxin and C. difficile toxin B, we localized the enzyme activity of the lethal toxin to the N terminus of the holotoxin and identified the region involved in protein substrate specificity. The toxin fragment of the N-terminal 546 amino acid residues of C. sordellii lethal toxin glucosylated Rho and Ras subfamily proteins, as the holotoxin did. Deletion of a further 30 amino acid residues from the C terminus of this active fragment drastically reduced glucotransferase activity and blocked glucohydrolase activity. Exchange of amino acid residues 364 through 516 of lethal toxin for those in the active toxin B fragment (1 to 546) allowed glucosylation of Ras subfamily proteins. In contrast, the chimera with amino acids 1 to 364 from toxin B, 365 to 468 from lethal toxin, and 469 to 546 from toxin B exhibited markedly reduced modification of Ras subfamily proteins, whereas modification of Rac and Cdc42 was hardly changed. The data indicate that the region of amino acid residues 364 through 516 primarily defines the substrate specificity of C. sordellii lethal toxin.

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Substrate specificity and regulation of the maize (Zea mays) leaf ADP: protein phosphotransferase catalysing phosphorylation/inactivation of pyruvate, orthophosphate dikinase

Biochemical Journal ◽

10.1042/bj2360579 ◽

1986 ◽

Vol 236 (2) ◽

pp. 579-584 ◽

Cited By ~ 20

Author(s):

R J A Budde ◽

S M Ernst ◽

R Chollet

Keyword(s):

Zea Mays ◽

Substrate Specificity ◽

Regulatory Protein ◽

Energy Charge ◽

Adenylate Energy Charge ◽

Protein Substrate ◽

Maize Leaf ◽

Pyruvate Orthophosphate Dikinase ◽

Orthophosphate Dikinase ◽

Regulatory Phosphorylation

The protein substrate specificity of the maize (Zea mays) leaf ADP: protein phosphotransferase (regulatory protein, RP) was studied in terms of its relative ability to inactivate/phosphorylate pyruvate, orthophosphate dikinase from Zea mays and the non-sulphur purple photosynthetic bacterium Rhodospirillum rubrum. The dimeric bacterial dikinase was inactivated by the maize leaf RP via phosphorylation, with a stoichiometry of approximately 1 mol of phosphate incorporated/mol of 92.7-kDa protomer. Inactivation required both ADP and ATP, with ADP being the specific donor for regulatory phosphorylation. The requirements for inactivation/phosphorylation in this heterologous system were identical with those previously established for the tetrameric maize leaf dikinase. The ADP-dependent maize leaf RP did not phosphorylate alternative protein substrates such as casein or phosvitin, and its activity was not affected by cyclic nucleotides, Ca2+ or calmodulin. The regulation of the maize leaf ADP: protein phosphotransferase was studied in terms of changes in adenylate energy charge and pyruvate concentration. The change in adenylate energy charge necessary to substantially inhibit phosphorylation of maize leaf dikinase was not suggestive of it being a physiological modulator of phosphotransferase activity. Pyruvate was a potent competitive inhibitor of regulatory phosphorylation (Ki = 80 microM), consistent with its interaction with the catalytic phosphorylated intermediate of dikinase, the true protein substrate for ADP-dependent phosphorylation/inactivation.

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Roles of the Tetratricopeptide Repeat Domain inO-GlcNAc Transferase Targeting and Protein Substrate Specificity

Journal of Biological Chemistry ◽

10.1074/jbc.m300036200 ◽

2003 ◽

Vol 278 (27) ◽

pp. 24608-24616 ◽

Cited By ~ 123

Author(s):

Sai Prasad N. Iyer ◽

Gerald W. Hart

Keyword(s):

Substrate Specificity ◽

Tetratricopeptide Repeat ◽

Protein Substrate ◽

Repeat Domain ◽

Tetratricopeptide Repeat Domain ◽

Glcnac Transferase

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Molecular dynamics investigations of BioH protein substrate specificity for biotin synthesis

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2015.1068223 ◽

2015 ◽

Vol 34 (5) ◽

pp. 1052-1060 ◽

Cited By ~ 5

Author(s):

Qiao Xue ◽

Ying-Lu Cui ◽

Qing-Chuan Zheng ◽

Hong-Xing Zhang

Keyword(s):

Molecular Dynamics ◽

Substrate Specificity ◽

Protein Substrate

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Difference in the Cytotoxic Effects of Toxin B from Clostridium difficile Strain VPI 10463 and Toxin B from Variant Clostridium difficile Strain 1470

Infection and Immunity ◽

10.1128/iai.01705-06 ◽

2006 ◽

Vol 75 (2) ◽

pp. 801-809 ◽

Cited By ~ 51

Author(s):

Johannes Huelsenbeck ◽

Stefanie Dreger ◽

Ralf Gerhard ◽

Holger Barth ◽

Ingo Just ◽

...

Keyword(s):

Clostridium Difficile ◽

Substrate Specificity ◽

Cytotoxic Effect ◽

Caspase 3 ◽

Cytopathic Effect ◽

Phosphatidyl Serine ◽

Toxin B ◽

Protein Substrate ◽

Cellular Effects ◽

Rbl Cells

ABSTRACT Glucosylation of RhoA, Rac1, and Cdc42 by Clostridium difficile toxin B from strain VPI 10463 (TcdB) results in actin reorganization (cytopathic effect) and apoptosis (cytotoxic effect). Toxin B from variant C. difficile strain 1470 serotype F (TcdBF) differs from TcdB with regard to substrate proteins, as it glucosylates Rac1 and R-Ras but not RhoA and Cdc42. In this study, we addressed the question of whether the cellular effects of the toxins depend on their protein substrate specificity. Rat basophilic leukemia (RBL) cells were synchronized using the thymidine double-block technique. We show that cells were most sensitive to the cytotoxic effect of TcdB in S phase, as analyzed in terms of phosphatidyl serine externalization, fragmentation of nuclei, and activation of caspase-3; in contrast, TcdBF induced only a marginal cytotoxic effect, suggesting that inactivation of RhoA (but not of Rac1) was required for the cytotoxic effect. The glucosylation of Rac1 was correlated to the cytopathic effect of either toxin, suggesting a close connection of the two effects. The cytotoxic effect of TcdB was executed by caspase-3, as it was responsive to inhibition by acetyl-Asp-Met-Gln-Asp-aldehyde (Ac-DMQD-CHO), an inhibitor of caspase-3. The viability of TcdB-treated RBL cells was reduced, whereas the viability of TcdBF-treated cells was unchanged, further confirming that inactivation of RhoA is required for the cytotoxic effect. In conclusion, the protein substrate specificity of the glucosylating toxins determines their biological activity.

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