(Received for publication, August 31, 1995)
From the
Escherichia coli heat-labile enterotoxin (LT) and the
related cholera toxin exert their effects on eukaryotic cells through
the ADP-ribosylation of guanine nucleotide-binding proteins of the
adenylate cyclase complex. The availability of the crystal structure
for LT has permitted the tentative identification of residues that lie
within or are vicinal to a presumptive NAD-binding
site and thus may play a role in substrate binding or catalysis. Using
a plasmid clone encoding the A subunit of LT, we have introduced
substitutions at such potential active-site residues and analyzed the
enzymatic properties of the resultant mutant analogs. Enzymatic
analyses, employing both transducin and agmatine as acceptor
substrates, revealed that substitutions at serine 61, glutamic acid
110, and glutamic acid 112 resulted in reduction of enzyme activity to
<10% of wild-type levels. Kinetic analyses indicated that alteration
of these sites affected the catalytic rate of the enzyme and had little
or no effect on the binding of either NAD
or agmatine.
Of the mutant analogs analyzed, only glutamic acid 112 appeared to
represent an essential catalytic residue as judged by the relative
effects on k
and k
/K
. The results
provide formal evidence that glutamic acid 112 of the A subunit of LT
represents a functional homolog or analog of catalytic glutamic acid
residues that have been identified in several other bacterial
ADP-ribosylating toxins and that it may play an essential role in
rendering NAD
susceptible to nucleophilic attack by an
incoming acceptor substrate.
Escherichia coli heat-labile enterotoxin (LT) ()is a member of a family of bacterial ADP-ribosylating
toxins that possess an A-B-type structure. LT is structurally very
similar to cholera toxin (CT) and is composed of an enzymatically
active A subunit (M
28,000) and five B subunits
that form a homopentameric B oligomer (M
55,000)
(reviewed in (1) and (2) ). The B pentamer is
responsible for binding of the toxins to gangliosides on the surface of
eukaryotic cells(3) , while the A subunit catalyzes the
transfer of ADP-ribose from NAD
to regulatory guanine
nucleotide-binding proteins (G
) of the adenylate
cyclase complex. ADP-ribosylation of G
results in
constitutive activation of adenylate cyclase, elevation of
intracellular cAMP levels, and, ultimately, the disruption of the
normal electrolytic balance in intestinal cells characteristic of
diarrheal disease (4) .
Apart from their role as important
virulence determinants, both LT and CT have been the subject of
considerable study in recent years because they are also potent
enhancers of mucosal immune
responses(5, 6, 7) . Therefore, there has
been considerable interest in elucidating the structure-function
relationships of LT and CT with respect to enzymatic and toxic
activity. Such efforts have been predicated on the notion that
identification of residues or motifs involved in the
ADP-ribosyltransferase activity will provide suitable targets for the
construction of genetically detoxified analogs for inclusion in
acellular vaccines and will further permit systematic investigation of
precise relationships between enzymatic activity, toxicity, and the
immunopotentiating activity. Several studies, using either random or
site-directed mutagenesis of the LT gene, have identified residues that
appear to be important to the ADP-ribosyltransferase activity of the A
subunit(8, 9, 10, 11, 12, 13) .
Among these residues are included Arg-7, Glu-110, and Glu-112.
Relatively conservative substitutions of these residues (e.g. R7K, E110D, and E112D) result in marked attenuation of
ADP-ribosyltransferase activity and in the formation of holotoxin
molecules that are largely devoid of toxic activity when assayed on
eukaryotic cells in vitro(11, 12) . Either
Glu-110 or Glu-112 of LT likely represents the functional equivalent of
active-site Glu residues that have been identified in other
ADP-ribosylating toxins including diphtheria toxin (DT), Pseudomonas aeruginosa exotoxin A (ETA), and pertussis toxin
(PT)(14, 15, 16) . Specific catalytic roles
for such Glu residues in the enzymatic activities of DT, ETA, and PT
were originally indicated by studies using UV-induced photolabeling
with NAD(17, 18, 19, 20) . The crystal
structures of DT, ETA, and PT have also shown that these glutamic acid
residues reside in grooves or clefts that appear to constitute the
NAD
-binding sites in this class of
toxins(21, 22, 23) . Kinetic analyses of
mutant analogs have demonstrated that such Glu residues possess a
catalytic role in DT, ETA, and
PT(14, 15, 16) . A presumptive
NAD
-binding site has also been identified in the
refined crystal structure of the A subunit of LT, and Arg-7, Glu-110,
and Glu-112 are found to reside within or very near this
site(2, 24) .
Recently, Pizza et al.(11) reported the effects of a number of site-directed
mutations in the A subunit of LT on the toxic and enzymatic activities
of the holotoxin. The mutations were designed to affect residues in or
vicinal to the NAD-binding site identified in the
crystal structure. The results confirmed that substitutions at Arg-7,
Glu-110, and Glu-112 markedly attenuate enzymatic and toxic activities.
A model was proposed in which Arg-7 is directly involved in
NAD
binding, while Glu-112 is involved in binding to
and/or orienting the incoming acceptor substrate for subsequent
catalysis. However, the mechanistic basis for this model has not been
investigated by kinetic analyses or other means. Using a rationale
based on primary and secondary structural comparisons to other toxins
and analysis of the proposed NAD
-binding site in the
crystal structure, we have constructed and analyzed the enzymatic
properties of various mutant analogs of the A subunit of LT. The
results provide evidence that Glu-112 has a catalytic role in
ADP-ribosyltransferase activity and is likely involved in the
hydrolysis of the glycosidic linkage between the ribose and
nicotinamide groups of NAD
.
Initial rate data for the
NAD:agmatine ADP-ribosyltransferase activity were
collected under conditions where either the NAD
or
agmatine concentration was held constant at saturating conditions and
the concentration of the other substrate was varied from
0.1 to
2.0 estimated K
. Kinetic parameters were
determined using Lineweaver-Burk plots of the initial velocity data.
Kinetic analyses of mutant analogs exhibiting very low levels of
activity were facilitated by simultaneously increasing the amount of
subunit analyzed, the time of the assay, and the specific radioactivity
of the NAD
employed as substrate all by
3-4-fold. Similar alterations in assay conditions were used to
measure the rates of NAD
glycohydrolysis in the
absence of acceptor substrate.
The mutagenic substitutions were introduced into the coding sequence for the protein designated rLTA, which possesses a 9-amino acid amino-terminal fusion peptide. We have previously shown that the presence of this peptide has little or no influence on enzymatic activity as judged by comparison to the activity exhibited by a purified recombinant subunit that lacks the fusion peptide(13) .
Figure 1: Purification of rLTA and mutant analogs. The genes encoding rLTA and the indicated mutant analogs were expressed in E. coli BL21(DE3), and the recombinant proteins were extracted and purified on Mono-Q HR 5/5 columns as described under ``Experimental Procedures.'' Aliquots containing 7.5 µg of protein were then subjected to SDS-PAGE using 12.5% resolving gels and stained with Coomassie Brilliant Blue R-250. The positions of the molecular mass standards are shown on the left and are given in kilodaltons.
Figure 2: Effect of limited trypsinolysis on rLTA and mutant analogs. The indicated recombinant A subunit preparations were solubilized in 50 mM Tris-HCl, pH 7.5, and incubated without(-) or with (+) trypsin at a trypsin/A subunit ratio of 1:100 (w/w) for 30 min at 30 °C. The reactions were terminated by the addition of an equal volume of double strength electrophoresis treatment buffer (30) and heating at 95 °C for 5 min. Aliquots containing 7.5 µg of protein were then electrophoresed on 12.5% resolving gels and electroblotted to polyvinylidene fluoride membranes. The A and A1 peptides were then detected using anti-A subunit antibodies as described under ``Experimental Procedures.'' The positions of the molecular mass standards are shown on the left and are given in kilodaltons.
Figure 3:
ADP-ribosyltransferase activity of rLTA
and mutant analogs. Purified rLTA and mutant analogs were incubated
with bovine ROS membranes,
[adenylate-P]NAD
and
other additions as described(12, 13) . The indicated
recombinant subunits were assayed at a final concentration of 25
µg/ml for 2 h at 30 °C. The labeled reaction products were then
separated by SDS-PAGE using 12.5% resolving gels. The gels were stained
with Coomassie Brilliant Blue R-250 and then dried onto Whatman No. 3MM
filter paper prior to autoradiography using Kodak X-Omat radiographic
film. The positions of the molecular mass standards are shown on the
left and are given in kilodaltons. The position of the
-subunit of
transducin (T
) is also shown. The autoradiographs shown
were obtained after 2.5-h exposures.
To provide
a more quantitative estimate of the relative activity of the mutants,
they were assayed for their ability to ADP-ribosylate a small guanidino
compound, agmatine, in the presence of rARF-I (Table 1). The
results generally confirmed the findings using ROS membranes and
further revealed that some of the mutants, notably rLTA/R54G,
rLTA/R54K, and rLTA/H70N, exhibited enhanced specific
NAD:agmatine ADP-ribosyltransferase activity. Initial
rate kinetic measurements using variable NAD
concentrations indicated that, in each of the cases in which
enhanced activity was observed, the primary effect was on k
(data not shown). The only mutant analogs that
appeared to possess significantly reduced enzymatic activity were
rLTA/S61T, rLTA/E110D, and rLTA/E112D. All three of these mutant
analogs exhibited <10% of the wild-type level of
NAD
:agmatine ADP-ribosyltransferase activity.
Several studies have now been published that have attempted to identify residues that are important to the ADP-ribosyltransferase activity or toxicity of LT by mutagenesis. Using random mutagenesis, Tsuji et al.(8, 9) and Harford et al.(10) reported that substitutions at Glu-112 (with Lys) and Ser-61 (with Phe), respectively, could abrogate toxicity. We have previously reported that crude extracts containing recombinant A subunit mutant analogs possessing substitutions at Arg-7, Glu-110, and Glu-112 exhibited markedly reduced ADP-ribosyltransferase activity(12) , and more recently, Pizza et al.(11) have confirmed these findings by examining the toxicity of holotoxins containing alterations at these and other positions. While substitution of Glu-110 and/or Glu-112 has been shown to attenuate enzymatic or toxic activity in several experimental systems, the mechanistic bases for the reductions using kinetic or other biophysical methods have not been investigated. We therefore sought to characterize in detail the enzymatic characteristics associated with alterations at residues that have been predicted by crystallographic analysis to be potentially important to the enzymatic activity of LT. Since ARF is known to enhance both the catalytic rate and substrate affinity of CT and, by inference, LT(35) , the availability of rARF-I in purified form (25) has permitted more detailed and accurate quantitative enzymatic analyses than had been performed in prior studies of mutant analogs(9, 11, 12) .
The enzymatically
active fragments of DT, ETA, and PT all contain a catalytically
important Glu residue, and in all three cases, the essential Glu was
initially shown to be at or near the NAD-binding site
by direct photolabeling with radiolabeled
NAD
(17, 18, 19, 20) .
A role in catalysis was established by kinetic analyses of mutant
analogs(14, 15, 16) . We have not yet been
able to efficiently photolabel a specific residue in the A subunit of
LT using unmodified NAD
and ultraviolet light,
presumably owing to the low affinity of the enzyme for NAD
(millimolar range) under presently used conditions. As noted,
previous studies showed that substitution of both Glu-110 and Glu-112
in the A subunit resulted in marked reductions in enzymatic
activity(11, 12) . The results of our kinetic analyses
strongly support a catalytic role for Glu-112 in the mechanism of the
ADP-ribosyltransferase reaction and indicate that Glu-110 is unlikely
to play a specific role in the reaction mechanism. Substitution of
Glu-112 with Asp results in a marked reduction of k
(by a factor of 100), with little or no effect on the K
for NAD
or agmatine. These
results are similar to those of Wilson et al.(14) using the enzymatically active A fragment of DT. In
the case of the DT A fragment, the K
value for
neither NAD
nor elongation factor 2 was affected by
substituting Asp for Glu-148, but the k
of the
reaction was reduced by a factor of 100. However, the NAD
glycohydrolase reaction catalyzed by the DT A fragment in the
absence of acceptor substrate was relatively unaffected by
substitutions at Glu-148. Coupled with the observation that the
glycohydrolase reaction catalyzed by DT proceeds at a rate that is
3 orders of magnitude less than the rate of ADP-ribosylation of
elongation factor 2, this finding has been interpreted to indicate that
the NAD
glycohydrolase reaction may occur through a
different mechanism than that of ADP-ribosylation of elongation factor
2 and perhaps involves strain or distortion effects on the
NAD
molecule that result in scission of the N-glycosidic bond(14) . Based on these findings,
Wilson et al.(14) proposed that the carboxyl group of
Glu-148 participates in catalysis both by acting as a general base in
the abstraction of a proton from the incoming diphthamide residue in a
displacement reaction and by maintaining the geometry of the active
site through hydrogen bonding interactions.
In contrast to DT, the
catalytic rate of NAD glycohydrolysis catalyzed by the
S1 subunit of PT is only 10-fold less than that of the ADP-ribosylation
of the
-subunit of transducin. Substitution of Glu-129 with Asp
results in a marked (by a factor of >200) decrease in NAD
glycohydrolase activity (16) . These findings have been
interpreted to indicate a catalytic role for Glu-129 in the
glycohydrolase reaction catalyzed by the S1 subunit, perhaps acting as
a general base in the stabilization of a developing oxycarbonium-like
intermediate in an S
2-type mechanism as proposed
by Locht and colleagues(16, 36) . Our finding that the
catalytic rate of the NAD
glycohydrolase activity of
the A subunit of LT is only 10-20-fold less than that of the
NAD
:agmatine ADP-ribosyltransferase activity suggests
that the reaction mechanism of LT is similar to that associated with
the S1 subunit of PT. The finding that substitution of Glu-112 also
results in a marked decrease in NAD
glycohydrolase
activity suggests that this residue and glutamic acid 129 of the S1
subunit have similar roles in the reaction mechanism. However, the
observation that the NAD
glycohydrolase activity of
the A subunit is less affected (by 3-fold) when compared with the
NAD
:agmatine ADP-ribosyltransferase activity by
substitution of Glu-112 might indicate a contribution of alternative
mechanisms, like that cited above for DT(14) , in the
hydrolysis of NAD
.
The similarity between the
enzymatic mechanisms of the A subunit of LT and the S1 subunit of PT is
also supported by the effects of mutations on other residues that
appear to be conserved among the two toxins. The active subunits of LT,
PT, and the mosquitocidal toxin from Bacillus sphaericus(37) can be aligned to reveal the apparent conservation of
several residues that have been shown by mutagenesis studies to be
important for the retention of enzymatic activity(38) . In the
A subunit of LT, these include His-44, Ser-61, and Glu-112. Various
substitutions at the positionally equivalent histidine (His-35) in the
S1 subunit of PT markedly reduce the k in both
the NAD
glycohydrolase and ADP-ribosyltransferase
reactions and have little or no effect on the K
for either NAD
or acceptor
substrates(38, 39) . We have found that substitution
of His-44 with Arg, Gln, or Asn in the A subunit of LT results in
substantial loss of activity when the mutant subunits are assayed in
unpurified or crude form; however, these results must be interpreted
with caution since these mutants also exhibit enhanced sensitivity to
limited trypsinolysis. (
)Accordingly, the potential role of
His-44 will await development of alternative purification schemes and
detailed kinetic analyses. Our results do, however, appear to formally
exclude any important role for His-70 in the enzymatic mechanism since
none of the three substitutions at this position decreased activity.
The current analyses also suggest that Ser-61 of the LT A subunit does
not play an essential role in the catalytic mechanism per se since the k
and k
/K
values for the
NAD
:agmatine ADP-ribosyltransferase reaction catalyzed
by rLTA/S61T were only reduced by a factor of 10, and the K
values for NAD
and agmatine
were relatively unaffected. A similar finding has been made with
respect to the role of the positionally equivalent Ser residue (Ser-52)
in the NAD
glycohydrolase reaction catalyzed by the S1
subunit of PT(16) . Furthermore, we have recently isolated a
mutant analog containing an Ala substitution at this position and have
found that it retains
10% of the wild-type
NAD
:agmatine ADP-ribosyltransferase activity. (
)Therefore, Ser-61 likely plays a role in maintaining the
overall geometry of the active site as this residue is observed to
participate in hydrogen bonding to Arg-9 in the crystal
structure(24) .
Domenighini et al.(40) have proposed a tentative model of the enzymatic
mechanism of LT based on the crystal structure of LT. In this model,
Arg-7 is proposed to participate directly in the binding of
NAD at the active site. The role of Arg-7 in binding
is supported by the observation that alteration of the positionally
equivalent residue in the S1 subunit of PT (Arg-9) results in
abrogation of UV-induced photolabeling with NAD
,
supporting the presumption that Arg-7 of the LT A subunit is involved
in the productive binding of NAD
rather than in
catalysis per se(41) . Mutagenic substitution of
His-21 of DT, which is positionally equivalent to Arg-7 of LT in the
two crystal structures, results in marked increases in the K
for NAD
, with little or no
effect on k
(42, 43) . Although
we have not been able to directly examine Arg-7 mutant analogs by
photolabeling or other means, we favor the interpretation that Arg-7,
like His-21 of DT, participates directly in NAD
binding. However, it should be noted that substitution of His-21
of DT with Arg reduces the affinity for NAD
substantially, suggesting potential mechanistic differences in
the roles of the positionally equivalent His and Arg residues in the
two toxins(42, 43) . In addition, substitution of
Arg-7 with Lys results in conformational perturbation of the A subunit
as judged by alteration of trypsin sensitivity and ability to support
complete assembly of the holotoxin (11, 12) , and
unlike Pizza et al.(11) , we have been unable to
isolate intact holotoxin molecules containing the Arg-7 to Lys
substitution. (
)Domenighini et al.(40) also proposed that Glu-112 of the A subunit of LT,
based on its position within the NAD
-binding cavity,
is involved in the interaction with the incoming acceptor substrate,
either through binding or by stabilizing the formation of a
nucleophilic transition state, similar to one proposed role for Glu-148
of DT(14) . As noted above, our results support the notion that
Glu-112 is a catalytic residue that likely participates in the
formation of a transitional form (oxycarbonium-like intermediate) of
NAD
that is capable of reacting with an incoming
nucleophile.
An important caveat concerning the identification of
potential active-site residues resides in the fact that the published
crystal structure of LT is that of a molecule that is essentially
enzymatically inactive (not proteolytically nicked, unreduced, without
bound ARF), although a more recent analysis indicates that the A
subunit of trypsin-cleaved LT has essentially the same structure of the
untreated molecule(44) . Therefore, the precise location and
orientation of various active-site amino acids in the enzymatically
active conformation are not likely to be accurately reflected in the
current structure. A more precise and informative picture of the
geometry of the active site will likely be revealed by the crystal
structure of the isolated A subunit since, as shown here and
elsewhere(13) , the A subunit does not require proteolysis for
expression of activity, and it can be maintained in an active
conformation by reduction and alkylation (35) . The specific
activities and kinetic parameters we have obtained for rLTA in the
NAD:agmatine ADP-ribosyltransferase reaction compare
favorably with those reported for LT, CT, and the conventionally
isolated CT A subunit(13, 35, 45) .
Accordingly, efforts to crystallize the purified recombinant A subunit
in reduced form are currently underway. Such efforts may also permit
characterization of the interaction of ARF with the A subunit since
expression of the ARF-binding site in LT requires prior activation by
proteolysis and reduction(46) .
W. C. dedicates this work to Dr. Eric Humphries, a valued colleague and friend, who most regrettably passed away.