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ADP-Ribosylargininyl reaction of cholix toxin is mediated through diffusible intermediates.

Sung VM, Tsai CL - BMC Biochem. (2014)

Bottom Line: Our studies on the enzymatic activity of cholix toxin catalytic fragment show that the transfer of ADP-ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of arginine residues proximal to the NAD+ binding pocket.Auto-ADP-ribosylation of cholix toxin appears to have negatively regulatory effect on ADP-ribosylation of exogenous substrate.Therefore, a diffusible strained form of NAD+ intermediate was proposed to react with arginine residues in a proximity dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston 02114, MA, USA. mvsung@gmail.com.

ABSTRACT

Background: Cholix toxin is an ADP-ribosyltransferase found in non-O1/non-O139 strains of Vibrio cholera. The catalytic fragment of cholix toxin was characterized as a diphthamide dependent ADP-ribosyltransferase.

Results: Our studies on the enzymatic activity of cholix toxin catalytic fragment show that the transfer of ADP-ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of arginine residues proximal to the NAD+ binding pocket. Multiple arginine residues, located near the catalytic site and at distal sites, can be the ADP-ribose acceptor in the auto-reaction. Kinetic studies of a model enzyme, M8, showed that a diffusible intermediate preferentially reacted with arginine residues in proximity to the NAD+ binding pocket. ADP-ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates. Auto-ADP-ribosylation of cholix toxin appears to have negatively regulatory effect on ADP-ribosylation of exogenous substrate. However, at the presence of both endogenous and exogenous substrates, ADP-ribosylation of exogenous substrates occurred more efficiently than that of endogenous substrates.

Conclusions: We discovered an ADP-ribosylargininyl activity of cholix toxin catalytic fragment from our studies in auto-ADP-ribosylation, which is mediated through diffusible intermediates. The lifetime of the hypothetical intermediate exceeds recorded and predicted lifetimes for the cognate oxocarbenium ion. Therefore, a diffusible strained form of NAD+ intermediate was proposed to react with arginine residues in a proximity dependent manner.

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Biotin signals on the self-modified wild type CTc is an ADP-ribosylation reaction. (A) Purified wild type CTc or inactive mutant CTc(YEDQ), was incubated with various concentrations of biotinyl-NAD+. The folds of fluorescence intensity increase between CTc and CTc(YEDQ) were shown on the top of each pair of bar graph. (B) Purified CTc or CTc(YEDQ) was incubated with various concentrations of [carbonyl-14C]-NAD+ at 37°C for 1 hour. Asterisk indicates the control group in which 293 cell lysate was added to CTc or CTc(YEDQ) to measure 14C incorporation into the TCA precipitants. Data were summarized from two separated experiments.
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Fig2: Biotin signals on the self-modified wild type CTc is an ADP-ribosylation reaction. (A) Purified wild type CTc or inactive mutant CTc(YEDQ), was incubated with various concentrations of biotinyl-NAD+. The folds of fluorescence intensity increase between CTc and CTc(YEDQ) were shown on the top of each pair of bar graph. (B) Purified CTc or CTc(YEDQ) was incubated with various concentrations of [carbonyl-14C]-NAD+ at 37°C for 1 hour. Asterisk indicates the control group in which 293 cell lysate was added to CTc or CTc(YEDQ) to measure 14C incorporation into the TCA precipitants. Data were summarized from two separated experiments.

Mentions: Excess free ADP-ribose (up to 125-fold above assay concentration for 32P-NAD+ or 636-fold for biotinyl-NAD+) did not significantly inhibit either 32P- or biotinyl-ADP-ribosylation signals in the auto-reaction of CTc, suggesting that a non-enzymatic pathway resulting from the initial generation of ADP-ribose by NAD+ glycohydrolase activity, followed by non-enzymatic addition, does not play a major role in contributing to the observed auto-ADP-ribosylation reaction (Figure 1F and G). However, we also observed increased biotin substitution of the inactive mutant CTc(YEDQ) that had been incubated with increasing amounts of biotinyl-NAD+, which may be contaminated with some reactive NAD+ intermediates or ADP-ribose from the synthesis of biotinyl-NAD+ to cause the increased background biotin signals without catalytic activity (Figure 2A). Biotinyl-NAD+ bears the biotin substitution on the adenosine moiety of NAD+, whereas [carbonyl-14C]-NAD+ has 14C labeling on the nicotinamide moiety. When biotinyl-NAD+ was substituted by [carbonyl-14C]-NAD+, there was no significant difference in 14C incorporation between active and inactive enzymes even when as high as 100 to 200 μM of [carbonyl-14C]-NAD+ was used in the auto-reactions (Figure 2B). In contrast, active enzyme CTc showed ~4-fold higher biotin substitution than inactive mutant CTc(YEDQ) when 100 μM biotinyl-NAD+ was used. Overall, these data suggest that the increase in biotin substitution caused by the enzymatic activity of CTc is an ADP-ribosylation reaction.Figure 2


ADP-Ribosylargininyl reaction of cholix toxin is mediated through diffusible intermediates.

Sung VM, Tsai CL - BMC Biochem. (2014)

Biotin signals on the self-modified wild type CTc is an ADP-ribosylation reaction. (A) Purified wild type CTc or inactive mutant CTc(YEDQ), was incubated with various concentrations of biotinyl-NAD+. The folds of fluorescence intensity increase between CTc and CTc(YEDQ) were shown on the top of each pair of bar graph. (B) Purified CTc or CTc(YEDQ) was incubated with various concentrations of [carbonyl-14C]-NAD+ at 37°C for 1 hour. Asterisk indicates the control group in which 293 cell lysate was added to CTc or CTc(YEDQ) to measure 14C incorporation into the TCA precipitants. Data were summarized from two separated experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4265445&req=5

Fig2: Biotin signals on the self-modified wild type CTc is an ADP-ribosylation reaction. (A) Purified wild type CTc or inactive mutant CTc(YEDQ), was incubated with various concentrations of biotinyl-NAD+. The folds of fluorescence intensity increase between CTc and CTc(YEDQ) were shown on the top of each pair of bar graph. (B) Purified CTc or CTc(YEDQ) was incubated with various concentrations of [carbonyl-14C]-NAD+ at 37°C for 1 hour. Asterisk indicates the control group in which 293 cell lysate was added to CTc or CTc(YEDQ) to measure 14C incorporation into the TCA precipitants. Data were summarized from two separated experiments.
Mentions: Excess free ADP-ribose (up to 125-fold above assay concentration for 32P-NAD+ or 636-fold for biotinyl-NAD+) did not significantly inhibit either 32P- or biotinyl-ADP-ribosylation signals in the auto-reaction of CTc, suggesting that a non-enzymatic pathway resulting from the initial generation of ADP-ribose by NAD+ glycohydrolase activity, followed by non-enzymatic addition, does not play a major role in contributing to the observed auto-ADP-ribosylation reaction (Figure 1F and G). However, we also observed increased biotin substitution of the inactive mutant CTc(YEDQ) that had been incubated with increasing amounts of biotinyl-NAD+, which may be contaminated with some reactive NAD+ intermediates or ADP-ribose from the synthesis of biotinyl-NAD+ to cause the increased background biotin signals without catalytic activity (Figure 2A). Biotinyl-NAD+ bears the biotin substitution on the adenosine moiety of NAD+, whereas [carbonyl-14C]-NAD+ has 14C labeling on the nicotinamide moiety. When biotinyl-NAD+ was substituted by [carbonyl-14C]-NAD+, there was no significant difference in 14C incorporation between active and inactive enzymes even when as high as 100 to 200 μM of [carbonyl-14C]-NAD+ was used in the auto-reactions (Figure 2B). In contrast, active enzyme CTc showed ~4-fold higher biotin substitution than inactive mutant CTc(YEDQ) when 100 μM biotinyl-NAD+ was used. Overall, these data suggest that the increase in biotin substitution caused by the enzymatic activity of CTc is an ADP-ribosylation reaction.Figure 2

Bottom Line: Our studies on the enzymatic activity of cholix toxin catalytic fragment show that the transfer of ADP-ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of arginine residues proximal to the NAD+ binding pocket.Auto-ADP-ribosylation of cholix toxin appears to have negatively regulatory effect on ADP-ribosylation of exogenous substrate.Therefore, a diffusible strained form of NAD+ intermediate was proposed to react with arginine residues in a proximity dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston 02114, MA, USA. mvsung@gmail.com.

ABSTRACT

Background: Cholix toxin is an ADP-ribosyltransferase found in non-O1/non-O139 strains of Vibrio cholera. The catalytic fragment of cholix toxin was characterized as a diphthamide dependent ADP-ribosyltransferase.

Results: Our studies on the enzymatic activity of cholix toxin catalytic fragment show that the transfer of ADP-ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of arginine residues proximal to the NAD+ binding pocket. Multiple arginine residues, located near the catalytic site and at distal sites, can be the ADP-ribose acceptor in the auto-reaction. Kinetic studies of a model enzyme, M8, showed that a diffusible intermediate preferentially reacted with arginine residues in proximity to the NAD+ binding pocket. ADP-ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates. Auto-ADP-ribosylation of cholix toxin appears to have negatively regulatory effect on ADP-ribosylation of exogenous substrate. However, at the presence of both endogenous and exogenous substrates, ADP-ribosylation of exogenous substrates occurred more efficiently than that of endogenous substrates.

Conclusions: We discovered an ADP-ribosylargininyl activity of cholix toxin catalytic fragment from our studies in auto-ADP-ribosylation, which is mediated through diffusible intermediates. The lifetime of the hypothetical intermediate exceeds recorded and predicted lifetimes for the cognate oxocarbenium ion. Therefore, a diffusible strained form of NAD+ intermediate was proposed to react with arginine residues in a proximity dependent manner.

Show MeSH
Related in: MedlinePlus