BACKGROUND
adp-ribosyltransferases  constitute a broadly distributed family of prokaryotic and eukaryotic enzymes that catalyze the transfer of an adp-ribose moiety from nad+ to one or more substrates.
the protein substrates of adprts are heterogeneous and include eukaryotic elongation factor  <dig>   <cit> , actin  <cit> , vimentin  <cit> , ezrin/radixin/moesin proteins  <cit> , various heterotrimeric and small monomeric gtpases  <cit> , the adapter proteins crk-i and crk-ii  <cit> , human α-defensin  and human β-defensin- <dig>   <cit> , and cell surface molecules  <cit> .
a family of adprts adp-ribosylate eef <dig> on a post-translationally modified histidine residue called diphthamide  <cit> .
other adprts transfer adp-ribose to arginine, cysteine, glutamate, or asparagine residues  <cit> .
many adprts also possess nad+ glycohydrolase activity attributed to the attack by hydroxyl ion on the activated nad+ transition state  <cit> .
a number of adprts exhibit auto-adp-ribosylation activity, including diphtheria toxin  <cit> , cholera toxin  <cit> , clostridium limosum c <dig> exoenzyme  <cit> , pseudomonas aeruginosa exoenzyme s  <cit> , escherichia coli heat-labile enterotoxin  <cit> , neisseria meningitidis adp-ribosylargininyl transferase, nare  <cit> , and several mammalian adprts  <cit> .
auto-adp-ribosylation of apdrts has been shown to have functional consequences for the enzyme.
for example, the auto-reaction of poly-adp-ribose polymerase  has been reported to reduce its dna-binding affinity, which in turn plays an important role in regulating the parp response to dna damage stimuli  <cit> .
similarly, auto-adp-ribosylation of mammalian adp-ribosyltransferase  <dig>  redirects the enzyme catalytic focus from nad+ glycohydrolase activity to adp-ribosyltransferase activity  <cit> .
auto-adp-ribosylation has also been shown to inhibit the activity of the c. limosum c <dig> exoenzyme, p. aeruginosa exoenzyme s, and neisseria meningitides adp-ribosylargininyl transferase, nare  <cit> .
cholix toxin   <cit>  and recently identified cholix toxin variants  <cit>  were identified by genomic sequencing and homology comparisons in vibrio cholera non-o1/non-o <dig> strains.
features of the primary sequences that were considered relevant to a phylogenetically conserved structural core supporting enzymatic function included an yxhg and an yxny motif and a low but significant degree of homology to p. aeruginosa exotoxin a  <cit> .
many of the enzymatic attributes of cholix toxin have been established by the expression, catalytic characterization and determination at high resolution of a three dimensional crystal structure of the toxin and its catalytic subunit  <cit> .
the results suggest that cholix toxin is a diphthamide-specific adp-ribosyltransferase with a general domain organization and topology similar to that of exotoxin a. members of the eef2-specific adprt class are both phylogenetically and enzymatically distinct from other adprts, and their common unique substrate, diphthamide, has been suggested to directly participate in the catalysis  <cit> .
this class of adprts has an elongated loop structure that is predicted or confirmed to significantly rearrange upon contact with eef <dig> and that likely mediates the majority of interactions between enzyme and substrate  <cit> .
data from crystallographic and kinetic studies suggest the adp-ribosylation of eef <dig> is mediated through a random third order sn <dig> reaction, though the ribosyl-diphthamide bond adopts the α conformation consistent with the stereo inversion expected of an sn <dig> mechanism  <cit> .
here we characterized the process of the auto-adp-ribosylation reaction of cholix toxin and evidenced that a metastable diffusible intermediate was generated upon the enzyme activation then diffused to react with arginine residues of the enzyme in a proximity dependent manner.
we also showed that wild type cholix toxin catalytic fragment  could adp-ribosylate oligo argininyl peptides and eef <dig>  mutant in which the post-translationally modified diphthamide at his <dig> was replaced by arginine.
we propose that this mechanism can be used to engineer adp-ribosyltransferases with alternative substrate specificity as long as there is an arginine residue or other adp-ribose acceptors close to the catalytic site of the enzyme.
RESULTS
an enzymatic pathway is involved in auto-adp-ribosylation
to characterize the enzymatic activity of cholix toxin we expressed the catalytic fragment under the control of the arabad promoter as a translational fusion to a secb pathway-dependent signal peptide and assessed the ability of the periplasmic fraction to carry out adp-ribosylation using biotinyl-nad+ as a biotinyl-adp-ribose donor.
initial experiments revealed the presence of an arabinose-inducible biotin-labeled band corresponding to the molecular weight of the catalytic fragment.
mutants y493a, e581q, and y493a/e581q  exhibited reduced biotinylation intensity compared to wild type and mutant e579q, suggesting the catalytic fragment was capable of utilizing itself as a substrate .
the biotin labeled or 32p-labeled purified enzymes were also observed , suggesting that host factors are not required.
the possibility that the mutant forms had undergone structural changes resulting in a loss of substrate potential was minimized by the finding that the circular dichroism spectra of wild type and mutant proteins were substantially identical .
the auto-adp-ribosylation and nad+ glycohydrolase activities of the purified enzymes were also assessed under comparable conditions by a fluorescence-based assay .
the results indicate that the auto-adp-ribosylation and nad+ glycohydrolase activities of ctc are highly concordant.
mutations on any one of the conserved residues involved in catalysis resulted in loss of the auto-adp-ribosylation activity .
these residues include e <dig>  the catalytic residue; y <dig> and y <dig>  two tyrosine residues binding to the aromatic group of nad+; h <dig>  providing the structural integrity of the catalytic site  <cit> .figure  <dig>
an enzymatic pathway is involved in auto-adp-ribosylation of ctc.
biotin signals are detected in the periplasmic fractions of e.coli lysate expressing wild type and mutant cholix toxin catalytic fragments .
the same blot was re-blotted with rabbit anti-ctc antibody to detect protein expression in each sample .
similar results are obtained by incubating purified protein with either biotinyl-nad+  or 32p-nad+
.
the blots shown are representative of multiple independent experiments.
the structures of these mutants were analyzed by circular dichroism  spectrometry.
nad+ glycohydrolase activity  and auto-adp-ribosylation activity  were quantified by 96-well plate based assays.
data are summarized from three independent experiments.
error bars show the standard deviation of the composite data.
asterisks indicate significant reduction of enzyme activity as compared to the wild type ctc with *, p-value <  <dig> ; **, p-value <  <dig> .
various concentrations of free adp-ribose were added to the auto-adp-ribosylation reaction.
as a control, the 32p-adp-ribosylation signals on ctc were removed by phosphodiesterase i treatment.
asterisk indicates significant reduction of 32p-auto-adp-ribosylation signal of the treated over the untreated samples with p-value <  <dig> .
data shown is representative of three independent experiments.
error-bars show the standard error of mean .
statistic analysis was done by student t-test.
excess amount of free adp-ribose was added to the adp-ribosylation buffer containing 3 μm purified wild type ctc and 50 μm biotinyl-nad+ at 37°c for 1 hour.
the biotinyl-adp-ribosylation signal was detected by irdye800cw-sa.
in all panels, arrowheads indicate the auto-adp-ribosylated ctc and arrows indicate the detection of ctc by anti-ctc antibody.
excess free adp-ribose  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 .
however, we also observed increased biotin substitution of the inactive mutant ctc 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 .
biotinyl-nad+ bears the biotin substitution on the adenosine moiety of nad+, whereas -nad+ has 14c labeling on the nicotinamide moiety.
when biotinyl-nad+ was substituted by -nad+, there was no significant difference in 14c incorporation between active and inactive enzymes even when as high as  <dig> to 200 μm of -nad+ was used in the auto-reactions .
in contrast, active enzyme ctc showed ~4-fold higher biotin substitution than inactive mutant ctc 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  <dig>
biotin signals on the self-modified wild type ctc is an adp-ribosylation reaction.
purified wild type ctc or inactive mutant ctc, was incubated with various concentrations of biotinyl-nad+.
the folds of fluorescence intensity increase between ctc and ctc were shown on the top of each pair of bar graph.
purified ctc or ctc was incubated with various concentrations of -nad+ at 37°c for 1 hour.
asterisk indicates the control group in which  <dig> cell lysate was added to ctc or ctc to measure 14c incorporation into the tca precipitants.
data were summarized from two separated experiments.
auto-adp-ribosylation favors intramolecular transfer
to understand if auto-adp-ribosylation of cholix toxin involves intra- or inter-molecular modification, a catalytically active higher molecular weight form of ctc was prepared by fusion of the catalytic fragment to an immunoglobulin constant domain , yielding an active enzyme with a molecular weight of 70 kda.
the resulting protein was mixed with mutant catalytic fragment in 1: <dig> molar ratio.
the catalytically active form of the enzyme, ig-ctc, was found to be preferentially substituted with biotinyl-adp-ribose .
a reduced level of substitution with biotinyl-adp-ribose was found for y493a mutant protein that had been incubated at high concentration with ig-ctc, which could be due to residual activity of the mutant enzyme or could reflect the occurrence of trans-adp-ribosylation at high substrate concentration.
similarly, when purified recombinant full-length wild type cholix toxin  was mixed with the catalytically inactive mutant, y493a, or the catalytically inactive full-length cholix toxin e581a mutant was mixed with the wild type ctc, the auto-adp-ribosylation of cholix toxin preferentially took place on the catalytically active form .
together, these data support the view that auto-adp-ribosylation of cholix toxin favors intramolecular transfer of the adp-ribose moiety.figure  <dig>
intramolecular transfer of adp-ribose to multiple arginines in the auto-reaction.
mixing marked enzymes experiments.
active ig-ctc was mixed with inactive mutant y493a ; active full-length cholix toxin, cxt , was mixed with mutant y493a ; inactive mutant cxt was mixed with active ctc  in auto-adp-ribosylation assays.
molar concentrations shown on the top of each blot are the concentrations of active enzyme.
the left blots  were detected by irdye800cw-sa; the same blots were re-probed with polyclonal rabbit anti-ctc antibody shown on right .
neutral hydroxylamine assays.
auto-adp-ribosylation reaction was carried out with periplasmic fractions of e. coli lysate expressing wild type or mutant ctc.
the products were subjected to  <dig>  m nh2oh  or  <dig>  m nacl treatment at 37°c for 2 hours.
the blot shown is representative of multiple experiments.
single amino acid substitution mutation studies.
a set of representative data from the detection of biotin signals on wild type or different mutants was shown in .
the fluorescence intensity of sa  was normalized with the coomassie blue intensity  to obtain the irdy-sa/cb ratio as a semi-quantitative auto-adp-ribosylation measurement of each enzyme.
data were summarized from four sets of experiments .
asterisk indicates significant reduction of auto-adp-ribosylation activity.
nad+ glycohydrolase activities of wild type and mutant enzymes are shown in .
multiple arginines are the target residues
adp-ribosyl adducts have different stabilities to neutral hydroxylamine: under conditions for which the half life of adp-ribosylated glutamate is about 3– <dig> minutes, the half life of adp-ribosylated arginine is about 120 minutes and the half lives of adp-ribosylated cysteine and asparagine are immeasurably large  <cit> .
as shown in the figure 3b, the biotin substitution of the wild type and two active mutants, e579q and e579r, showed no reduction following exposure to  <dig>  m nacl but fell by half after two hours of  <dig>  m neutral hydroxylamine treatment at 37°c.
under these conditions there were little or no changes in the quantity or mobility of proteins as a result of either exposure.
these data are consistent with the view that a significant fraction of biotinyl-adp-ribose substitutions are the result of modification of arginine residues.
since the adp-ribosyl-glutamate linkage is labile in the neutral hydroxylamine treatment, we substituted two glutamate residues near the nad+ binding pocket with lysine residues to create mutants e484k and/or e486k.
none of these substitution mutations reduced the biotin signals on the auto-adp-ribosylated enzymes .
we therefore focus our study on the modification of arginine target residues.
to identify candidate residues for the auto-adp-ribosylation,  <dig> arginine residues in the catalytic fragment were mutated to alanine residues.
the extent of auto-adp-ribosylation was significantly reduced after incubation of the r506a, r519a, r525a, r530a, r535a, r540a, or r543a mutant proteins with biotinyl-nad+ substrate , indicating these arginine residues may be the targets of auto-adp-ribosylation.
however, some mutant proteins also exhibited lower levels of nad+ glycohydrolase activity than wild type catalytic fragment  especially in the case of the r506a, r525a and r530a mutant proteins.
therefore, we replaced arg <dig>  arg <dig>  and arg <dig> with glutamine or lysine, which has greater steric bulk than alanine .
the nad+ glycohydrolase activity of r506q, r506k, r525q, or r525k was detectable at a level similar to that of other mutants and auto-adp-ribosylation was also detected, suggesting other arginine residues than these can be adp-ribosylated.
all the attempts to replace arg <dig> resulted in unstable enzymes with very poor activity and a low level of protein expression, indicating the enzyme is structurally sensitive to arg <dig> modification.
with the exception of arg <dig> mutants, none of which were active, none of the single amino acid substitutions completely abolished auto-adp-ribosylation, leads to the hypothesis that multiple arginine residues could serve as adp-ribose acceptors.
identification of arginine target residues by 2-d electrophoresis and ms/ms analysis
to support this hypothesis, we analyzed the auto-adp-ribosylation of ctc using nad+ as adp-ribose donor by two-dimensional  electrophoresis.
ctc has an isoelectric point  of  <dig> .
one adp-ribose addition to this fragment shifts its pi ~ <dig>  ph units.
following auto-reaction of the catalytic fragment, multiple spots shifted toward the acidic ph from the spot of ctc were detected by 2-d electrophoresis compared to the sample without incubation with nad+ , indicating multiple residues being modified in the same protein.
an additional spot found in the sample prior to incubation with nad+ may result from auto-adp-ribosylation in vivo, or from other post-translational modifications that occurred in the e.coli strain from which ctc was expressed.
to map the location of the adp-ribosylated arginines, the auto-adp-ribosylated ctc was digested with lysc, predicted to cleave the ctc into three major peptides: peptide 423– <dig> , peptide 489– <dig>  and peptide 509– <dig> .
the lysc digested auto-adp-ribosylated ctc was resolved in ph 3– <dig> matrices followed by  <dig> % tris-tricine peptide gel analysis to separate the fragments.
two fragments  were detected in gel spots b and a, respectively.
peptide 489– <dig> was too small to be observed in the peptide gel.
however, a partial digest product spanning residues 489– <dig> was detected from which multiple shifted spots were detected .
at least three modified spots arising from the unmodified peptide 509– <dig> were detected in coomassie blue stained 2-d peptide gels .
these spots were missing from gels prepared from the ctc which was not incubated with nad+.
peptide 509– <dig> contains several arginine residues  identified by reverse mutagenesis studies to be the targets for auto-adp-ribosylation of ctc.figure  <dig>
detection of multiple adp-ribosylarginines by two-dimensional  electrophoresis.
the amino acids sequences of ctc fused with outer membrane signal peptide was shown in .
ctc was incubated with or without 250 μm nad+
.
ctc incubated with or without nad+ was further digested with lysc prior to peptide 2-d electrophoresis .
amino acid sequences of three major predicted proteolytic products are shown in .
circle i indicates the partially digested peptide 489– <dig>  circle ii indicates spots shifted from the auto-adp-ribosylated peptide 509– <dig>  spots a and b are peptides 509– <dig> and 423– <dig>  respectively.
we also confirmed the adp-ribosylarginine modifications by ms/ms analysis.
the adp-ribose moiety of adp-ribosylated peptide is labile to the ms or ms/ms detection process.
a two-step collision method was performed to remove the modification group  with an in-source decay done in the tandem mass spectrometer  <cit> .
the product from the first collision was then subject to the second collision to fragmentize the peptide chain with the small marker moiety to locate and determine the amino acid sequences.
the combination of selection candidate peptides by the precursor ion scanning with adp-r-carbodiimide as the marker ion and further fragmentation of the ion of the adp-r-carbodiimide-derived peptides provided highly specific and reliable data applicable for ms/ms database searching for de novo peptide sequences.
the adp-ribosylargininyl peptide yields a product peptide in which arginine is replaced with ornithine  after first collision.
from spot a , we sequenced two orn peptides, yglpt/orn/aerd and argvml/orn/vyiprasle, indicating that the corresponding arginine positions arg <dig> and arg <dig> were adp-ribosylated .
from the circled spots of figure 4b and circles i and ii of figure 4d, four orn peptide signals were detected to be orn/fy/orn/tntple , rfy/orn/tntple , orn/fyrtntple , and hitqvighslpl/orn/neaftgpe .
the orn peptide containing arg <dig> was unexpectedly detected in this study.
the arg <dig> position is sensitive to modification and the buried side-chain of arg <dig> makes it unlikely to be adp-ribosylated in its native conformation.
we therefore reason the modification of arg <dig> occurred artificially during the overnight iso-electrofocusing step when the enzyme was exposed to the 2-d sample buffer.
the urea and detergent in the sample buffer could expose the side chain of arg <dig> from its native position to react with the diffusible intermediate.
although we removed free nad+ from the sample by gel filtration chromatography at the end of auto-adp-ribosylation reaction prior to 2-d analysis, we may not have completely removed the enzyme bound fractions of nad+.
arg <dig> is located close to the nad+ binding pocket geometrically in the native 3-d structure of ctc.
identification of arginine target residues by direct and reverse mutagenesis
in our efforts to identify the auto-adp-ribosylation target residues, a series of composite lysine to arginine replacement mutants were created as shown in table  <dig>  in which each successive mutant contained all the modifications of its parental template.
mutant m <dig> was constructed by introducing the r506k and r566k mutations into wild type catalytic fragment.
m <dig> was used as a template to make mutant m <dig>  which has additional r426k, r473k and r629k mutations.
arg <dig> was retained in all mutants because the protein integrity and catalytic activity are sensitive to its replacement.
the mutants were purified and analyzed for their auto-adp-ribosylation and nad+ glycohydrolase activities .
mutant m <dig> exhibited a reduction of biotin signal compared to wild type enzyme, implicating arg <dig> and/or arg <dig> as potential targets.
similarly, the degree of biotin substitution was decreased in m5- <dig>  m <dig>  and m <dig> compared to the parental m <dig> template, implicating arg <dig> and arg <dig> as potential targets.
the reduction of biotinylation from m5- <dig> to m <dig> could be attributed to the r535k mutation and the reduction from m <dig> to m <dig> could be attributed to the r525k mutation.
introduction of r479k into the m <dig> template to make mutant m <dig> resulted in apparent abolition of biotinylation, suggesting arg <dig> is an important target.
however, when the r479k mutation was introduced into the context of mutant m <dig> to form m <dig> mutant, significant biotinylation was detected, indicating arg <dig> is not the sole adp-ribose acceptor.
the loss of substrate activity by mutant m <dig> coincided with a restoration of nad+ glycohydrolase activity, suggesting the principal explanation for the diminished degree of biotinylation seen in this mutant is not extinction of catalytic activity .
based on these data, arg <dig>  arg <dig>  arg <dig>  arg <dig>  arg <dig>  arg <dig>  arg <dig> are potential target residues for auto-adp-ribosylation.table  <dig>
cholix toxin catalytic fragment variants contain arginine  to lysine  replacement mutations
r to k mutants
r to k replacement mutations on the wild type ctc
ǂ
auto-adp-ribosylation activity
¶
nad
+
glycohydrolase activity
ǂ data show the mean fluorescence of intensity of biotin signals on the auto-adp-ribosylated enzyme band detected by irdye800-sa.
¶data show the specific activity of each enzyme as described in methods.
*wild type cholix toxin catalytic fragment  contains r <dig>  r <dig>  r <dig>  r <dig>  r <dig>  r <dig>  r <dig>  r <dig>  r <dig>  r <dig>  r <dig>  r <dig>  r <dig>  r <dig>
mutant m <dig> exhibited nearly undetectable auto-adp-ribosylation and the background biotinyl signals of m <dig> were similar to those of the inactive mutant, m <dig> .
m <dig> mutant contains  <dig> arginines to lysines replacement mutations except arg <dig>  arg <dig> has a buried  side chain, which makes it less likely to be the target of auto-adp-ribosylation in its native conformation.
we therefore chose m <dig> as template to generate lysine  to arginine  reverse mutants.
among 13 k to r reverse mutants, m <dig>  m <dig>  m <dig>  m <dig>  m <dig> and m <dig> were all found to regain biotinylation signal intensity , indicating multiple arginines can serve as adp-ribose acceptors in the auto-adp-ribosylation of m <dig> derived mutants.
together, we identified arg <dig>  arg <dig>  arg <dig>  arg <dig>  arg <dig>  arg <dig> and arg <dig> to be the target residues for auto-adp-ribosylation.figure  <dig>
auto-adp-ribosylation prefers arginines as target residues.
mutant m <dig> contains  <dig> arginines  to lysines  replacement mutations except arg <dig>   the same amount of m <dig> and its catalytically inactive form m <dig> were incubated with various concentrations of biotinyl-nad+ in the auto-adp-ribosylation reaction.
the arrow indicates the biotinylated bands.
mutant m <dig> was used as template for the k to r reverse mutation to determine which arginine could be auto-adp-ribosylated.
the arginines present in the m <dig> and m <dig> derived mutant enzymes are indicated with their numerical positions in the full-length recombinant cholix toxin shown at the bottom of panel b.  variation of acceptor residues were carried out by replacing lys <dig> or lys <dig> on the mutant m <dig> with arginine , glutamine , asparagine , or cysteine .
the circular dichroism of these m <dig> derived mutants were shown in  and .
auto-adp-ribosylation was performed with wild type or mutant cholix toxin catalytic fragments and detected by western blotting shown in .
arrowheads indicate biotinyl-auto-adp-ribosylation of the catalytic fragments.
arrows indicate detection of the catalytic fragments by anti-ctc antibody for protein loading control.
diffusible intermediates
the locations of these arginine target residues are not restricted near the nad+ binding pocket but also found at distal sites of the enzyme .
in conjunction with the finding of intramolecular transfer of adp-ribose to the active enzyme itself, we propose a mechanism by which a diffusible intermediate is created upon enzymatic activation and diffuses to react with arginine residues.
to further support this mechanism, we determined the auto-adp-ribosylation rate of individual arginine residues using m <dig> k to r reverse mutants.
each m <dig> k to r reverse mutant contains arg <dig> and one additional arginine residue.
using the method described in kinetic experiments, we compared the auto-adp-ribosylation rates of six selected arginine target residues located at the proximal and distal sites of the nad+ binding pocket .
m <dig> containing arg <dig> target residue located at the end of the nad+ binding loop had the highest auto-adp-ribosylation rate, ~16-20-fold faster than the auto-adp-ribosylation rates of m <dig> and m <dig>  under the assumption that these enzymes should have similar km,biotin-nad+ to make the correlation of the reaction rates with the distances of the acceptor residues in a simplified diffusion model, we normalized the vmax of m <dig>  m <dig>  and m <dig> with their corresponding km, biotin-nad+, then the normalized auto-adp-ribosylation rate of arg <dig> is approximately 4 ~ 5-fold faster than those of arg <dig> and arg <dig>  which are located on the substrate recognition loop near the nad+ binding pocket.
the auto-adp-ribosylation rates of arg <dig>  arg <dig> and arg <dig>  located at the distal or the opposite face of the nad+ binding pocket, were low and highly variable among the inter- and intra-experimental repeats.
although the km,biotin-nad+ of m <dig> is 2-fold less than m <dig>  the normalized auto-adp-ribosylation rate  of m <dig> is ~200-fold lower than that of m <dig>  suggesting the electronic environment in the solution and the stability of the diffusible intermediate may affect the measured modification rates more pronouncedly in the auto-adp-ribosylation of the three distal arginines.
these data support that the auto-adp-ribosylation is mediated through a diffusible intermediate that reacts with its target residues in a primarily proximity-controlled mechanism.table  <dig>
kinetic analysis of auto-adp-ribosylation of various arginines on m <dig> derived mutants
km, biotin-nad
vmax
m8
m8
m8
m8
m8
m8
*n.d., cannot be determined.
the values represent the mean ± standard error from  <dig> different experiments with two to three repeats in each experiment using graphpad prism  <dig> .
to test what other amino acids the diffusible intermediate would react with, the lys <dig> or lys <dig> in m <dig> mutants were substituted with other potential adp-ribose acceptors including cysteine, asparagine and glutamine.
most of these substitutions yielded significant secondary structure changes in the mutants.
mutant m <dig> showed a cd spectrum similar to m <dig> whereas m <dig> showed a cd spectrum similar to m <dig>  suggesting these mutants share similar structures .
however, neither m <dig> nor m <dig> showed higher biotin substitution than m <dig> or m <dig> in the auto-adp-ribosylation .
these data are consistent with previous reports that arginine is the preferred target residue for auto-adp-ribosylation  <cit> .
human adprh hydrolysis analysis
human adp-ribosylarginine hydrolase  has been shown to preferentially hydrolyze the α-anomer of adp-ribosylarginine to release adp-ribose from adp-ribosylated protein  <cit> .
pseudomonas aeruginosa exotoxin t  is an adp-ribosyltransferase which specifically adp-ribosylates arginine residues on target proteins in the presence of a host co-factor, fas  <cit> .
a minimal four amino acid peptide derived from crk-i/ii adaptor protein fused to the c-terminal domain of clostridium perfringens enterotoxin , named exot substrate, was shown to be specifically adp-ribosylated by exot/fas complex .
to understand the intermediate step of the auto-adp-ribosylation of cholix toxin, we compared the activity of hadprh on substrates formed by cholix toxin auto-activity or exot action on its substrate.
figure 6a and b show that hadprh was able to effectively hydrolyze the biotinylated adp-ribosylarginine from the adp-ribosylated exot substrate  whereas only 20-40% reduction of the auto-adp-ribosylation of wild type ctc was observed after 30 minutes of hydrolysis reaction.
upon increasing hydrolysis incubation time, the biotin signals on the auto-adp-ribosylated wild type ctc increased whereas the biotin-signals on the adp-ribosylated exot substrate decreased, suggesting the removal of adp-ribose from the auto-adp-ribosylated ctc may cause the enzyme to become more active.
wild type ctc has multiple arginines to serve as adp-ribose acceptors.
we speculated the increasing biotin substitution after hadprh treatment could be attributed to more arginines being adp-ribosylated at different sites of the enzyme.
we therefore examined the hadprh hydrolysis of the auto-adp-ribosylated m <dig>  m <dig> and m <dig>  in which only two arginine residues are present in each mutant enzyme.
the biotin signal of each auto-adp-ribosylated m <dig> k to r mutant was reduced upon incubation with hadprh though these mutants displayed different degrees of sensitivity to hadprh hydrolysis .
m <dig> and m <dig> exhibited a 10-20% of reduction in the auto-adp-ribosylation signals after 120 minutes of incubation with hadprh whereas m <dig> and m <dig> showed 40-50% reductions, possibly indicating steric factors may affect the accessibility of hadprh to certain adp-ribosylarginine residues.figure  <dig>
auto-adp-ribosylation results a mixture of α
- and
β-anomeric adp-ribosylargininyl adducts.
auto-adp-ribosylated wild type ctc or m <dig> derived mutants or the adp-ribosylated exot substrate was incubated with purified hadprh for various periods of time.
the biotinyl-adp-ribosylation remaining was detected by blotting with irdye800cw-sa and quantified by odyssey infrared imaging system.
the data shown in  are representative blots from multiple experiments.
five independent experiments were quantified and summarized in .
data from  <dig> different experiments of hydrolysis of auto-adp-ribosylated m8k525r, m8k543r and m8k566r were summarized in .
the error bar shows standard error of mean .
hydrolysis index is the ratio of normalized biotin signals remained on the hadprh treated versus untreated substrates.
auto-adp-ribosylation of catalytic fragments of exotoxin a  and cholix toxin  was carried out with various concentrations of biotinyl-nad+ in the adp-ribosylation buffer containing 3 μm of purified enzymes shown in top panel.
the bottom panel shows the coomassie blue stained bands for protein loading control.
free adp-ribose, generated by hadprh hydrolysis activity, reacting with lysine to form schiff-base adducts did not attribute to the increased resistance of auto-adp-ribosylated ctc to hadprh hydrolysis because no increases of resistance was observed in m <dig>  m <dig>  m <dig> nor in exot substrate.
adp-ribosylation of exot substrate at 37°c resulted in a small proportion of adp-ribosylated exot substrate  becoming more resistant to hadprh hydrolysis than the exot substrate that was adp-ribosylated at 25°c.
however, no difference was observed in the rates of hydrolysis between adp-ribosylated exot at 37°c and 25°c, suggesting no spontaneous anomerization occurred during the hadprh hydrolysis reaction at 37°c.
thus, the possibility of spontaneous anomerization of α- to β-ribosyl linkage in adp-ribosylarginine at 37°c, observed in choleragen  <cit> , can not completely explain the degree of resistance to hadprh hydrolysis seen in the auto-adp-ribosylated ctc.
together, these data suggest that adp-ribosylation carried by exot/fas complex is an sn2-like reaction; whereas auto-adp-ribosylation of ctc has mixed stereochemistry consistent with an sn1-like reaction mediated through diffusible intermediates.
auto-adp-ribosylation model
to understand what this diffusible intermediate was, we first ruled out the possibility of the diffusible intermediate to be the adp-ribose generated by the nad+ glycohydrolase activity of the enzyme by free adp-ribose competition assays shown in figure 1f and g. moreover, adp-ribose has been shown to primarily interact with lysine, not arginine, residues at neutral ph.
decreases of biotin signals on a series of composed arginine to lysine substitution mutants was observed whereas lysine to arginine reverse mutants restored the biotin signals on the auto-adp-ribosylated enzyme, indicating arginine is the primary target residue for the auto-adp-ribosylation of ctc.
however, we cannot exclude that lysine residue may contribute to some level of biotin signals appeared on the m <dig> mutant .
the identification of the distal arginine residues, like arg <dig>  arg <dig> and arg <dig>  being auto-adp-ribosylated, suggests that this diffusible intermediate should be relatively stable in aqueous solution.
the estimated half life of oxocarbenium ion, another well described intermediate, in aqueous solution is about  <dig> to 100 ps  <cit> , which is too short to accommodate the time required for the intermediate to diffuse out of nad+ binding pocket to react with the arginine target residues located at the distal sites before it reacts with water.
literature estimates for the nadh diffusion coefficient,  <dig> ×10−6 cm2 s− <dig>  <cit> , predict that on the order of  <dig> to 400 ns would be required for the intact nad+ molecule to diffuse  <dig> to 10 nm.
because rotational diffusion is typically orders of magnitude faster than translational diffusion and the predicted rotational relaxation time  of a protein with the approximate dimensions of ctc is about 15 ns  <cit> , a combination of rotational and translational diffusions of both nad+ and the enzyme would allow the distal sites on the opposite face of the enzyme to be reached within tens of nanoseconds.
we thus hypothesize that the intermediate is a strained form of reactive nad+.
it is known that rate acceleration by enzymes could at least in part be due to introduction of strain into the part of the substrate which is undergoing reaction.
the rate enhancement which is found upon introduction of strain into the substrate molecule by distortion can be very profound.
this type of rate acceleration is thought to increase the energy of the ground state rather than to decrease the peak of the activation energy curve  <cit> .
one obvious question is how this strained nad+ intermediate was formed.
we previously observed pseudomonas exotoxin a has much lower auto-adp-ribosylation activity than cholix toxin when similar levels of nad+ substrate were used in the auto-adp-ribosylation reactions.
pseudomonas aeruginosa exotoxin a, another member of the diphthamide-dependent adp-ribosyltransferase family, shares 30% identity to cholix toxin and has similar numbers of arginines in the catalytic fragments .
if biotinyl-nad+ by itself is capable of reacting with arginines, the same concentration of biotinyl-nad+ used in the auto-reaction of exotoxin a and cholix toxin should yield similar level of biotin signals on both enzymes.
figure 6d shows that exotoxin a  requires a much higher concentration of biotinyl-nad+ to reach a similar level of auto-adp-ribosylation signal as cholix toxin .
km,ε-nad+ of the enzyme by definition is the substrate concentration required to reach half of the maximal velocity of its nad+ glycohydrolase activity.
previously, oppenheimer proposed a model for the inductive stabilization of an oxocarbenium ion intermediate via an interaction between the 2′-hydroxyl and an active site carboxylate of an anionic residue at neutral ph, such as diphtheria toxin and exotoxin a, in an enzyme-mediated nad+ hydrolysis mechanism  <cit> .
cholix toxin, like the other members of dt family, has glutamic acid  as its catalytic residue.
glutamic acid has a pka of  <dig> .
at neutral ph, its carboxyl group is predominately negatively charged.
here we hypothesize that a strained nad+ intermediate is pre-protonated at the catalytic site and released from the site before nicotinamide fully dissociated from the ribose while the enzyme is actively processing the substrate with its maximum speed .
the strained nad+ intermediate then rotates to interact with arginine residues in proximity.
this would provide an explanation for which exotoxin a required higher concentration of nad+ substrate than cholix toxin did to reach similar level of auto-adp-ribosylation when similar numbers of arginine target residues were available on both enzymes.
following the release of this pre-protonated nad+ intermediate, the guanidinium group of arginine side chain  approaching the partially negatively charged 2′-hydroxyl of the strained nad+ intermediate leads to the complete dissociation of nicotinamide to form an oxocarbenium cation with an electrophilic c <dig> atom which then immediately reacts with the partially negatively charged nitrogen  of the guanidinium group of the target arginine  to finish the nucleophilic substitution reaction.figure  <dig>
hypothetical model for auto-adp-ribosylation.
formation of the diffusible strained nad+ intermediate.
at ph  <dig> , the negatively charged carboxylate of the catalytic residue interacting with the 2′ hydroxyl group induced partial negative charges at the 2′hydroxyl position and formation of pseudo ribose diol to cause pre-protonation at c1′.
this pre-protonated form of nad+ is usually stabilized by the solvent molecules in its surroundings.
we propose a mechanism in which this pre-protonated nad+ intermediate gets released from the catalytic site and diffuses out of the nad+ binding pocket prior to dissociation of nicotinamide when enzyme is busy in processing high concentration of nad+.
transfer of adp-ribosyl moiety from strained nad+ to arginine residue.
this pre-protonated nad+ intermediate approaches arginine residue through the positive charged side chain of arginine and interacts with the partially negatively charged pseudo ribose diol .
guanidinium group of arginine side chain contains three nitrogens.
when one nitrogen is positively charged to interact with the ribose diol group, the induced partially negatively charged nitrogen  interacts with the electrophilic c <dig> center of the n-ribose to cause dissociation of nicotinamide and formation of oxocarbenium ion which then immediately reacts with the partially negatively charged nitrogen on the guanidinium group of arginine to facilitate the transfer of adp-ribose to the target arginine residue .
figure  <dig> shows poly-l-arginines could enhance the auto-adp-ribosylation of ctc up to 7–fold at the concentration of 200 ng/ml.
poly arginines are commonly found around the substrate binding domains of the adp-ribosyltransferases known as arginine fingers.
these arginines are located closely to the nad+ binding pocket.
the location of the poly arginines may alternatively provide an environment to facilitate the auto-adp-ribosylation of the enzyme in the presence of nad+ and absence of the exogenous substrate, like eef <dig> for cholix toxin.figure  <dig>
poly-l-arginines enhanced biotin signals on the auto-adp-ribosylated wild type ctc.
various concentrations of free poly-l-arginines  or l-arginine  were added to the auto-adp-ribosylation reaction of ctc and incubated at 37°c for 1 hr.
the biotinyl-auto-adp-ribosylation signals were detected by western blotting and presented as fold of change compared to the no amino acid added sample.
the data shown is representative of three different experiments.
adp-ribosylation of endogenous versus exogenous substrates
similar to several other adp-ribosyltransferases  <cit> , 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 eef <dig> .
cholix toxin and exotoxin a are both characterized as diphthamide-dependent adp-ribosyltransferases which modify eef <dig> in nature.
we also found that ctc could modify exogenous oligo-arginine peptides .
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 eef <dig> or re <dig> c cell lysate, containing a mutated eef <dig> without diphthamide modification, in the adp-ribosylation reactions.
the auto-adp-ribosylation of the input enzyme was only found in highest concentration  of ctc, whereas the adp-ribosylation of wild type eef <dig> 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  <dig>  μm of the enzyme .
we further incubated purified flag-tagged eef <dig> or flag-tagged eef <dig>  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 eef <dig> was much faster than that of auto-adp-ribosylation of the enzyme or adp-ribosylation of the flag-tagged eef <dig> mutant, in which diphthamide was replaced with an arginine residue .
diphthamide, which has imidazole-like structure on the modified histidine of eef <dig>  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  <cit> .
we also observed low concentration of imidazole could enhance the biotinyl-adp-ribosylation signals on the modified substrates .
these findings suggest that diphthamide can act as a catalyst to make adp-ribosylation of eef <dig> 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  <dig>
adp-ribosylation of endogenous versus exogenous substrates.
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  or control  were used in the nad+ glycohydrolase activity assays and adp-ribosylation of eef <dig> in  <dig> lysate.
his-tagged oligo-l-arginine or oligo-l-asparagine peptides were incubated with purified recombinant wild type ctc, catalytically defective mutant  or catalytically active mutant .
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.
various concentrations of catalytic fragments of cholix toxin or exotoxin a  was incubated with cho or re <dig> c cell lysate at 37°c for 1 hr.
the biotin signals on the adp-ribosylated eef <dig> 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-eef <dig> antibodies shown in the middle panel.
the bottom panel shows the coomassie blue stained gel for protein loading control.
to detect auto-adp-ribosylation at the presence of exogenous substrates, excess amount of ctc  was incubated with purified flag-tagged wild type eef <dig>  or flag-tagged eef <dig>  mutant  at the presence of 50 μm biotinyl-nad+ for various periods of time.
CONCLUSIONS
here we show that auto-adp-ribosylation reaction of cholix toxin primarily involves the intramolecular transfer of adp-ribosyl moiety to multiple arginine residues located around the nad+ binding pocket as well as distal sites on the opposite face of the enzyme.
this process is mediated through diffusible intermediates generated upon enzyme activation.
kinetic studies of m <dig> derived variants show the auto-adp-ribosylation rates of the arginines at the proximal sites of the nad+ binding pocket is significantly faster than those of the arginines located at the distal sites, supporting the diffusion mechanism.
owing to the extremely short half life of free oxocarbenium ion in aqueous solution, we hypothesize that the diffusible intermediate is a form of strained nad+ which is pre-protonated at the catalytic site and released prior to the dissociation of nicotinamide to react with proximal arginine residues.
this study elucidates the existence of a diffusible strained nad+ intermediate that is different from the strained nad+ alleviation model of the adp-ribosylarginine reaction in the ita toxin previously described  <cit> .
we propose this mechanism can be applied to engineer artificial adp-ribosyltransferases with alternative substrates by bringing substrates with arginine residues  close to the nad+ binding pocket to react with diffusible intermediates generated by the adp-ribosyltransferases.

 adp-ribosyltransferases constitute a broadly distributed family of prokaryotic and eukaryotic enzymes that catalyze the transfer of an adp-ribose moiety from nad+ to one or more substrates. the protein substrates of adprts are heterogeneous and include eukaryotic elongation factor, actin, vimentin, ezrin/radixin/moesin proteins, various heterotrimeric and small monomeric gtpases, the adapter proteins crk-i and crk-ii, human α-defensin and human β-defensin-, and cell surface molecules. a family of adprts adp-ribosylate eef on a post-translationally modified histidine residue called diphthamide. other adprts transfer adp-ribose to arginine, cysteine, glutamate, or asparagine residues. many adprts also possess nad+ glycohydrolase activity attributed to the attack by hydroxyl ion on the activated nad+ transition state. a number of adprts exhibit auto-adp-ribosylation activity, including diphtheria toxin, cholera toxin, clostridium limosum c exoenzyme, pseudomonas aeruginosa exoenzyme s, escherichia coli heat-labile enterotoxin, neisseria meningitidis adp-ribosylargininyl transferase, nare, and several mammalian adprts. auto-adp-ribosylation of apdrts has been shown to have functional consequences for the enzyme. for example, the auto-reaction of poly-adp-ribose polymerase has been reported to reduce its dna-binding affinity, which in turn plays an important role in regulating the parp response to dna damage stimuli. similarly, auto-adp-ribosylation of mammalian adp-ribosyltransferase redirects the enzyme catalytic focus from nad+ glycohydrolase activity to adp-ribosyltransferase activity. auto-adp-ribosylation has also been shown to inhibit the activity of the c. limosum c exoenzyme, p. aeruginosa exoenzyme s, and neisseria meningitides adp-ribosylargininyl transferase, nare. cholix toxin and recently identified cholix toxin variants were identified by genomic sequencing and homology comparisons in vibrio cholera non-o1/non-o strains. features of the primary sequences that were considered relevant to a phylogenetically conserved structural core supporting enzymatic function included an yxhg and an yxny motif and a low but significant degree of homology to p. aeruginosa exotoxin a. many of the enzymatic attributes of cholix toxin have been established by the expression, catalytic characterization and determination at high resolution of a three dimensional crystal structure of the toxin and its catalytic subunit. the results suggest that cholix toxin is a diphthamide-specific adp-ribosyltransferase with a general domain organization and topology similar to that of exotoxin a. members of the eef2-specific adprt class are both phylogenetically and enzymatically distinct from other adprts, and their common unique substrate, diphthamide, has been suggested to directly participate in the catalysis. this class of adprts has an elongated loop structure that is predicted or confirmed to significantly rearrange upon contact with eef and that likely mediates the majority of interactions between enzyme and substrate. data from crystallographic and kinetic studies suggest the adp-ribosylation of eef is mediated through a random third order sn reaction, though the ribosyl-diphthamide bond adopts the α conformation consistent with the stereo inversion expected of an sn mechanism. here we characterized the process of the auto-adp-ribosylation reaction of cholix toxin and evidenced that a metastable diffusible intermediate was generated upon the enzyme activation then diffused to react with arginine residues of the enzyme in a proximity dependent manner. we also showed that wild type cholix toxin catalytic fragment could adp-ribosylate oligo argininyl peptides and eef mutant in which the post-translationally modified diphthamide at his was replaced by arginine. we propose that this mechanism can be used to engineer adp-ribosyltransferases with alternative substrate specificity as long as there is an arginine residue or other adp-ribose acceptors close to the catalytic site of the enzyme.  an enzymatic pathway is involved in auto-adp-ribosylation to characterize the enzymatic activity of cholix toxin we expressed the catalytic fragment under the control of the arabad promoter as a translational fusion to a secb pathway-dependent signal peptide and assessed the ability of the periplasmic fraction to carry out adp-ribosylation using biotinyl-nad+ as a biotinyl-adp-ribose donor. initial experiments revealed the presence of an arabinose-inducible biotin-labeled band corresponding to the molecular weight of the catalytic fragment. mutants y493a, e581q, and y493a/e581q exhibited reduced biotinylation intensity compared to wild type and mutant e579q, suggesting the catalytic fragment was capable of utilizing itself as a substrate. the biotin labeled or 32p-labeled purified enzymes were also observed, suggesting that host factors are not required. the possibility that the mutant forms had undergone structural changes resulting in a loss of substrate potential was minimized by the finding that the circular dichroism spectra of wild type and mutant proteins were substantially identical. the auto-adp-ribosylation and nad+ glycohydrolase activities of the purified enzymes were also assessed under comparable conditions by a fluorescence-based assay. the results indicate that the auto-adp-ribosylation and nad+ glycohydrolase activities of ctc are highly concordant. mutations on any one of the conserved residues involved in catalysis resulted in loss of the auto-adp-ribosylation activity. these residues include e the catalytic residue; y and y two tyrosine residues binding to the aromatic group of nad+; h providing the structural integrity of the catalytic site.figure an enzymatic pathway is involved in auto-adp-ribosylation of ctc. biotin signals are detected in the periplasmic fractions of e.coli lysate expressing wild type and mutant cholix toxin catalytic fragments. the same blot was re-blotted with rabbit anti-ctc antibody to detect protein expression in each sample. similar results are obtained by incubating purified protein with either biotinyl-nad+ or 32p-nad+. the blots shown are representative of multiple independent experiments. the structures of these mutants were analyzed by circular dichroism spectrometry. nad+ glycohydrolase activity and auto-adp-ribosylation activity were quantified by 96-well plate based assays. data are summarized from three independent experiments. error bars show the standard deviation of the composite data. asterisks indicate significant reduction of enzyme activity as compared to the wild type ctc with *, p-value < ; **, p-value < . various concentrations of free adp-ribose were added to the auto-adp-ribosylation reaction. as a control, the 32p-adp-ribosylation signals on ctc were removed by phosphodiesterase i treatment. asterisk indicates significant reduction of 32p-auto-adp-ribosylation signal of the treated over the untreated samples with p-value < . data shown is representative of three independent experiments. error-bars show the standard error of mean. statistic analysis was done by student t-test. excess amount of free adp-ribose was added to the adp-ribosylation buffer containing 3 μm purified wild type ctc and 50 μm biotinyl-nad+ at 37°c for 1 hour. the biotinyl-adp-ribosylation signal was detected by irdye800cw-sa. in all panels, arrowheads indicate the auto-adp-ribosylated ctc and arrows indicate the detection of ctc by anti-ctc antibody. excess free adp-ribose 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. however, we also observed increased biotin substitution of the inactive mutant ctc 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. biotinyl-nad+ bears the biotin substitution on the adenosine moiety of nad+, whereas -nad+ has 14c labeling on the nicotinamide moiety. when biotinyl-nad+ was substituted by -nad+, there was no significant difference in 14c incorporation between active and inactive enzymes even when as high as to 200 μm of -nad+ was used in the auto-reactions. in contrast, active enzyme ctc showed ~4-fold higher biotin substitution than inactive mutant ctc 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 biotin signals on the self-modified wild type ctc is an adp-ribosylation reaction. purified wild type ctc or inactive mutant ctc, was incubated with various concentrations of biotinyl-nad+. the folds of fluorescence intensity increase between ctc and ctc were shown on the top of each pair of bar graph. purified ctc or ctc was incubated with various concentrations of -nad+ at 37°c for 1 hour. asterisk indicates the control group in which cell lysate was added to ctc or ctc to measure 14c incorporation into the tca precipitants. data were summarized from two separated experiments. auto-adp-ribosylation favors intramolecular transfer to understand if auto-adp-ribosylation of cholix toxin involves intra- or inter-molecular modification, a catalytically active higher molecular weight form of ctc was prepared by fusion of the catalytic fragment to an immunoglobulin constant domain, yielding an active enzyme with a molecular weight of 70 kda. the resulting protein was mixed with mutant catalytic fragment in 1: molar ratio. the catalytically active form of the enzyme, ig-ctc, was found to be preferentially substituted with biotinyl-adp-ribose. a reduced level of substitution with biotinyl-adp-ribose was found for y493a mutant protein that had been incubated at high concentration with ig-ctc, which could be due to residual activity of the mutant enzyme or could reflect the occurrence of trans-adp-ribosylation at high substrate concentration. similarly, when purified recombinant full-length wild type cholix toxin was mixed with the catalytically inactive mutant, y493a, or the catalytically inactive full-length cholix toxin e581a mutant was mixed with the wild type ctc, the auto-adp-ribosylation of cholix toxin preferentially took place on the catalytically active form. together, these data support the view that auto-adp-ribosylation of cholix toxin favors intramolecular transfer of the adp-ribose moiety.figure intramolecular transfer of adp-ribose to multiple arginines in the auto-reaction. mixing marked enzymes experiments. active ig-ctc was mixed with inactive mutant y493a; active full-length cholix toxin, cxt, was mixed with mutant y493a; inactive mutant cxt was mixed with active ctc in auto-adp-ribosylation assays. molar concentrations shown on the top of each blot are the concentrations of active enzyme. the left blots were detected by irdye800cw-sa; the same blots were re-probed with polyclonal rabbit anti-ctc antibody shown on right. neutral hydroxylamine assays. auto-adp-ribosylation reaction was carried out with periplasmic fractions of e. coli lysate expressing wild type or mutant ctc. the products were subjected to  m nh2oh or  m nacl treatment at 37°c for 2 hours. the blot shown is representative of multiple experiments. single amino acid substitution mutation studies. a set of representative data from the detection of biotin signals on wild type or different mutants was shown in. the fluorescence intensity of sa was normalized with the coomassie blue intensity to obtain the irdy-sa/cb ratio as a semi-quantitative auto-adp-ribosylation measurement of each enzyme. data were summarized from four sets of experiments. asterisk indicates significant reduction of auto-adp-ribosylation activity. nad+ glycohydrolase activities of wild type and mutant enzymes are shown in. multiple arginines are the target residues adp-ribosyl adducts have different stabilities to neutral hydroxylamine: under conditions for which the half life of adp-ribosylated glutamate is about 3– minutes, the half life of adp-ribosylated arginine is about 120 minutes and the half lives of adp-ribosylated cysteine and asparagine are immeasurably large. as shown in the figure 3b, the biotin substitution of the wild type and two active mutants, e579q and e579r, showed no reduction following exposure to  m nacl but fell by half after two hours of  m neutral hydroxylamine treatment at 37°c. under these conditions there were little or no changes in the quantity or mobility of proteins as a result of either exposure. these data are consistent with the view that a significant fraction of biotinyl-adp-ribose substitutions are the result of modification of arginine residues. since the adp-ribosyl-glutamate linkage is labile in the neutral hydroxylamine treatment, we substituted two glutamate residues near the nad+ binding pocket with lysine residues to create mutants e484k and/or e486k. none of these substitution mutations reduced the biotin signals on the auto-adp-ribosylated enzymes. we therefore focus our study on the modification of arginine target residues. to identify candidate residues for the auto-adp-ribosylation, arginine residues in the catalytic fragment were mutated to alanine residues. the extent of auto-adp-ribosylation was significantly reduced after incubation of the r506a, r519a, r525a, r530a, r535a, r540a, or r543a mutant proteins with biotinyl-nad+ substrate, indicating these arginine residues may be the targets of auto-adp-ribosylation. however, some mutant proteins also exhibited lower levels of nad+ glycohydrolase activity than wild type catalytic fragment especially in the case of the r506a, r525a and r530a mutant proteins. therefore, we replaced arg arg and arg with glutamine or lysine, which has greater steric bulk than alanine. the nad+ glycohydrolase activity of r506q, r506k, r525q, or r525k was detectable at a level similar to that of other mutants and auto-adp-ribosylation was also detected, suggesting other arginine residues than these can be adp-ribosylated. all the attempts to replace arg resulted in unstable enzymes with very poor activity and a low level of protein expression, indicating the enzyme is structurally sensitive to arg modification. with the exception of arg mutants, none of which were active, none of the single amino acid substitutions completely abolished auto-adp-ribosylation, leads to the hypothesis that multiple arginine residues could serve as adp-ribose acceptors. identification of arginine target residues by 2-d electrophoresis and ms/ms analysis to support this hypothesis, we analyzed the auto-adp-ribosylation of ctc using nad+ as adp-ribose donor by two-dimensional electrophoresis. ctc has an isoelectric point of. one adp-ribose addition to this fragment shifts its pi ~ ph units. following auto-reaction of the catalytic fragment, multiple spots shifted toward the acidic ph from the spot of ctc were detected by 2-d electrophoresis compared to the sample without incubation with nad+, indicating multiple residues being modified in the same protein. an additional spot found in the sample prior to incubation with nad+ may result from auto-adp-ribosylation in vivo, or from other post-translational modifications that occurred in the e.coli strain from which ctc was expressed. to map the location of the adp-ribosylated arginines, the auto-adp-ribosylated ctc was digested with lysc, predicted to cleave the ctc into three major peptides: peptide 423–, peptide 489– and peptide 509–. the lysc digested auto-adp-ribosylated ctc was resolved in ph 3– matrices followed by % tris-tricine peptide gel analysis to separate the fragments. two fragments were detected in gel spots b and a, respectively. peptide 489– was too small to be observed in the peptide gel. however, a partial digest product spanning residues 489– was detected from which multiple shifted spots were detected. at least three modified spots arising from the unmodified peptide 509– were detected in coomassie blue stained 2-d peptide gels. these spots were missing from gels prepared from the ctc which was not incubated with nad+. peptide 509– contains several arginine residues identified by reverse mutagenesis studies to be the targets for auto-adp-ribosylation of ctc.figure detection of multiple adp-ribosylarginines by two-dimensional electrophoresis. the amino acids sequences of ctc fused with outer membrane signal peptide was shown in. ctc was incubated with or without 250 μm nad+. ctc incubated with or without nad+ was further digested with lysc prior to peptide 2-d electrophoresis. amino acid sequences of three major predicted proteolytic products are shown in. circle i indicates the partially digested peptide 489– circle ii indicates spots shifted from the auto-adp-ribosylated peptide 509– spots a and b are peptides 509– and 423– respectively. we also confirmed the adp-ribosylarginine modifications by ms/ms analysis. the adp-ribose moiety of adp-ribosylated peptide is labile to the ms or ms/ms detection process. a two-step collision method was performed to remove the modification group with an in-source decay done in the tandem mass spectrometer. the product from the first collision was then subject to the second collision to fragmentize the peptide chain with the small marker moiety to locate and determine the amino acid sequences. the combination of selection candidate peptides by the precursor ion scanning with adp-r-carbodiimide as the marker ion and further fragmentation of the ion of the adp-r-carbodiimide-derived peptides provided highly specific and reliable data applicable for ms/ms database searching for de novo peptide sequences. the adp-ribosylargininyl peptide yields a product peptide in which arginine is replaced with ornithine after first collision. from spot a, we sequenced two orn peptides, yglpt/orn/aerd and argvml/orn/vyiprasle, indicating that the corresponding arginine positions arg and arg were adp-ribosylated. from the circled spots of figure 4b and circles i and ii of figure 4d, four orn peptide signals were detected to be orn/fy/orn/tntple, rfy/orn/tntple, orn/fyrtntple, and hitqvighslpl/orn/neaftgpe. the orn peptide containing arg was unexpectedly detected in this study. the arg position is sensitive to modification and the buried side-chain of arg makes it unlikely to be adp-ribosylated in its native conformation. we therefore reason the modification of arg occurred artificially during the overnight iso-electrofocusing step when the enzyme was exposed to the 2-d sample buffer. the urea and detergent in the sample buffer could expose the side chain of arg from its native position to react with the diffusible intermediate. although we removed free nad+ from the sample by gel filtration chromatography at the end of auto-adp-ribosylation reaction prior to 2-d analysis, we may not have completely removed the enzyme bound fractions of nad+. arg is located close to the nad+ binding pocket geometrically in the native 3-d structure of ctc. identification of arginine target residues by direct and reverse mutagenesis in our efforts to identify the auto-adp-ribosylation target residues, a series of composite lysine to arginine replacement mutants were created as shown in table  in which each successive mutant contained all the modifications of its parental template. mutant m was constructed by introducing the r506k and r566k mutations into wild type catalytic fragment. m was used as a template to make mutant m which has additional r426k, r473k and r629k mutations. arg was retained in all mutants because the protein integrity and catalytic activity are sensitive to its replacement. the mutants were purified and analyzed for their auto-adp-ribosylation and nad+ glycohydrolase activities. mutant m exhibited a reduction of biotin signal compared to wild type enzyme, implicating arg and/or arg as potential targets. similarly, the degree of biotin substitution was decreased in m5- m and m compared to the parental m template, implicating arg and arg as potential targets. the reduction of biotinylation from m5- to m could be attributed to the r535k mutation and the reduction from m to m could be attributed to the r525k mutation. introduction of r479k into the m template to make mutant m resulted in apparent abolition of biotinylation, suggesting arg is an important target. however, when the r479k mutation was introduced into the context of mutant m to form m mutant, significant biotinylation was detected, indicating arg is not the sole adp-ribose acceptor. the loss of substrate activity by mutant m coincided with a restoration of nad+ glycohydrolase activity, suggesting the principal explanation for the diminished degree of biotinylation seen in this mutant is not extinction of catalytic activity. based on these data, arg arg arg arg arg arg arg are potential target residues for auto-adp-ribosylation.table cholix toxin catalytic fragment variants contain arginine to lysine replacement mutations r to k mutants r to k replacement mutations on the wild type ctc ǂ auto-adp-ribosylation activity ¶ nad + glycohydrolase activity ǂ data show the mean fluorescence of intensity of biotin signals on the auto-adp-ribosylated enzyme band detected by irdye800-sa. ¶data show the specific activity of each enzyme as described in methods. *wild type cholix toxin catalytic fragment contains r r r r r r r r r r r r r r mutant m exhibited nearly undetectable auto-adp-ribosylation and the background biotinyl signals of m were similar to those of the inactive mutant, m. m mutant contains arginines to lysines replacement mutations except arg arg has a buried side chain, which makes it less likely to be the target of auto-adp-ribosylation in its native conformation. we therefore chose m as template to generate lysine to arginine reverse mutants. among 13 k to r reverse mutants, m m m m m and m were all found to regain biotinylation signal intensity, indicating multiple arginines can serve as adp-ribose acceptors in the auto-adp-ribosylation of m derived mutants. together, we identified arg arg arg arg arg arg and arg to be the target residues for auto-adp-ribosylation.figure auto-adp-ribosylation prefers arginines as target residues. mutant m contains arginines to lysines replacement mutations except arg the same amount of m and its catalytically inactive form m were incubated with various concentrations of biotinyl-nad+ in the auto-adp-ribosylation reaction. the arrow indicates the biotinylated bands. mutant m was used as template for the k to r reverse mutation to determine which arginine could be auto-adp-ribosylated. the arginines present in the m and m derived mutant enzymes are indicated with their numerical positions in the full-length recombinant cholix toxin shown at the bottom of panel b. variation of acceptor residues were carried out by replacing lys or lys on the mutant m with arginine, glutamine, asparagine, or cysteine. the circular dichroism of these m derived mutants were shown in and. auto-adp-ribosylation was performed with wild type or mutant cholix toxin catalytic fragments and detected by western blotting shown in. arrowheads indicate biotinyl-auto-adp-ribosylation of the catalytic fragments. arrows indicate detection of the catalytic fragments by anti-ctc antibody for protein loading control. diffusible intermediates the locations of these arginine target residues are not restricted near the nad+ binding pocket but also found at distal sites of the enzyme. in conjunction with the finding of intramolecular transfer of adp-ribose to the active enzyme itself, we propose a mechanism by which a diffusible intermediate is created upon enzymatic activation and diffuses to react with arginine residues. to further support this mechanism, we determined the auto-adp-ribosylation rate of individual arginine residues using m k to r reverse mutants. each m k to r reverse mutant contains arg and one additional arginine residue. using the method described in kinetic experiments, we compared the auto-adp-ribosylation rates of six selected arginine target residues located at the proximal and distal sites of the nad+ binding pocket. m containing arg target residue located at the end of the nad+ binding loop had the highest auto-adp-ribosylation rate, ~16-20-fold faster than the auto-adp-ribosylation rates of m and m under the assumption that these enzymes should have similar km,biotin-nad+ to make the correlation of the reaction rates with the distances of the acceptor residues in a simplified diffusion model, we normalized the vmax of m m and m with their corresponding km, biotin-nad+, then the normalized auto-adp-ribosylation rate of arg is approximately 4 ~ 5-fold faster than those of arg and arg which are located on the substrate recognition loop near the nad+ binding pocket. the auto-adp-ribosylation rates of arg arg and arg located at the distal or the opposite face of the nad+ binding pocket, were low and highly variable among the inter- and intra-experimental repeats. although the km,biotin-nad+ of m is 2-fold less than m the normalized auto-adp-ribosylation rate of m is ~200-fold lower than that of m suggesting the electronic environment in the solution and the stability of the diffusible intermediate may affect the measured modification rates more pronouncedly in the auto-adp-ribosylation of the three distal arginines. these data support that the auto-adp-ribosylation is mediated through a diffusible intermediate that reacts with its target residues in a primarily proximity-controlled mechanism.table kinetic analysis of auto-adp-ribosylation of various arginines on m derived mutants km, biotin-nad vmax m8 m8 m8 m8 m8 m8 *n.d., cannot be determined. the values represent the mean ± standard error from different experiments with two to three repeats in each experiment using graphpad prism. to test what other amino acids the diffusible intermediate would react with, the lys or lys in m mutants were substituted with other potential adp-ribose acceptors including cysteine, asparagine and glutamine. most of these substitutions yielded significant secondary structure changes in the mutants. mutant m showed a cd spectrum similar to m whereas m showed a cd spectrum similar to m suggesting these mutants share similar structures. however, neither m nor m showed higher biotin substitution than m or m in the auto-adp-ribosylation. these data are consistent with previous reports that arginine is the preferred target residue for auto-adp-ribosylation. human adprh hydrolysis analysis human adp-ribosylarginine hydrolase has been shown to preferentially hydrolyze the α-anomer of adp-ribosylarginine to release adp-ribose from adp-ribosylated protein. pseudomonas aeruginosa exotoxin t is an adp-ribosyltransferase which specifically adp-ribosylates arginine residues on target proteins in the presence of a host co-factor, fas. a minimal four amino acid peptide derived from crk-i/ii adaptor protein fused to the c-terminal domain of clostridium perfringens enterotoxin, named exot substrate, was shown to be specifically adp-ribosylated by exot/fas complex. to understand the intermediate step of the auto-adp-ribosylation of cholix toxin, we compared the activity of hadprh on substrates formed by cholix toxin auto-activity or exot action on its substrate. figure 6a and b show that hadprh was able to effectively hydrolyze the biotinylated adp-ribosylarginine from the adp-ribosylated exot substrate whereas only 20-40% reduction of the auto-adp-ribosylation of wild type ctc was observed after 30 minutes of hydrolysis reaction. upon increasing hydrolysis incubation time, the biotin signals on the auto-adp-ribosylated wild type ctc increased whereas the biotin-signals on the adp-ribosylated exot substrate decreased, suggesting the removal of adp-ribose from the auto-adp-ribosylated ctc may cause the enzyme to become more active. wild type ctc has multiple arginines to serve as adp-ribose acceptors. we speculated the increasing biotin substitution after hadprh treatment could be attributed to more arginines being adp-ribosylated at different sites of the enzyme. we therefore examined the hadprh hydrolysis of the auto-adp-ribosylated m m and m in which only two arginine residues are present in each mutant enzyme. the biotin signal of each auto-adp-ribosylated m k to r mutant was reduced upon incubation with hadprh though these mutants displayed different degrees of sensitivity to hadprh hydrolysis. m and m exhibited a 10-20% of reduction in the auto-adp-ribosylation signals after 120 minutes of incubation with hadprh whereas m and m showed 40-50% reductions, possibly indicating steric factors may affect the accessibility of hadprh to certain adp-ribosylarginine residues.figure auto-adp-ribosylation results a mixture of α - and β-anomeric adp-ribosylargininyl adducts. auto-adp-ribosylated wild type ctc or m derived mutants or the adp-ribosylated exot substrate was incubated with purified hadprh for various periods of time. the biotinyl-adp-ribosylation remaining was detected by blotting with irdye800cw-sa and quantified by odyssey infrared imaging system. the data shown in are representative blots from multiple experiments. five independent experiments were quantified and summarized in. data from different experiments of hydrolysis of auto-adp-ribosylated m8k525r, m8k543r and m8k566r were summarized in. the error bar shows standard error of mean. hydrolysis index is the ratio of normalized biotin signals remained on the hadprh treated versus untreated substrates. auto-adp-ribosylation of catalytic fragments of exotoxin a and cholix toxin was carried out with various concentrations of biotinyl-nad+ in the adp-ribosylation buffer containing 3 μm of purified enzymes shown in top panel. the bottom panel shows the coomassie blue stained bands for protein loading control. free adp-ribose, generated by hadprh hydrolysis activity, reacting with lysine to form schiff-base adducts did not attribute to the increased resistance of auto-adp-ribosylated ctc to hadprh hydrolysis because no increases of resistance was observed in m m m nor in exot substrate. adp-ribosylation of exot substrate at 37°c resulted in a small proportion of adp-ribosylated exot substrate becoming more resistant to hadprh hydrolysis than the exot substrate that was adp-ribosylated at 25°c. however, no difference was observed in the rates of hydrolysis between adp-ribosylated exot at 37°c and 25°c, suggesting no spontaneous anomerization occurred during the hadprh hydrolysis reaction at 37°c. thus, the possibility of spontaneous anomerization of α- to β-ribosyl linkage in adp-ribosylarginine at 37°c, observed in choleragen, can not completely explain the degree of resistance to hadprh hydrolysis seen in the auto-adp-ribosylated ctc. together, these data suggest that adp-ribosylation carried by exot/fas complex is an sn2-like reaction; whereas auto-adp-ribosylation of ctc has mixed stereochemistry consistent with an sn1-like reaction mediated through diffusible intermediates. auto-adp-ribosylation model to understand what this diffusible intermediate was, we first ruled out the possibility of the diffusible intermediate to be the adp-ribose generated by the nad+ glycohydrolase activity of the enzyme by free adp-ribose competition assays shown in figure 1f and g. moreover, adp-ribose has been shown to primarily interact with lysine, not arginine, residues at neutral ph. decreases of biotin signals on a series of composed arginine to lysine substitution mutants was observed whereas lysine to arginine reverse mutants restored the biotin signals on the auto-adp-ribosylated enzyme, indicating arginine is the primary target residue for the auto-adp-ribosylation of ctc. however, we cannot exclude that lysine residue may contribute to some level of biotin signals appeared on the m mutant. the identification of the distal arginine residues, like arg arg and arg being auto-adp-ribosylated, suggests that this diffusible intermediate should be relatively stable in aqueous solution. the estimated half life of oxocarbenium ion, another well described intermediate, in aqueous solution is about to 100 ps, which is too short to accommodate the time required for the intermediate to diffuse out of nad+ binding pocket to react with the arginine target residues located at the distal sites before it reacts with water. literature estimates for the nadh diffusion coefficient, ×10−6 cm2 s−, predict that on the order of to 400 ns would be required for the intact nad+ molecule to diffuse to 10 nm. because rotational diffusion is typically orders of magnitude faster than translational diffusion and the predicted rotational relaxation time of a protein with the approximate dimensions of ctc is about 15 ns, a combination of rotational and translational diffusions of both nad+ and the enzyme would allow the distal sites on the opposite face of the enzyme to be reached within tens of nanoseconds. we thus hypothesize that the intermediate is a strained form of reactive nad+. it is known that rate acceleration by enzymes could at least in part be due to introduction of strain into the part of the substrate which is undergoing reaction. the rate enhancement which is found upon introduction of strain into the substrate molecule by distortion can be very profound. this type of rate acceleration is thought to increase the energy of the ground state rather than to decrease the peak of the activation energy curve. one obvious question is how this strained nad+ intermediate was formed. we previously observed pseudomonas exotoxin a has much lower auto-adp-ribosylation activity than cholix toxin when similar levels of nad+ substrate were used in the auto-adp-ribosylation reactions. pseudomonas aeruginosa exotoxin a, another member of the diphthamide-dependent adp-ribosyltransferase family, shares 30% identity to cholix toxin and has similar numbers of arginines in the catalytic fragments. if biotinyl-nad+ by itself is capable of reacting with arginines, the same concentration of biotinyl-nad+ used in the auto-reaction of exotoxin a and cholix toxin should yield similar level of biotin signals on both enzymes. figure 6d shows that exotoxin a requires a much higher concentration of biotinyl-nad+ to reach a similar level of auto-adp-ribosylation signal as cholix toxin. km,ε-nad+ of the enzyme by definition is the substrate concentration required to reach half of the maximal velocity of its nad+ glycohydrolase activity. previously, oppenheimer proposed a model for the inductive stabilization of an oxocarbenium ion intermediate via an interaction between the 2′-hydroxyl and an active site carboxylate of an anionic residue at neutral ph, such as diphtheria toxin and exotoxin a, in an enzyme-mediated nad+ hydrolysis mechanism. cholix toxin, like the other members of dt family, has glutamic acid as its catalytic residue. glutamic acid has a pka of. at neutral ph, its carboxyl group is predominately negatively charged. here we hypothesize that a strained nad+ intermediate is pre-protonated at the catalytic site and released from the site before nicotinamide fully dissociated from the ribose while the enzyme is actively processing the substrate with its maximum speed. the strained nad+ intermediate then rotates to interact with arginine residues in proximity. this would provide an explanation for which exotoxin a required higher concentration of nad+ substrate than cholix toxin did to reach similar level of auto-adp-ribosylation when similar numbers of arginine target residues were available on both enzymes. following the release of this pre-protonated nad+ intermediate, the guanidinium group of arginine side chain approaching the partially negatively charged 2′-hydroxyl of the strained nad+ intermediate leads to the complete dissociation of nicotinamide to form an oxocarbenium cation with an electrophilic c atom which then immediately reacts with the partially negatively charged nitrogen of the guanidinium group of the target arginine to finish the nucleophilic substitution reaction.figure hypothetical model for auto-adp-ribosylation. formation of the diffusible strained nad+ intermediate. at ph , the negatively charged carboxylate of the catalytic residue interacting with the 2′ hydroxyl group induced partial negative charges at the 2′hydroxyl position and formation of pseudo ribose diol to cause pre-protonation at c1′. this pre-protonated form of nad+ is usually stabilized by the solvent molecules in its surroundings. we propose a mechanism in which this pre-protonated nad+ intermediate gets released from the catalytic site and diffuses out of the nad+ binding pocket prior to dissociation of nicotinamide when enzyme is busy in processing high concentration of nad+. transfer of adp-ribosyl moiety from strained nad+ to arginine residue. this pre-protonated nad+ intermediate approaches arginine residue through the positive charged side chain of arginine and interacts with the partially negatively charged pseudo ribose diol. guanidinium group of arginine side chain contains three nitrogens. when one nitrogen is positively charged to interact with the ribose diol group, the induced partially negatively charged nitrogen interacts with the electrophilic c center of the n-ribose to cause dissociation of nicotinamide and formation of oxocarbenium ion which then immediately reacts with the partially negatively charged nitrogen on the guanidinium group of arginine to facilitate the transfer of adp-ribose to the target arginine residue. figure  shows poly-l-arginines could enhance the auto-adp-ribosylation of ctc up to 7–fold at the concentration of 200 ng/ml. poly arginines are commonly found around the substrate binding domains of the adp-ribosyltransferases known as arginine fingers. these arginines are located closely to the nad+ binding pocket. the location of the poly arginines may alternatively provide an environment to facilitate the auto-adp-ribosylation of the enzyme in the presence of nad+ and absence of the exogenous substrate, like eef for cholix toxin.figure poly-l-arginines enhanced biotin signals on the auto-adp-ribosylated wild type ctc. various concentrations of free poly-l-arginines or l-arginine were added to the auto-adp-ribosylation reaction of ctc and incubated at 37°c for 1 hr. the biotinyl-auto-adp-ribosylation signals were detected by western blotting and presented as fold of change compared to the no amino acid added sample. the data shown is representative of three different experiments. adp-ribosylation of endogenous versus exogenous substrates similar to several other adp-ribosyltransferases, 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 eef. cholix toxin and exotoxin a are both characterized as diphthamide-dependent adp-ribosyltransferases which modify eef in nature. we also found that ctc could modify exogenous oligo-arginine peptides. 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 eef or re c cell lysate, containing a mutated eef without diphthamide modification, in the adp-ribosylation reactions. the auto-adp-ribosylation of the input enzyme was only found in highest concentration of ctc, whereas the adp-ribosylation of wild type eef 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  μm of the enzyme. we further incubated purified flag-tagged eef or flag-tagged eef 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 eef was much faster than that of auto-adp-ribosylation of the enzyme or adp-ribosylation of the flag-tagged eef mutant, in which diphthamide was replaced with an arginine residue. diphthamide, which has imidazole-like structure on the modified histidine of eef 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. we also observed low concentration of imidazole could enhance the biotinyl-adp-ribosylation signals on the modified substrates. these findings suggest that diphthamide can act as a catalyst to make adp-ribosylation of eef 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 adp-ribosylation of endogenous versus exogenous substrates. 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 or control were used in the nad+ glycohydrolase activity assays and adp-ribosylation of eef in lysate. his-tagged oligo-l-arginine or oligo-l-asparagine peptides were incubated with purified recombinant wild type ctc, catalytically defective mutant or catalytically active mutant. 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. various concentrations of catalytic fragments of cholix toxin or exotoxin a was incubated with cho or re c cell lysate at 37°c for 1 hr. the biotin signals on the adp-ribosylated eef 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-eef antibodies shown in the middle panel. the bottom panel shows the coomassie blue stained gel for protein loading control. to detect auto-adp-ribosylation at the presence of exogenous substrates, excess amount of ctc was incubated with purified flag-tagged wild type eef or flag-tagged eef mutant at the presence of 50 μm biotinyl-nad+ for various periods of time.  here we show that auto-adp-ribosylation reaction of cholix toxin primarily involves the intramolecular transfer of adp-ribosyl moiety to multiple arginine residues located around the nad+ binding pocket as well as distal sites on the opposite face of the enzyme. this process is mediated through diffusible intermediates generated upon enzyme activation. kinetic studies of m derived variants show the auto-adp-ribosylation rates of the arginines at the proximal sites of the nad+ binding pocket is significantly faster than those of the arginines located at the distal sites, supporting the diffusion mechanism. owing to the extremely short half life of free oxocarbenium ion in aqueous solution, we hypothesize that the diffusible intermediate is a form of strained nad+ which is pre-protonated at the catalytic site and released prior to the dissociation of nicotinamide to react with proximal arginine residues. this study elucidates the existence of a diffusible strained nad+ intermediate that is different from the strained nad+ alleviation model of the adp-ribosylarginine reaction in the ita toxin previously described. we propose this mechanism can be applied to engineer artificial adp-ribosyltransferases with alternative substrates by bringing substrates with arginine residues close to the nad+ binding pocket to react with diffusible intermediates generated by the adp-ribosyltransferases. 
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, m showed that a diffusible intermediate preferentially reacted with arginine residues in proximity to the nad+ binding pocket. therefore, a diffusible strained form of nad+ intermediate was proposed to react with arginine residues in a proximity dependent manner. we discovered an adp-ribosylargininyl activity of cholix toxin catalytic fragment from our studies in auto-adp-ribosylation, which is mediated through diffusible intermediates. auto-adp-ribosylation of cholix toxin appears to have negatively regulatory effect on adp-ribosylation of exogenous substrate. cholix toxin is an adp-ribosyltransferase found in non-o1/non-o strains of vibrio cholera. the catalytic fragment of cholix toxin was characterized as a diphthamide dependent adp-ribosyltransferase. adp-ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates. however, at the presence of both endogenous and exogenous substrates, adp-ribosylation of exogenous substrates occurred more efficiently than that of endogenous substrates.
 cholix toxin is an adp-ribosyltransferase found in non-o1/non-o strains of vibrio cholera. the catalytic fragment of cholix toxin was characterized as a diphthamide dependent adp-ribosyltransferase.  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, m 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.  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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cholix toxinadp-ribosylationauto-adp-ribosylationarginineissue-copyright-statement© the author 2014 