(Received for publication, August 18, 1994; and in revised form, November 28, 1994)
From the
Sequence similarities between the enzymatic region of
poly-ADP-ribose polymerase and the corresponding region of
mono-ADP-ribosylating bacterial toxins suggest similarities in active
site structure and catalytic mechanism. Glu of the human
polymerase aligns with the catalytic glutamic acid of the toxins, and
replacement of this residue with Gln, Asp, or Ala caused major
reductions in synthesis of enzyme-linked poly-ADP-ribose. Replacement
of any of 3 other nearby Glu residues had little effect. The
Glu
mutations produced similar changes in activity in the
carboxyl-terminal 40-kDa catalytic fragment fused to maltose-binding
protein: E988Q and E988A reduced polymer elongation >2000-fold, and
E988D
20-fold. Smaller changes were seen in chain initiation. The
mutations had little effect on the K
of
NAD, indicating a predominantly catalytic function for
Glu
. The results support the concept of similar active
sites of the polymerase and the ADP-ribosylating toxins. Glu
may function in polymer elongation similarly to the toxins'
active site glutamate, as a general base to activate the attacking
nucleophile (in the case of the polymerase, the 2`-OH of the terminal
adenosine group of a nascent poly-ADP-ribose chain).
Poly-ADP-ribose polymerase (PARP), ()a DNA-binding
protein found in most eukaryotes, is activated by DNA strand breaks in vitro or in vivo to make long, branched glycosidic
polymers of ADP-ribose from
NAD(1, 2, 3, 4, 5) . Much
of the polymer is covalently attached to PARP itself(6) , but
certain other proteins such as histones have also been found to have
poly-ADP-ribose attached(7) . Although PARP has been suggested
to have a role in DNA repair, much remains to be learned about its
physiological functions.
The polymerase consists of a single polypeptide chain (113 kDa for the human enzyme, hPARP) and contains three functional domains(8) . The amino-terminal domain binds DNA and has two zinc fingers that are required for recognizing DNA strand breaks and activating the enzyme (9, 10, 11) . The carboxyl-terminal catalytic domain poly-ADP-ribosylates the central, so-called automodification domain(8) . The automodification domain has been predicted to have a leucine zipper (12) and may be responsible for the dimerization of PARP(13) . A carboxyl-terminal 40-kDa tryptic fragment of PARP containing the catalytic domain has ADP-ribosylation activity that is unaffected by DNA and approximately equal in magnitude to that of the intact enzyme in the absence of DNA(14) .
The ADP-ribosylation reactions catalyzed by PARP bear resemblances to those catalyzed by the ADP-ribosylating bacterial toxins, such as diphtheria and cholera toxins(15, 16) . All of these enzymes catalyze transfer of the ADP-ribose moiety of NAD to a substrate protein. In the toxins, the site of attachment of ADP-ribose is typically a specific side chain on a host cell regulatory protein, whereas PARP initiates poly-ADP-ribose chains by ADP-ribosylating acidic side chains on its own automodification domain. Following the initiation step, PARP effects chain elongation by ADP-ribosylating the 2`-OH group of terminal adenosines of poly-ADP-ribose chains, and it catalyzes branch formation by ADP-ribosylating the 2`-OH group of internal ribose moieties within the poly-ADP-ribose chain. PARP also shares with toxins the ability to catalyze NAD hydrolysis, a reaction of no known physiological significance for these proteins. Like some ADP-ribosylating toxins, the polymerase requires a macromolecular cofactor for enzymic activation. Both cholera toxin (16, 17) and Pseudomonas aeruginosa exoenzyme S (18) are activated by specific host proteins, whereas PARP is activated by DNA.
The ADP-ribosylation activity of many, and perhaps
all, ADP-ribosylating toxins is dependent on a key catalytic glutamic
acid residue. Photoaffinity labeling with NAD first identified
Glu as an active site residue in diphtheria toxin (DT) (19, 20) , and functionally equivalent glutamic acids
were subsequently found in other toxins, Glu
in P. aeruginosa exotoxin A(21, 22) ,
Glu
in pertussis
toxin(23, 24, 25) , and Glu
in
the Clostridium limosum C3-like
ADP-ribosyltransferase(26) . Where tested, mutations of these
residues severely affected ADP-ribosyltransferase activity and
toxicity(23, 25, 27, 28, 29, 30, 31) ,
while causing little change in substrate binding. Thus, a largely
catalytic role is indicated for the active site glutamic acids. The
crystallographic structures of exotoxin A(32) , Escherichia
coli heat-labile toxin(33) , DT(34) , and
pertussis toxin (35) have shown strong similarities in the
active site fold among the ADP-ribosylating toxins and the presence of
glutamic acid in the position corresponding to Glu
of DT.
Domenighini et al.(36) reported sequence
similarities between the enzymatic region of PARP and the catalytic
domains of several of the ADP-ribosylating toxins, in particular DT and P. aeruginosa exotoxin A. Although the percent identity was
low, nearly all of the active site residues of these toxins were
conserved in PARP, including the active site glutamic acid,
corresponding to Glu of hPARP. To test the possibility
that Glu
has a role in catalyzing formation of
poly-ADP-ribose and to probe the similarities in active site structure
between PARP and the toxins, we have made a series of site-directed
replacements for this and certain other residues in hPARP, expressed
the mutant proteins in E. coli, purified them, and
characterized their changes in activity.
The report of Domenighini et al.(36) led us
to construct a refined alignment of the catalytic region of known
vertebrate and insect PARP sequences with that of DT and P. aeruginosa exotoxin A (Fig. 1) (42) . An
alignment in which most of the major active site residues of these
toxins were conserved, including Glu and His
(corresponding to Glu
and His
,
respectively, of DT) was achieved with introduction of only a minimal
number of gaps. Besides Glu
, the 3 other glutamic acids
in the catalytic domain of hPARP (Glu
, Glu
,
and Glu
) are conserved in the polymerase from various
species, but not in the toxins.
Figure 1:
Amino
acid sequence alignment of PARP from various species with diphtheria
toxin and exotoxin A from P. aeruginosa. Toxin
alignment(22) : DTA, DT fragment A; ETA, P. aeruginosa exotoxin A. PARP alignment: PARP.hum, human(67) ; PARP.mus,
mouse(68) ; PARP.rat, rat (partial
sequence)(69) ; PARP.bov, bovine(70) ; PARP.chk, chicken(71) ; PARP.xen, Xenopus
laevis (EMBL: XLPARPG, accession no. Z12139 (B. M., Saulier-le
Drean, personal communication); PARP.sar, Sarcophaga
peregrina(72) ; PARP.dro, Drosophila
melanogaster(12) . The PARP alignment is based on an
alignment generated with PILEUP (Wisconsin Genetics Computer
Group,(73) ). For each sequence group, identities to the
consensus sequence are shown as dots (.). Gaps inserted to
allow alignment within sequence groups are shown as dashes(-).
Gaps inserted to allow alignment between sequence groups are designated
by slashes (). Residues shared between PARP (including at
least six of the eight PARP sequences) and one or both of the toxin
sequences are designated by shading. Asterisks (*) represent toxin active site residues. Human PARP
residues altered by site-directed mutagenesis are indicated by triangles (), and residues mutated by Simonin et
al.(14, 58) are designated by V.
Using oligonucleotide-directed
mutagenesis, we replaced Glu with aspartic acid,
glutamine, and alanine, Glu
, Glu
, and
Glu
with glutamine, and His
with alanine.
Initially, we tested extracts of the lon
E. coli
strain ME8417 expressing the mutant PARP proteins for ability to
incorporate radiolabel from [
P]NAD into
trichloroacetic acid-insoluble material during a 5-min incubation. The
3 Glu
mutants showed a reduction in incorporation of 45-
to 1100-fold, with E988A showing the greatest reduction in
incorporation and E988Q the least (Table 1). The H862A mutation
also caused a major reduction, comparable to that of E988A. In
contrast, the E931Q, E923Q, and E883Q mutants displayed little or no
change in activity.
The mutant forms of PARP were purified by
chromatography on phosphocellulose, and the products formed by each
during incubation with [P]NAD were
characterized. The radiolabeled products were cleaved from the enzyme
with weak base, fractionated by electrophoresis on SDS-polyacrylamide
gels, and analyzed by autoradiography with a PhosphorImager (Fig. 2). In contrast to the wild-type protein, in which large
amounts of high molecular weight polymer were formed, the E988Q mutant
incorporated only mono-ADP-ribose (trace amounts of low molecular
weight oligo-ADP-ribose could be detected in the 2 h sample, but only
by careful inspection of the autoradiograph). Mono-ADP-ribose was also
the major product of the E988A mutant, but small amounts of low
molecular weight oligo-ADP-ribose (n
5) were seen at late
time points. Among the 3 Glu
mutants characterized, only
the E988D mutant made long poly-ADP-ribose chains. Polymer elongation
occurred at a greatly reduced rate in this mutant, however, and
full-length polymer was seen only after prolonged incubation (1-2
h, in contrast to wild-type PARP, which produced full-length polymer
within 5 min). Processive elongation by the E988D mutant is implied by
the fact that long, rather than short, oligomer chains predominate at
late time points, despite this mutant's low overall activity. The
products made by the control mutants (E931Q, E923Q, and E883Q) were
indistinguishable from those made by wild-type PARP (data not shown).
The H862A mutant made a mixture of mono- and short oligo-ADP-ribose
chains (n
10).
Figure 2:
ADP-ribosylation product time courses of
phosphocellulose-purified PARP. A, PARP (2 µg) was
incubated with [P]NAD under standard assay
conditions in a volume of 120 µl. At time intervals (noted in min),
20-µl aliquots were withdrawn and trichloroacetic
acid-precipitated. Samples were then hydrolyzed with 0.1 N NaOH, 20 mM EDTA, neutralized, and run on 15%
polyacrylamide, 0.1% SDS Tris-borate EDTA gels as described under
``Experimental Procedures.'' The dried gels were analyzed
using a PhosphorImager. B, plot of PhosphorImager scanning
data from samples in Fig. 3A. Wild-type (
), E988Q
(
), E988D (
), and E988A (
). Inset shows
Glu
mutant data at higher
resolution.
Figure 3:
Susceptibility of the E988Q
PARP-(mono-ADP-ribose) bond to hydrolysis by hydroxylamine.
Phosphocellulose-purified E988Q PARP (20 µg) was ADP-ribosylated
under standard conditions with 50 µM [P]NAD, and precipitated by addition of an
equal volume 40% trichloroacetic acid. The trichloroacetic acid pellet
was resuspended in 0.1 N HEPES pH 7.0, 0.5% SDS and aliquots
were mixed with an equal volume 2 M NH
OH pH 7.0
(
), or H
O (
), and incubated for various time
points. Reactions were terminated by spotting to trichloroacetic
acid/ether-saturated paper and unincorporated counts removed with
repeated 5% trichloroacetic acid washes. Residual (unreleased)
ADP-ribosylation was measured by scintillation counting.
The E988Q mutant exhibited rapid
initial incorporation of mono-ADP-ribose, differing little from the
wild-type protein (Fig. 2B). This suggested little
effect of the mutation on initiation, despite the large effect on
elongation. In contrast, with the E988A mutant there was a major defect
in initiation as well as elongation. The E988D mutant showed an
intermediate level of activity, with defects in both initiation and
elongation. Like the wild-type enzyme, all of the Glu mutants were dependent on DNA for ADP-ribosylation activity.
The identity of the E988Q product as mono-ADP-ribose was established
by its comigration with authentic [P]ADP-ribose
in polyacrylamide gels (Fig. 2) and its comigration with
unlabeled ADP-ribose on two-dimensional thin layer chromatograms (data
not shown). In addition, digestion of the product with snake venom
phosphodiesterase yielded
P-labeled AMP, as determined by
two-dimensional thin layer chromatography (data not shown). The bond
linking the E988Q mono-ADP-ribose product to PARP was sensitive to
hydrolysis by hydroxylamine at pH 7.0 (Fig. 3), consistent with
the ester bond reported to link poly-ADP-ribose to the wild-type
enzyme(39) . Approximately two mono-ADP-ribosyl groups/PARP
monomer were incorporated by the E988Q mutant at saturation, which is
approximately equal to the number of poly-ADP-ribose attachment sites
we found with wild-type PARP. In E. coli, a 90 kDa form of the
enzyme resulting from aberrant chain initiation at an internal
methionine is produced in addition to the wild-type 113 kDa form. Both
forms were ADP-ribosylated to an equal extent in the E988Q mutant,
although the 90 kDa form has been shown to have only basal
ADP-ribosylating activity(37) . The value of approximately two
poly-ADP-ribose chains (or mono-ADP-ribose groups, for the E988Q
mutant) per monomer differs from an earlier report of approximately 15
sites of polymer attachment on PARP(41) . The basis of this
discrepancy is unknown.
We introduced the Glu mutations into the minimal catalytic fragment of PARP in order to
study the effect of these replacements on enzymic activity in the
absence of other factors that might complicate analysis (e.g. activation by DNA, or the presence of the 90 kDa form). This
fragment comprises the carboxyl-terminal 40 kDa of the 51-kDa PARP
catalytic domain(8, 14) and has
ADP-ribosyltransferase activity that is independent of DNA and
approximately equal in magnitude to that of the intact enzyme in the
absence of DNA (about 1/500 that of the fully activated enzyme; 14). To
facilitate purification of the fragment, we attached maltose-binding
protein (MBP) to its amino terminus using the polymerase chain reaction (43, 44) . Cleavage of the resulting fusion protein,
termed MBP40 (82 kDa) at a factor Xa protease site at the fusion
junction yielded the expected MBP fragment of 42 kDa and PARP fragment
of 40 kDa (data not shown). Fortuitously, the MBP40 fusion protein
proved to be
10-fold more active than the 40-kDa catalytic
fragment alone, due to the ability of the PARP catalytic domain to
ADP-ribosylate the MBP portion of the fusion protein. The intact MBP40
fusion protein was therefore routinely used instead of the 40-kDa
fragment in basal activity measurements of the Glu
mutants.
Following initial studies demonstrating that the
effects of the Glu mutations in MBP40 were similar to
those in hPARP, we determined the K
for NAD for
each mutant form of MBP40. The K
for NAD of the
PARP catalytic fragment has been reported to be roughly 50
µM(14) , and published values for intact PARP
range between 50 and 100 µM NAD (37, 45) As shown in Table 2, none of the
Glu
mutants showed major alterations in K
. Values for the catalytic coefficient, k
/K
, were dramatically
reduced relative to wild-type MBP40, however. The E988Q and E988D
mutants were decreased by
35- and 50-fold, respectively, and the
E988A by
400-fold. These results are consistent with the notion
that Glu
plays a catalytic role in formation of
poly-ADP-ribose. It should be noted that the values obtained were
calculated from total ADP-ribose incorporation under the assumption of
equal K
values for polymer initiation and
elongation. There may be a difference in these values, however, since
the K
for NAD of the mono-ADP-ribosylating
(elongation deficient) E988Q mutant appears to be slightly lower than
that of the wild-type enzyme.
Catalysis of the initiation reaction
for poly-ADP-ribose synthesis, in which (ADP-ribose)-protein esters are
formed, could conceivably differ from that of the elongation and
branching reactions, which produce polyglycosides of ADP-ribose. We
therefore characterized the products of the MBP40 fusion proteins more
fully in order to explore the role(s) of Glu in polymer
elongation and initiation further and to seek information about its
possible role in polymer branching. Wild-type and mutant MBP40 fusion
proteins were incubated with [
P]NAD, and the
trichloroacetic acid-precipitable fraction was collected, washed, and
hydrolyzed with base. After neutralization, the products were treated
with snake venom phosphodiesterase and separated by two-dimensional
thin layer chromatography. Measurements of label in phosphoribosyl-AMP
(main chain product), AMP (product of the protein-distal chain
terminus), and diphosphoribosyl-AMP (branch product), provided the
basis for estimating chain length, number of polymer chains/enzyme, and
degree of branching, according to the method of Kawaichi et
al.(41) .
The Glu replacements caused
only modest effects on the rate of initiation, as judged by the number
of polymer chains/enzyme (Table 3). The E988Q mutant was reduced
in initiation only
3-fold, and the E988D and E988A mutants by
20- and 30-fold, respectively. These results are consistent with
the kinetics of polymer formation by the full-length mutant PARP
proteins (see Fig. 2). Chain elongation, on the other hand, as
estimated from the phosphoribosyl-AMP product, was dramatically reduced
in the E988Q and E988A mutants (2800- and 2200-fold, respectively), but
decreased only
20-fold by the E988D substitution. Estimates of
branching efficiency from the diphosphoribosyl product could not be
obtained from the E988Q and E988A proteins because they made only short
polymer and in trace amounts. In the E988D mutant, branch, as a
proportion of total poly-ADP-ribose synthesized, was only slightly
lower than in the wild-type enzyme (0.5 and 0.8% for the mutant and
wild-type, respectively).
The findings that both conservative and nonconservative
replacements for Glu caused major reductions in
elongation of poly-ADP-ribose chains in PARP (and the mutation of
His
to Ala caused a similar reduction) are consistent
with the suggestion that PARP and the ADP-ribosylating toxins have
similar active site structures and folds. The alignment alone provides
substantial support for this proposal (Fig. 1). In DT, which
appears to have the closest sequence similarity to PARP among the
toxins, His
, Tyr
, Tyr
, and
Glu
, which are near each other within the NAD site, are
conserved in the alignment with hPARP as His
,
Tyr
, Tyr
, and Glu
,
respectively. All of these residues are identical in PARP from various
species, and there is also significant conservation among neighboring
residues, particularly near the histidine, the first tyrosine, and the
glutamate. In the crystallographic structure of DT, His
,
Tyr
, and Tyr
are believed to form the
nicotinamide subsite, and Glu
has been shown to be
important in catalyzing ADP-ribose transfer(48) . In addition,
Phe
in hPARP, which is also conserved among diverse
species, aligns with Trp
of DT, another determinant of NAD
affinity(47) .
In this study we focused on Glu of hPARP because its putative homolog in DT, Glu
,
plays a major catalytic role and appears to be the only functional
active site residue universally conserved among the ADP-ribosylating
toxins. The E988Q and E988D mutations demonstrated the importance of
the presence and precise spatial location of a side chain carboxylate
at this site for poly-ADP-ribose chain elongation. Thus, the Gln mutant
was almost devoid of elongation activity (2800-fold reduction), whereas
the Asp mutant was reduced only about 20-fold. These mutations had
relatively little effect on chain initiation, however, a 3-fold
reduction for the Gln and 20-fold for the Asp, indicating a different,
and less important, function for Glu
in initiation. The
Ala substitution caused reductions in elongation and initiation
comparable to those of the E988Q and E988D mutants, respectively.
In
DT (and by implication, aeruginosa exotoxin A) ADP-ribosylation of the
diphthamide residue of EF-2 proceeds via an ordered sequential
mechanism in which NAD must bind to the active site cleft of the
catalytic fragment before the second substrate EF-2 can bind and the
reaction take place(48) . Replacing Glu of DT
with Gln, Asp, or Ser showed that this residue is essential for the
ADP-ribosylation of EF-2, but relatively unimportant for
NAD-glycohydrolysis(46) . On the basis of kinetics measurements
for the two reactions, together with knowledge of the K
for NAD and the stereochemistry of the ADP-ribose-protein
linkage, the ADP-ribosylation and NAD-glycohydrolase reactions are
believed to proceed via a direct SN
displacement
mechanism(15, 49, 50) . In this model, the
nicotinamide group of NAD would be displaced by the incoming
nucleophile, namely the diphthamide moiety of EF-2 for the
ADP-ribosylation reaction or water for NAD-glycohydrolysis. The
dramatic reduction in ADP-ribosylation activity that occurs when
Glu
is mutated may be explained by the carboxyl group of
Glu
serving as a general base that activates the incoming
nucleophile, diphthamide(50) . This mechanism is consistent
with the fact that mutation of Glu
has little effect on
NAD affinity or the NAD-glycohydrolase reaction. In other
ADP-ribosylating toxins, the homologs of Glu
of DT are
assumed to serve similar functions in activating the incoming
nucleophile that serves as the attachment point for ADP-ribose.
For
hPARP we propose that Glu functions in a similar manner
to catalyze elongation of poly-ADP-ribose chains (Fig. 4) by
hydrogen bonding to properly position and to activate, through its
action as a general base, the 2`-OH of the terminal adenosine group of
a nascent poly-ADP-ribose chain. This activated species would carry out
the nucleophilic attack on the nicotinamide-ribose bond of NAD to form
an (ADP-ribose)-(ADP-ribose) glycoside with an
linkage (see
below). Similar catalytic and positioning roles through hydrogen
bonding have been proposed for active site carboxyl groups in other
systems(51, 52, 53) . Consistent with this
prediction, the E988D mutant was the only mutation at position 988 that
retained significant polymer elongation activity. Trace elongation
activity of the E988A mutant might result from the uncovering of
another acidic group near the PARP active site by the smaller alanine
side chain, as observed in the case of an alanine substitution of a
catalytic glutamic acid of ricin, a toxin with endoglycosidase activity (54) .
Figure 4:
Model for
the role of Glu in poly-ADP-ribose synthesis. A,
initiation of poly-ADP-ribose. Glu
facilitates
nucleophilic attack on the nicotinamide-ribose bond by hydrogen bonding
to, and positioning, the substrate (automodification domain) acidic
side chain. B, elongation of poly-ADP-ribose. Glu
catalyzes the nucleophilic attack on the nicotinamide-ribose bond
by activating the 2`-OH of the terminal adenosine of a polymer chain. MOD, PARP automodification domain; CAT, PARP
catalytic domain.
In PARP one would predict a priori that the
precise role of Glu in the initiation reaction is
different from its role in the elongation reaction, given that acidic
side chain(s) on the automodification domain serve as sites of chain
attachment. Glu
could serve in initiation by hydrogen
bonding to position these substrate acidic groups of the
automodification domain in the PARP active site cleft, thereby
facilitating nucleophilic attack by these groups on the
nicotinamide-ribose bond. The substrate carboxyl groups presumably
function as intrinsic nucleophiles, and being predominantly ionized at
neutral pH, they would not require activation by Glu
(Fig. 4). This proposal is consistent with the relatively
small (3-fold) reduction in initiation activity seen in the E988Q
mutant and somewhat larger effects seen with the other Glu
mutants. Glutamine has been shown to activate incoming
nucleophiles such as water via hydrogen bonding in other
instances(50) . Significantly, PARP also hydrolyzes NAD
(NAD-glycohydrolase activity) concomitant with polymer synthesis, which
may reflect a competition between the initiation of new polymer chains
and hydrolysis of NAD by water.
The notion that PARP and the
ADP-ribosylating toxins share a common mechanism of catalysis is
supported by, among other factors, the stereochemistry of the
ADP-ribosylation products. All ADP-ribosylating toxins examined cause
inversion of configuration at the anomeric carbon of the nicotinamide
ribose, from -linkage in NAD to
between the ADP-ribose and
the substrate protein(49) . Similarly, the
(ADP-ribose)-(ADP-ribose) glycosidic bonds made by PARP are
in
configuration(55, 56, 57) . The configuration
of the ester linkage between the initial ADP-ribose of poly-ADP-ribose
and the protein is not known. It has been proposed that an SN
mechanism may account for this inversion in the case of
DT(49) . Although it is possible that an enzyme-catalyzed
inversion of configuration can occur by an SN
mechanism,
there are few examples, presumably because of steric and electronic
considerations regarding the substrate nucleophile and leaving
groups(58) . Additionally, NAD-glycohydrolases, most of which
use an SN
mechanism, are additionally able to catalyze base
exchange and methanolysis of NAD, whereas neither PARP nor the toxins
catalyze either reaction. Finally, poly-ADP-ribose chains are elongated
in a manner that is analogous to mono-ADP-ribosylation by the toxins:
that is, by sequential addition of new ADP-ribose residues to the
protein-distal termini of growing polymer
chains(59, 75) . (
)A proximal growth model
proposed earlier (60) is apparently incorrect.
After this
study was initiated, another model for PARP structure and function was
put forth by Simonin et al.(14, 61) , in
which PARP was proposed to resemble a family of glutamic acid
dehydrogenases, in particular the Clostridium symbosium glutamic acid dehydrogenase, whose crystal structure was recently
determined(62) . The homology reported between PARP and the
glutamate dehydogenases is limited, however, and many gaps were used in
aligning PARP residues with those conserved among dehydrogenases.
Although these authors found that mutations of several residues
conserved between PARP and the dehydrogenases have an effect on the
ADP-ribosylation activity of PARP (14, 61) , a toxin
model for PARP structure can also explain the results of these same
mutations. For instance, 2 of the PARP residues mutated by Simonin et al., Lys and Asp
, are located
in important parts of a toxin-like structure. Lys
is
conserved with the toxins, and mutations of this residue in DT
(Arg
) result in a loss of activity. (
)Likewise,
although Asp
is not conserved with the toxins, mutations
at this position also result in a loss of activity (Trp
of DT; 47). Our finding that an alanine substitution for
His
in hPARP results in a large loss of ADP-ribosylation
activity is consistent with results obtained with substitutions of
His
in DT(63) . Conversely, the dehydrogenase
model does not predict a role for Glu
, which would be on
the outer edge of a
-strand, away from the active site cleft. If
this were the true physical context of Glu
, it would be
more difficult to rationalize a large, and specific, effect on polymer
synthesis of a glutamine replacement.
It should be noted that the
toxins do not use a Rossman fold to bind NAD, and the structure of the C. symbosium glutamate dehydrogenase, or any other
dehydrogenase, does not resemble the structure of the catalytic domain
of either DT or P. aeruginosa exotoxin A. A crystallographic
structure of the PARP catalytic domain will be required to show which
physical model is correct. The presence of Glu in the
active site cleft of PARP would be strong evidence in favor of a
mechanism of ADP-ribosylation similar to that of the ADP-ribosylating
toxins.
If PARP is ultimately shown to have an active site fold similar to that of the ADP-ribosylating toxins, this will raise interesting questions regarding the evolutionary lineages of the various ADP-ribosyltransferases. The ADP-ribosyltransferases are likely to be an ancient class of enzymes, as evidenced by the regulation of nitrogen metabolism by ADP-ribosylation in the bacterium Rhodospirillum rubrum(64) and the poly-ADP-ribosylating activities reported in dinoflagellates (65) and a thermophilic archaebacterium, Sulfobolus sulfataricus(66) . While the ADP-ribosylating toxins may have evolved directly from precursor ADP-ribosyltransferases in bacteria, it has been suggested, on the basis of the strong specificity of diphtheria toxin for the diphthamide residue of elongation factor-2, that such toxins may have originated from the ``capture'' and modification of eukaryotic genes encoding endogenous regulatory proteins(74) . Additional sequence data from eukaryotic and prokaryotic ADP-ribosyltransferases will aid in resolving such questions.