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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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ADP-ribosylation of endogenous versus exogenous substrates. (A-B) Cholix toxin catalytic fragments were pre-incubated with or without 50 μM NAD+ at 37°C for 1 hour. Free NAD+ was removed by gel filtration chromatography. The recovered enzymes were quantified. Equal concentrations of auto-ADP-ribosylated CTc (pre-incubated with NAD+) or control (non-auto-ADP-ribosylated CTc, processed through auto-ADP-ribosylation reaction without NAD+) were used in the NAD+ glycohydrolase activity assays and ADP-ribosylation of eEF2 in 293 lysate. (C) His-tagged oligo-L-arginine or oligo-L-asparagine peptides were incubated with purified recombinant wild type CTc, catalytically defective mutant (Y493A) or catalytically active mutant (E579Q). The samples were analyzed by a 96-well plate based ADP-ribosylation assay. Data shown are composite from two experiments with triplicates within-plate replicates. (D)Various concentrations of catalytic fragments of cholix toxin or exotoxin A (PEA) was incubated with CHO or Re1.22c cell lysate at 37°C for 1 hr. The biotin signals on the ADP-ribosylated eEF2 and auto-ADP-ribosylated enzymes were detected by IRDye800CW-SA shown on the top panel. The same blot was stripped and re-probed with anti-CTc and anti-eEF2 antibodies shown in the middle panel. The bottom panel shows the Coomassie Blue stained gel for protein loading control. (E) To detect auto-ADP-ribosylation at the presence of exogenous substrates, excess amount of CTc (6 μM) was incubated with purified flag-tagged wild type eEF2 (0.2 μM) or flag-tagged eEF2 (H715R) mutant (0.2 μM) at the presence of 50 μM biotinyl-NAD+ for various periods of time.
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Fig9: ADP-ribosylation of endogenous versus exogenous substrates. (A-B) Cholix toxin catalytic fragments were pre-incubated with or without 50 μM NAD+ at 37°C for 1 hour. Free NAD+ was removed by gel filtration chromatography. The recovered enzymes were quantified. Equal concentrations of auto-ADP-ribosylated CTc (pre-incubated with NAD+) or control (non-auto-ADP-ribosylated CTc, processed through auto-ADP-ribosylation reaction without NAD+) were used in the NAD+ glycohydrolase activity assays and ADP-ribosylation of eEF2 in 293 lysate. (C) His-tagged oligo-L-arginine or oligo-L-asparagine peptides were incubated with purified recombinant wild type CTc, catalytically defective mutant (Y493A) or catalytically active mutant (E579Q). The samples were analyzed by a 96-well plate based ADP-ribosylation assay. Data shown are composite from two experiments with triplicates within-plate replicates. (D)Various concentrations of catalytic fragments of cholix toxin or exotoxin A (PEA) was incubated with CHO or Re1.22c cell lysate at 37°C for 1 hr. The biotin signals on the ADP-ribosylated eEF2 and auto-ADP-ribosylated enzymes were detected by IRDye800CW-SA shown on the top panel. The same blot was stripped and re-probed with anti-CTc and anti-eEF2 antibodies shown in the middle panel. The bottom panel shows the Coomassie Blue stained gel for protein loading control. (E) To detect auto-ADP-ribosylation at the presence of exogenous substrates, excess amount of CTc (6 μM) was incubated with purified flag-tagged wild type eEF2 (0.2 μM) or flag-tagged eEF2 (H715R) mutant (0.2 μM) at the presence of 50 μM biotinyl-NAD+ for various periods of time.

Mentions: Similar to several other ADP-ribosyltransferases [20,21,23], if we pre-incubated CTc with NAD+ to allow auto-ADP-ribosylation to occur prior to analysis, auto-ADP-ribosylation of the CTc suppressed its NAD+ glycohydrolase activity and ADP-ribosyltransferase activity to modify eEF2 (Figure 9A and B). Cholix toxin and exotoxin A are both characterized as diphthamide-dependent ADP-ribosyltransferases which modify eEF2 in nature. We also found that CTc could modify exogenous oligo-arginine peptides (Figure 9C). To understand how the enzyme ADP-ribosylates exogenous substrates in the presence of endogenous substrate, we mixed various amounts of purified catalytic fragments of cholix toxin or exotoxin A with CHO cell lysate, containing diphthamide-modified eEF2 or Re1.22c cell lysate, containing a mutated eEF2 without diphthamide modification, in the ADP-ribosylation reactions. The auto-ADP-ribosylation of the input enzyme was only found in highest concentration (1.2 μM) of CTc, whereas the ADP-ribosylation of wild type eEF2 has reached saturated signals with much lower concentrations of the enzymes. In this experimental setting, auto-ADP-ribosylation of exotoxin A was not detected even with 1.2 μM of the enzyme (Figure 9D). We further incubated purified flag-tagged eEF2(wt) or flag-tagged eEF2(H715R), with excess concentration of CTc to observe the kinetics of auto-ADP-ribosylation in the presence of exogenous substrates. The kinetics of ADP-ribosylation of flag-tagged eEF2 was much faster than that of auto-ADP-ribosylation of the enzyme or ADP-ribosylation of the flag-tagged eEF2(H715R) mutant, in which diphthamide was replaced with an arginine residue (Figure 9E). Diphthamide, which has imidazole-like structure on the modified histidine of eEF2, was shown to directly contact NAD+, and suggested to be involved in triggering the cleavage of NAD+ and interacting with the oxocarbenium intermediate during the nucleophilic substitution reaction [31]. We also observed low concentration of imidazole could enhance the biotinyl-ADP-ribosylation signals on the modified substrates (Additional file 4). These findings suggest that diphthamide can act as a catalyst to make ADP-ribosylation of eEF2 much more efficient than the auto-ADP-ribosylation reaction of cholix toxin. Moreover, at the presence of both endogenous and exogenous substrates, the binding of the exogenous substrate would segregate the arginine residues around the NAD+ binding pocket of the enzyme from interacting with the reactive strained NAD+ intermediate. Several arginine residues around the NAD+ binding pocket involved in substrate recognition and binding are also target residues of auto-ADP-ribosylation. Therefore, if the ADP-ribosylation of exogenous substrate results in lethal effect or triggers downstream of signal transduction pathways of the modified substrate, the negatively regulatory effect of the auto-ADP-ribosylation may be neglected and this is most likely to be true for most of bacteria toxins.Figure 9


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

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

ADP-ribosylation of endogenous versus exogenous substrates. (A-B) Cholix toxin catalytic fragments were pre-incubated with or without 50 μM NAD+ at 37°C for 1 hour. Free NAD+ was removed by gel filtration chromatography. The recovered enzymes were quantified. Equal concentrations of auto-ADP-ribosylated CTc (pre-incubated with NAD+) or control (non-auto-ADP-ribosylated CTc, processed through auto-ADP-ribosylation reaction without NAD+) were used in the NAD+ glycohydrolase activity assays and ADP-ribosylation of eEF2 in 293 lysate. (C) His-tagged oligo-L-arginine or oligo-L-asparagine peptides were incubated with purified recombinant wild type CTc, catalytically defective mutant (Y493A) or catalytically active mutant (E579Q). The samples were analyzed by a 96-well plate based ADP-ribosylation assay. Data shown are composite from two experiments with triplicates within-plate replicates. (D)Various concentrations of catalytic fragments of cholix toxin or exotoxin A (PEA) was incubated with CHO or Re1.22c cell lysate at 37°C for 1 hr. The biotin signals on the ADP-ribosylated eEF2 and auto-ADP-ribosylated enzymes were detected by IRDye800CW-SA shown on the top panel. The same blot was stripped and re-probed with anti-CTc and anti-eEF2 antibodies shown in the middle panel. The bottom panel shows the Coomassie Blue stained gel for protein loading control. (E) To detect auto-ADP-ribosylation at the presence of exogenous substrates, excess amount of CTc (6 μM) was incubated with purified flag-tagged wild type eEF2 (0.2 μM) or flag-tagged eEF2 (H715R) mutant (0.2 μM) at the presence of 50 μM biotinyl-NAD+ for various periods of time.
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Fig9: ADP-ribosylation of endogenous versus exogenous substrates. (A-B) Cholix toxin catalytic fragments were pre-incubated with or without 50 μM NAD+ at 37°C for 1 hour. Free NAD+ was removed by gel filtration chromatography. The recovered enzymes were quantified. Equal concentrations of auto-ADP-ribosylated CTc (pre-incubated with NAD+) or control (non-auto-ADP-ribosylated CTc, processed through auto-ADP-ribosylation reaction without NAD+) were used in the NAD+ glycohydrolase activity assays and ADP-ribosylation of eEF2 in 293 lysate. (C) His-tagged oligo-L-arginine or oligo-L-asparagine peptides were incubated with purified recombinant wild type CTc, catalytically defective mutant (Y493A) or catalytically active mutant (E579Q). The samples were analyzed by a 96-well plate based ADP-ribosylation assay. Data shown are composite from two experiments with triplicates within-plate replicates. (D)Various concentrations of catalytic fragments of cholix toxin or exotoxin A (PEA) was incubated with CHO or Re1.22c cell lysate at 37°C for 1 hr. The biotin signals on the ADP-ribosylated eEF2 and auto-ADP-ribosylated enzymes were detected by IRDye800CW-SA shown on the top panel. The same blot was stripped and re-probed with anti-CTc and anti-eEF2 antibodies shown in the middle panel. The bottom panel shows the Coomassie Blue stained gel for protein loading control. (E) To detect auto-ADP-ribosylation at the presence of exogenous substrates, excess amount of CTc (6 μM) was incubated with purified flag-tagged wild type eEF2 (0.2 μM) or flag-tagged eEF2 (H715R) mutant (0.2 μM) at the presence of 50 μM biotinyl-NAD+ for various periods of time.
Mentions: Similar to several other ADP-ribosyltransferases [20,21,23], if we pre-incubated CTc with NAD+ to allow auto-ADP-ribosylation to occur prior to analysis, auto-ADP-ribosylation of the CTc suppressed its NAD+ glycohydrolase activity and ADP-ribosyltransferase activity to modify eEF2 (Figure 9A and B). Cholix toxin and exotoxin A are both characterized as diphthamide-dependent ADP-ribosyltransferases which modify eEF2 in nature. We also found that CTc could modify exogenous oligo-arginine peptides (Figure 9C). To understand how the enzyme ADP-ribosylates exogenous substrates in the presence of endogenous substrate, we mixed various amounts of purified catalytic fragments of cholix toxin or exotoxin A with CHO cell lysate, containing diphthamide-modified eEF2 or Re1.22c cell lysate, containing a mutated eEF2 without diphthamide modification, in the ADP-ribosylation reactions. The auto-ADP-ribosylation of the input enzyme was only found in highest concentration (1.2 μM) of CTc, whereas the ADP-ribosylation of wild type eEF2 has reached saturated signals with much lower concentrations of the enzymes. In this experimental setting, auto-ADP-ribosylation of exotoxin A was not detected even with 1.2 μM of the enzyme (Figure 9D). We further incubated purified flag-tagged eEF2(wt) or flag-tagged eEF2(H715R), with excess concentration of CTc to observe the kinetics of auto-ADP-ribosylation in the presence of exogenous substrates. The kinetics of ADP-ribosylation of flag-tagged eEF2 was much faster than that of auto-ADP-ribosylation of the enzyme or ADP-ribosylation of the flag-tagged eEF2(H715R) mutant, in which diphthamide was replaced with an arginine residue (Figure 9E). Diphthamide, which has imidazole-like structure on the modified histidine of eEF2, was shown to directly contact NAD+, and suggested to be involved in triggering the cleavage of NAD+ and interacting with the oxocarbenium intermediate during the nucleophilic substitution reaction [31]. We also observed low concentration of imidazole could enhance the biotinyl-ADP-ribosylation signals on the modified substrates (Additional file 4). These findings suggest that diphthamide can act as a catalyst to make ADP-ribosylation of eEF2 much more efficient than the auto-ADP-ribosylation reaction of cholix toxin. Moreover, at the presence of both endogenous and exogenous substrates, the binding of the exogenous substrate would segregate the arginine residues around the NAD+ binding pocket of the enzyme from interacting with the reactive strained NAD+ intermediate. Several arginine residues around the NAD+ binding pocket involved in substrate recognition and binding are also target residues of auto-ADP-ribosylation. Therefore, if the ADP-ribosylation of exogenous substrate results in lethal effect or triggers downstream of signal transduction pathways of the modified substrate, the negatively regulatory effect of the auto-ADP-ribosylation may be neglected and this is most likely to be true for most of bacteria toxins.Figure 9

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