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Characterization of the enzymatic component of Clostridium perfringens iota-toxin
M Nagahama1, Y Sakaguchi, K Kobayashi
1Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.
Abstract:
The iota(a) component (i(a)) of Clostridium perfringens ADP ribosylates nonmuscle beta/gamma actin and skeletal muscle alpha-actin. Replacement of Arg-295 in i(a) with alanine led to a complete loss of NAD(+)-glycohydrolase (NADase) and ADP-ribosyltransferase (ARTase); that of the residue with lysine caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them. Substitution of alanine for Glu-378 and Glu-380 caused a complete loss of NADase and ARTase. However, exchange of Glu-378 to aspartic acid or glutamine resulted in little effect on NADase activity but a drastic reduction in ARTase activity (<0.1% of the wild-type activity). Exchange of Glu-380 to aspartic acid caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them; that of the residue to glutamine caused a complete loss of ARTase activity. Replacement of Ser-338 with alanine resulted in 0.7 to 2.3% wild-type activities, and that of Ser-340 and Thr-339 caused a reduction in these activities of 5 to 30% wild-type activities. The kinetic analysis showed that Arg-295 and Ser-338 also play an important role in the binding of NAD(+) to i(a), that Arg-295, Glu-380, and Ser-338 play a crucial role in the catalytic rate of NADase activity, and that these three amino acid residues and Glu-378 are essential for ARTase activity. The effect of amino acid replacement in i(a) on ARTase activity was similar to that on lethal and cytotoxic activities, suggesting that lethal and cytotoxic activities in i(a) are dependent on ARTase activity.
Insights
Clostridium perfringens iota component (Ia) ADP-ribosylates actin. Key amino acid residues Arg-295, Glu-378, Glu-380, Ser-338, Ser-340, and Thr-339 are crucial for NADase and ARTase activities, impacting Ia
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Clostridium perfringens produces the iota toxin, a binary toxin.
- The iota toxin consists of an enzymatic component (Ia) and a binding component (Ib).
- The Ia component possesses ADP-ribosyltransferase (ARTase) and NAD(+)-glycohydrolase (NADase) activities, crucial for its cytotoxic effects.
Purpose of the Study:
- To elucidate the roles of specific amino acid residues in the catalytic activity of the iota(a) component (Ia).
- To determine the contribution of these residues to NADase and ARTase functions.
- To correlate the enzymatic activities with the cytotoxic and lethal effects of the toxin.
Main Methods:
- Site-directed mutagenesis was employed to replace key amino acid residues within the Ia component.
- Enzymatic assays were performed to measure NADase and ARTase activities of the mutated Ia.
- Kinetic analysis was used to understand the binding and catalytic mechanisms.
Main Results:
- Replacement of Arg-295, Glu-378, Glu-380, and Ser-338 significantly impaired or abolished NADase and ARTase activities.
- Specific substitutions (e.g., Glu-378 to Asp/Gln, Glu-380 to Asp/Gln) differentially affected NADase and ARTase functions.
- Arg-295 and Ser-338 were found to be important for NAD(+) binding, while Arg-295, Glu-380, and Ser-338 were critical for NADase catalysis.
- Arg-295, Glu-378, Glu-380, and Ser-338 were identified as essential for ARTase activity.
Conclusions:
- Specific amino acid residues (Arg-295, Glu-378, Glu-380, Ser-338) are critical for the NADase and ARTase activities of Clostridium perfringens Ia.
- The ARTase activity of Ia is directly linked to its lethal and cytotoxic effects.
- Understanding these structure-function relationships can inform the development of therapeutic strategies against C. perfringens infections.