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Updated: Aug 16, 2026
A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli
Published on: December 9, 2017
Change of the donor substrate specificity of Clostridium difficile toxin B by site-directed mutagenesis
Thomas Jank1, Dirk J Reinert, Torsten Giesemann
1Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Freiburg, Germany.
Abstract:
The large cytotoxins of Clostridia species glycosylate and thereby inactivate small GTPases of the Rho family. Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin use UDP-glucose as the donor for glucosylation of Rho/Ras GTPases. In contrast, alpha-toxin from Clostridium novyi N-acetylglucosaminylates Rho GTPases by using UDP-N-acetylglucosamine as a donor substrate. Based on the crystal structure of C. difficile toxin B, we studied the sugar donor specificity of the toxins by site-directed mutagenesis. The changing of Ile-383 and Gln-385 in toxin B to serine and alanine, respectively, largely increased the acceptance of UDP-N-acetylglucosamine as a sugar donor for modification of RhoA. The K(m) value was reduced from 960 to 26 mum for the double mutant. Accordingly, the potential of the double mutant of toxin B to hydrolyze UDP-N-acetylglucosamine was higher than that for UDP-glucose. The changing of Ile-383 and Gln-385 in the lethal toxin of C. sordellii allowed modification of Ras in the presence of UDP-N-acetyl-glucosamine and reduced the acceptance of UDP-glucose as a donor for glycosylation. Vice versa, the changing of the equivalent residues in C. novyi alpha-toxin from Ser-385 and Ala-387 to isoleucine and glutamine, respectively, reversed the donor specificity of the toxin from UDP-N-acetylglucosamine to UDP-glucose. These data demonstrate that two amino acid residues are crucial for the co-substrate specificity of clostridial glycosylating toxins.
Insights
Two amino acid residues critically control the sugar donor specificity in Clostridia glycosylating toxins. Mutating specific residues in toxins like Clostridium difficile toxin B and alpha-toxin alters their substrate preference between UDP-glucose and UDP-N-acetylglucosamine.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Clostridia species produce large cytotoxins that inactivate Rho family GTPases through glycosylation.
- Different Clostridia toxins utilize distinct sugar donors, such as UDP-glucose or UDP-N-acetylglucosamine, for this modification.
Purpose of the Study:
- To investigate the role of specific amino acid residues in determining the sugar donor specificity of Clostridia glycosylating toxins.
- To understand how altering these residues affects the toxins' ability to glycosylate Rho/Ras GTPases.
Main Methods:
- Site-directed mutagenesis based on the crystal structure of Clostridium difficile toxin B.
- Enzyme kinetics (K(m) values) to assess substrate acceptance and hydrolysis.
- Analysis of toxin activity with different sugar donors (UDP-glucose and UDP-N-acetylglucosamine).
Main Results:
- Mutating Ile-383 and Gln-385 in toxin B significantly increased its acceptance of UDP-N-acetylglucosamine, reducing K(m) from 960 to 26 μM.
- Equivalent mutations in Clostridium sordellii lethal toxin altered its donor preference.
- Reversing mutations in Clostridium novyi alpha-toxin switched its donor specificity from UDP-N-acetylglucosamine to UDP-glucose.
Conclusions:
- Two specific amino acid residues are crucial determinants of co-substrate specificity in clostridial glycosylating toxins.
- These findings provide insights into the molecular mechanisms underlying toxin activity and potential targets for therapeutic intervention.
