©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Active Site Mutations of Pseudomonas aeruginosa Exotoxin A
ANALYSIS OF THE HIS RESIDUE (*)

(Received for publication, October 3, 1994)

Xiang Y. Han Darrell R. Galloway (§)

From the Department of Microbiology, The Ohio State University, Columbus, Ohio 43210-1292

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Pseudomonas aeruginosa exotoxin A (ETA) is a member of the family of bacterial ADP-ribosylating toxins which use NAD as the ADP-ribose donor. By analogy to diphtheria and pertussis toxins, the His residue of ETA has been proposed to be one of the critical residues within the active site of the toxin. In this study the role of the His residue was explored through site-directed mutagenesis which resulted in the production of ETA proteins containing Ala, Asn, and Phe substitutions at the 440 position. The His-substituted ETA proteins were purified and analyzed. All substitutions at the 440 site displayed severely reduced ADP-ribosylation activity (>1000-fold). However, NAD glycohydrolase activity remained intact and in the case of ETAH440N actually increased 10-fold. NAD binding is not affected by substitutions at the 440 site as indicated by similar K values for the ETA variants tested. Conformational integrity of the mutant toxins appears to be largely unaffected as assessed by analysis with a conformation-sensitive monoclonal antibody as well as sensitivity to proteinase digestion. In view of the location of His residue within or close to the proposed NAD-binding site, these results suggest that His may be a catalytic residue involved in the transfer of the ADP-ribose moiety to the EF-2 substrate.


INTRODUCTION

Pseudomonas aeruginosa exotoxin A (ETA) (^1)and diphtheria toxin (DT) are two well characterized members of a group of bacterial protein toxins known as ADP-ribosyltransferases, based upon their enzymatic mode of action. Both toxins catalyze the transfer of the ADP-ribose moiety of NAD into covalent modification of the eucaryotic protein elongation factor 2 (EF-2). The result is paralysis of the function of the EF-2 protein and the resultant inhibition of protein synthesis in the target cell which the toxin has entered(1, 2) .

The three-dimensional structure of the 613-amino acid ETA protein has been resolved to the 3-Å level(3) . This study and others (3, 4, 5, 6) have led to an assignment of the functional domains within the ETA structure. According to this scheme, residues 405-613 (domain III) and 365-404 (domain Ib) form the catalytic domain responsible for the ADP-ribosyltransferase activity as well as associated NAD glycohydrolase activity. The N-terminal domains Ia (residues 1-252) and II (residues 253-364) are involved in binding to the ETA receptor and for translocation across the endosomal membrane of sensitive cells, respectively.

Recently the three-dimensional structure of diphtheria toxin has been resolved(7) . Although immunological cross-reactivity has not been demonstrated between the intact ETA and DT molecules, considerable sequence homology exists within the enzymatic domains of these two toxins(8, 9) . The NAD-binding site in both ETA and DT have been proposed on the basis of computer modeling studies(9, 10) . Essentially, the NAD site consists of a hydrophobic cavity composed of residues with aromatic side chains, such as Tyr (Tyr, DT), Tyr (Tyr, DT), Trp (Trp, DT), Trp (Trp, DT), and His (His, DT). The hydrophobicity of the pocket favors the binding of the nicotinamide and adenine portions of NAD due to aromatic ring-stacking as well as hydrogen bonding interactions. Two alternative NAD binding arrangements have been proposed. In one arrangement the nicotinamide ring of NAD faces Tyr while the adenine ring faces Trp, or vice versa in the alternative arrangement. The His residue which is positioned at the bottom of the cavity projects its imidazole ring into the cavity and rotates freely in order to facilitate its involvement in hydrogen bonding. Several hydrophilic residues such as Glu (Glu, DT), Glu (Glu, DT), Glu, Asp (Asn, DT), and Asp (Asp, DT) form the edge of the NAD binding cavity and participate in H-bonding with the two ribose moieties of the NAD molecule.

This inspection of the ETA active site residues suggests an important role for the His residue. The importance of His is also implicated by its strict conservation in the active sites of other ADP-ribosylating toxins, e.g. DT (His), pertussis toxin (His), cholera toxin (His), and Escherichia coli heat-labile enterotoxin (His). The role of His in DT has been analyzed recently in two independent studies using site-specific substitution with a variety of amino acids(11, 12) . Results of both studies indicate that His is involved in NAD binding, NAD glycohydrolysis, and ADP-ribosylation of EF-2. Likewise, the analogous His residue of pertussis toxin has been found to be critical for NAD binding and glycohydrolysis(13) .

In order to further define and establish the function of the ETA His residue, we utilized site-directed mutagenesis to insert various amino acids in place of the histidine at position 440. The mutated toxins (ETAH440A, ETAH440N, and ETAH440F) exhibit dramatic reductions of ADP-ribosyltransferase activity as well as a severe reduction of in vitro cytotoxicity. However, NAD glycohydrolase activity appears to be undiminished in the case of the His substitutions studied thus far.


EXPERIMENTAL PROCEDURES

Oligonucleotide-directed Mutagensis and Toxin Production

An overlap extension polymerase chain reaction (PCR) method (14, 15) was used to introduce oligonucleotide primer-directed substitutions at position 440 within the ETA sequence. The wild-type ETA gene (toxA) in plasmid pGW601 (16) served as a template for the PCR process. Two pairs of primers were used to direct the synthesis of two fragments with a CAC (His) to GCA (Ala), TTC (Phe), or AAC (Asn) mutation in each (Table 1). Both fragments overlapped by 17 base pairs and in the presence of two far end primers (primers 1901-1918 and 2400-2381), the PCR process produced a 500-base pair fragment encoding the desired His substitutions (Table 1). A 380-base pair ApaI/XhoI fragment of the 500-base pair PCR product was introduced into pGW601 by substitution with the corresponding wild-type sequence. The resultant plasmids, pXH102 (ETAH440A), pXH105 (ETAH440N), and pXH106 (ETAH440F) were sequenced in order to verify the presence of the correct substitutions (Sequenase version 2.0, U. S. Biochemicals Corp.).



In order to obtain suitable amounts of the altered toxins for study, the toxA gene in each plasmid was subcloned in the following manner for expression in the ToxA-negative P. aeruginosa host strain PA103A which carries an insertionally inactivated toxA gene. (^2)A 1.3-kilobase pair EcoRI/BglII fragment from each plasmid was subsequently cloned into pGW580 (16) by fragment replacement, resulting in plasmids pXH202, pXH205, and pXH206 which carry the toxA gene under control of its own promoter. In each case, the toxA gene was subsequently extracted using a 2.7-kilobase pair EcoRI/HindIII fragment and inserted into pRO1614, a broad-host range plasmid(18) . The resultant plasmids, pXH302 (ETAH440A), pXH305 (ETAH440N), and pXH306 (ETAH440F) were used to transform P. aeruginosa strain PA103A, a non-toxigenic derivative of strain PA103. Transformation of PA103A was carried out according to a previously described procedure (18) and transformants were selected on Pseudomonas Isolation Agar in the presence of 300 µg ml carbenicillin (Sigma). Procedures such as plasmid isolation, endonuclease digestion, transformation of E. coli cells, and agarose gel electrophoresis of DNA fragments or plasmids, were carried out according to the methods described by Sambrook et al.(19) .

Wild-type ETA was purified from P. aeruginosa strain PA103 transformed with a plasmid (pGW28) containing the toxR gene which encodes for a transcriptional activator known to enhance toxin production, as described previously(20) . The point-mutated ETA proteins were purified from the Tox PA103A strain transformed with the appropriate plasmid carrying the mutated toxA gene. All toxins were purified to homogeneity as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Coomassie Blue staining(21) .

Conformation-sensitive ELISA

An enzyme-linked immunosorbent assay format (ELISA) was used to determine conformational changes in the mutant exotoxin proteins. The procedure was carried out at 25 °C according to a published protocol (22) with modifications. Purified monoclonal antibody (TC-1, IgG(2)) which has previously been shown to be sensitive to conformational changes in toxin (23) was coated onto Immulon II plates (Dynatech, Alexandria, VA) in 10 mM phosphate-buffered saline (PBS), pH 7.3, for 20 h. Following washing and blocking with 1% bovine serum albumin, purified exotoxin A in PBS containing 1% bovine serum albumin and 0.1% Tween 20 was added and incubated with the immobilized mAb for 2 h. The exotoxin samples were either pretreated with 8 M urea in the presence of 130 mM dithiothreitol (DTT) in order to activate the toxin prior to incubation with the immobilized mAb or left untreated. Toxin molecules captured by the immobilized mAb were detected following the sequential additions of rabbit anti-toxin followed by goat anti-rabbit IgG-horseradish peroxidase-conjugated antibody. ortho-Phenylenediamine served as the substrate for the horseradish peroxidase conjugate and the resulting absorbance was recorded at 490 nm.

In Vitro Protein Synthesis Assay

The cytotoxicity of wild-type and mutant ETA was determined by measuring the inhibition of protein synthesis using mouse L-929 cells cultured in the presence of trace amounts of [^3H]leucine (ICN Radiochemicals, Irvine, CA), as described previously(20) . 10^5 cells were seeded into 24-well culture plates and incubated overnight in RPMI medium (Whittaker Laboratories, Walkersville, MD) supplemented with 10% fetal calf serum. Following incubation, the cells were washed once in leucine-free medium and then incubated in medium to which toxin had been added. Following a 1-h exposure to toxin, the cells were washed once again with leucine-free medium and incubated for an additional hour in leucine-free RPMI. Finally, [^3H]leucine (0.3 µCi) was added during the last hour of incubation in order to allow [^3H]leucine uptake into the cells and incorporation into newly synthesized protein. In the last step, cells were washed thoroughly and lysed in the presence of 0.1 M NaOH. The cell lysates were collected by filtration and protein-incorporated [^3H]leucine was measured by liquid scintillation counting. Lack of [^3H]leucine incorporation into cellular proteins is indicative of the inhibition of protein synthesis as a result of exposure to functional toxin.

ADP-ribosyltransferase Assay

The ability of ETA to carry out the ADP-ribosyltransferase reaction was evaluated as described originally by Iglewski and Kabat (1) and modified by Galloway et al. (20) using [adenine-^14C]NAD (Amersham Corp., Arlington Heights, IL) and wheat germ EF-2. Briefly, 0.1 µg of wild-type ETA or 1 µg of mutated ETA was diluted to a final volume of 10 µl in either 10 mM PBS, pH 7.3, or in activation buffer consisting of PBS containing 3 M urea, 30 mM DTT. The diluted toxin was then added to 42 µl of assay buffer (80 mM Tris-HCl, pH 8.0, 70 mM DTT, 1 mM EDTA) containing 41 pmol of radiolabeled NAD and approximately 1 nmol of wheat germ EF-2. The reaction mixture was allowed to incubate at a specified temperature between 0 °C (ice bath) and 37 °C for 1 (ETA) or 4 h (mutated ETA). The reaction was stopped by the addition of 100 µl of 10% trichloroacetic acid. The precipitated ADP-ribosebulletEF-2 complex was collected onto Whatman (Whatman Inc.) glass microfilter membranes which were washed with 5% trichloroacetic acid, dried and counted using liquid scintillation methodology.

NAD Glycohydrolase Assay

The NAD glycohydrolase activity of toxin was determined according to a method described by Barbieri et al.(24) . Toxin (1 µg) was diluted in 50 mM Tris-HCl, pH 8.0, buffer containing a final concentration of 3 M urea plus 30 mM DTT (activated) and incubated in a 25-µl reaction mixture containing 80 pmol of [nicotinamide-^3H]NAD (1.26 Ci/mmol), 100 mM Tris-HCl, pH 8.0, 20 mM DTT, and 1 mg/ml egg albumin. The reaction was extended to 4 h at 15 °C and stopped by the addition of 10 µl of 1 M boric acid. Free radioactive nicotinamide was extracted using 250 µl of water-saturated ethyl acetate and counted. Urea/DTT buffer controls were included in order to determine background levels of radioactivity. For steady-state kinetic analysis, serial [^3H]NAD concentrations were used in the assay.

Proteinase Sensitivity Analysis

Native ETA or His-substituted ETA (30 µg, in PBS, pH 7.3) were subjected to limited digestion by trypsin (2 µg, EC 3.4.21.4, Sigma) at 25 °C for varying periods before the reaction was stopped by the addition of soybean trypsin inhibitor (2 µg, Sigma). The resulting digests were evaluated using 15% SDS-PAGE analysis and Coomassie Blue staining.


RESULTS

ADP-ribosyltransferase Analysis

Using the purified wild-type and His-substituted toxins (see Fig. 4) we first evaluated the effect of the His substitutions on the primary enzymatic activity associated with exotoxin A, namely the ability to ADP-ribosylate eucaryotic EF-2. Our initial results indicated a substantial loss in ADP-ribosyltransferase activity associated with substitution at the His site (data not shown). The assay was repeated under conditions of lower temperature (15 °C) and a longer incubation period (4 h) in order to obtain measurable counts for evaluation. We have recently observed that 15 °C is an optimum temperature for this assay. (^3)As shown in Table 2, each of the 440-substituted toxins demonstrated a substantial reduction (>1000-fold) in ADP-ribosyltransferase activity, regardless of whether the toxins had been previously activated or not. These results establish that His occupies a critical position within the active site of the toxin.


Figure 4: Trypsin digestion profiles of purified ETA and His-substituted ETA. Purified toxins (1.5 µg/µl) were digested in the presence of trypsin (0.08 µg/µl) for either 30 or 60 min at 25 °C. The reactions were stopped by the addition of soybean trypsin inhibitor and the resulting digests analyzed using 15% SDS-PAGE and Coomassie Blue staining. Lanes 2, 5, 8, and 11 represent undigested toxin; lanes 3, 6, 9, and 12 represent 30-min digests; and lanes 4, 7, 10, and 13 represent 60-min digests; lanes 14 and 15 represent trypsin (3 µg) and soybean trypsin inhibitor (3 µg), respectively.





An alternative method for the evaluation of ADP-ribosyltransferase activity involves measuring the ability of toxin to inhibit protein synthesis in toxin-sensitive cells. When functional ETA is incorporated into susceptible mouse L-929 fibroblast cells, ADP-ribosylation of EF-2 results in the inhibition of protein synthesis which can be observed by a reduction in [^3H]leucine incorporation into newly synthesized protein. As an example, ETAH440A was analyzed in this fashion and it can be seen by the results presented in Fig. 1that a substantial reduction in the inhibition of protein synthesis occurs, similar to that observed for the His mutant (ETAH426L) as described previously(20) . The His residue has previously been shown to occupy a critical site which may be involved in the interaction with the EF-2 substrate(20, 25) .


Figure 1: Inhibition of protein synthesis in mouse L-929 fibroblasts. Mouse L-929 fibroblast cells (10^5 cells/well/24-well plate) were cultured in the presence of trace amounts of [^3H]leucine in RPMI medium followed by a 1-h exposure to ETA or mutated ETA at the indicated concentrations. The cells were placed in fresh medium to which 0.3 µCi of [^3H]leucine had been added and incubated an additional hour. The cells were harvested, washed repeatedly, and lysed in the presence of 0.1 N NaOH. Lysates were collected by filtration and counted using liquid scintillation methodology. The inset indicates the concentration of ETA that resulted in 50% inhibition of [^3H]leucine incorporation into protein.



NAD Glycohydrolase Activity Analysis

In the absence of EF-2, ETA hydrolyzes the NAD substrate into nicotinamide and ADP-ribose at a slow, but measurable rate(26, 27) . In order to determine whether the 440-substituted mutant toxins contain a functional defect in their ability to interact with NAD or EF-2, or both, the NAD glycohydrolase activities of the wild-type and mutant toxins were compared (Fig. 2). According to published protocols the NAD glycohydrolase activity is generally measured at 37 °C(24, 28) . However, we have observed that 15 °C is the optimal temperature for this assay (data not shown) and therefore our assays have been performed at this temperature. The results of our analysis are shown in Fig. 2and kinetic analysis of the data is presented in Table 3. In comparison with the wild-type toxin, all 440-substituted toxins show similar K(m) values (<2-fold), implying functional NAD binding capability. Furthermore, the K value of the wild-type toxin is similar to those of ETAH440A and ETAH440F. However, K for ETAH440N shows a significant (10-fold) increase. The catalytic efficiency (K/K(m)) for ETAH440N is approximately 5-fold higher than that of the wild-type toxin. These data indicate that while substitutions at site 440 have severely reduced ADP-ribosyltransferase activity, NAD glycohydrolase activity is not adversely affected. Since there is no evidence to suggest that ADP-ribosyltransferase and NAD glycohydrolase activities are catalyzed by separate sites within the ETA structure, it is likely that the inefficient ADP-ribosyltransferase activity associated with these substitutions at the 440 position are caused by defects in the catalytic mechanism or by inefficient interactions with the EF-2 substrate. The increased NAD glycohydrolase activity associated with ETAH440N may be caused by a number of mechanisms, including regional conformational changes within the proposed NAD-binding site. A conformational assessment of the toxins was therefore carried out.


Figure 2: Comparative analysis of the NAD glycohydrolase activities of wild-type and His-substituted ETA. In each case 1 µg of purified ETA was activated by treatment with urea/DTT and then assayed for NAD hydrolysis as described under ``Experimental Procedures.'' Assays were carried out at 15 °C for a 4-h period using the concentrations of [^3H]NAD indicated (10-1800 µM).





Conformational Analysis of ETA

It has previously been shown that ETA is produced in a nonfunctional configuration which must be activated in order to demonstrate ADP-ribosyltransferase activity(29) . We have previously described the use of a monoclonal antibody designated TC-1 which is able to distinguish between the inactive and active configurations of ETA by binding to an epitope within the 419-432 sequence which assumes an alpha helical conformation within domain III of the toxin(23) . The TC-1 mAb binds to the epitope which is apparently exposed when ETA is in the active configuration and fails to bind to the inactive configuration of ETA. Therefore the TC-1 antibody can be used as a probe of unfolding or conformational change of the toxin molecule. In this study mAb TC-1 was used to analyze possible conformational changes resulting from substitutions at the 440 site. Immunoblotting analysis indicated similar binding to the unfolded toxin molecules regardless of substitution at site 440 (data not shown). Using a capture ELISA format we analyzed the ability of the conformation-sensitive TC-1 mAb to bind to both the activated (unfolded) and non-activated forms of wild-type and mutant ETA, as shown in Fig. 3. Our results indicate that all the activated configurations of toxin bind the TC-1 mAb equally well (as supported by the immunoblotting analysis). The non-activated toxins do not bind the TC-1 mAb as readily, as expected. However, non-activated ETAH440F exhibits an intermediate degree of TC-1 binding (Fig. 3), suggesting possible destabilization or conformational alteration of the ETA structure as a result of placement of a phenylalanine residue at position 440. The presence of a bulky aromatic ring contributed by the Phe residue within the 440 site may result in local conformational shifts or destabilization which produces a conformational change at the 419-432 helix.


Figure 3: ELISA-based capture assay for conformational analysis of activated and non-activated ETA and His-substituted ETA. Individual wells of Immulon II (Dynatech, Alexandria, VA) 96-well microtiter plates were coated with purified IgG of the TC-1 mAb (2 µg/ml). Activated and non-activated toxins were added and incubated for 2 h at 25 °C. TC-1-bound toxin was subsequently detected using high-titered rabbit anti-ETA antibody and donkey anti-rabbit IgG-horseradish peroxidase conjugate as described under ``Experimental Procedures.''



In a separate analysis of conformational differences among the 440-substituted toxins we used proteinase sensitivity as an indication of conformational integrity. Our analysis of trypsin sensitivity (Fig. 4) of the modified ETA proteins indicates that the His substitutions do not render ETA more sensitive to trypsin digestion than the wild-type ETA. These results suggest that the substitutions at the 440 site do not result in destabilization of the ETA structure.


DISCUSSION

In this study we have demonstrated that the His residue of ETA plays a critical role in the ADP-ribosylation of elongation factor 2. Previous studies have proposed that His of exotoxin A, His of DT, and His of pertussis toxin are functionally homologous residues within the NAD-binding sites of their respective proteins(8, 9) . Analysis of the three-dimensional structures of ETA and DT reveal that ETA His and DT His occupy essentially identical positions within the active sites of these toxins(3, 7) . Using a site-directed mutagenesis approach in which the ETA His residue is replaced by Ala, Asn, or Phe provides evidence that substitution of the histidine at the 440 position reduces the ADP-ribosyltransferase activity of the toxin more than 1000-fold (Table 2). This result is substantiated by the further observation that a replacement of the histidine at the 440 position substantially reduces the ability of the toxin to inhibit protein synthesis in an in vitro assay (Fig. 1). In view of the apparent analogy of ETA His with His of DT these results are not surprising.

Significantly, and in contrast to the findings recently reported for both DT and PT (11, 12, 13) alteration of the ETA His residue does not appear to eliminate or reduce the NAD glycohydrolase activity associated with the toxin. In fact, substitution of an Asn residue at this position results in an apparent 10-fold increase in the NAD glycohydrolase activity (Fig. 2, Table 3). Analysis of the kinetics of the NAD glycohydrolase reaction indicates that the catalytic efficiency (K/K(m)) of this reaction is similar for both the Ala and Phe substitutions, but increases 5-fold in the case of Asn substitution (Table 3). However, similar K(m) values for both the wild-type and mutant toxins implies that the NAD binding function remains intact (Table 3). These results are supported by fluoresence quenching experiments which indicate that NAD binding in the mutant toxins is unaffected by substitutions at the 440 position (data not shown). The increased NAD glycohydrolase activity associated with the Asn substitution in ETA may result from the effects of the polar side chain of the residue. It seems unlikely that the Asn side chain is directly involved in the formation of hydrogen bonds with NAD during cleavage of the N-glycosidic bond, as appears to be the case in DT, since by analogy with DT substitution with Asn at this position should result in some retention of ADP-ribosyltransferase activity. Similarly, the nonpolar Ala and Phe substitutions in ETA should result in reduced NAD glycohydrolase activities as observed in the case of DT if the same hydrogen bonding model applies. Our findings suggest that ETA His does not appear to follow the model proposed for DT His. Rather, Asn substitution may facilitate NAD glycohydrolysis in some indirect fashion. One possibility is that the polar side chain may allow the ready access of water molecules in order to accept the ADP-ribose moiety following cleavage of the N-glycosidic bond through the involvement of as yet unidentified residues. Another possibility is regional conformational changes which could allow more efficient NAD glycohydrolysis to occur.

Similar site-directed mutagenesis studies have recently been reported for the analysis of the His residue of diphtheria toxin (11, 12) . In these studies it was reported that substitution of His with Arg, Leu, Ala, and Gln significantly reduces both ADP-ribosyltransferase and NAD glycohydrolase activities. The reduction in these activities was shown to be due to reduced NAD binding as revealed by fluoresence quenching analysis. Interestingly, an Asn substitution only reduced the ADP-ribosyltransferase activity 3-fold. This conservation of function may be due to the ability of the amide -nitrogen to form hydrogen bonds. Consequently Blanke et al.(11) proposed that the primary function of His is to form a hydrogen bond with the nicotinamide carboxymide of NAD. This interaction would facilitate formation of the initial DTbulletNAD complex, orient the N-glycosidic bond of NAD for nucleophilic attack by the incoming diphthamide residue of EF-2, and stabilize the transition state intermediate of this reaction. Similar mutagenesis studies of the His residue of pertussis toxin also indicate the involvement of this residue in NAD binding and glycohydrolysis(13) .

However, the results of the present study do not support a direct role for the ETA His residue in NAD binding or hydrolysis. Our results do indicate that His is required for the ADP-ribosyltransferase reaction and therefore suggest that the His residue must either be involved in the interaction with EF-2 or participate in the catalysis of the reaction. It is difficult to measure the interaction with EF-2 directly. However, direct contact of His with the incoming EF-2 substrate is unlikely since His is located on the floor of a hydrophobic cavity within the active site of ETA(3) . Furthermore, a role for the His residue in maintaining an appropriate conformation for interaction with EF-2 seems unlikely in view of the fact that sterically conservative substitutions of Ala and Asn do not maintain ADP-ribosyltransferase activity. In view of these considerations it may be that His plays a catalytic role in the ADP-ribosylation reaction. This hypothesis is supported by the location of the residue within the active site (30) and by its close spatial relationship with Glu, a crucial catalytic residue(27) . Glu was initially identified using an NAD-photolabeling technique (31) and upon further analysis a catalytic function was suggested for this residue(32, 33) . These studies demonstrated that deletion of Glu, or substitutions with Asp or Cys at this site all abolished ADP-ribosyltransferase activity and cytotoxicity, yet produced little or no change in NAD binding or glycohydrolysis. In conjunction with studies of Glu of DT, a functional homologue of Glu, it was found that both the negative charge and the chain length of the carboxyl group of ETA Glu (or DT Glu) are crucial with respect to catalytic function(17, 27) . Therefore it was proposed that the carboxylate serves to deprotonate the incoming diphthamide on EF-2, thereby activating the nitrogen at position 1 of the diphthamide imidazole ring which then imposes a nucleophilic attack on the N-glycosidic bond of NAD in a direct displacement mechanism(30) . In some respects the functional features of Glu appear to be similar to those of His, as demonstrated in the present study. The fact that the Asn substitution severely reduces catalysis of the ADP-ribosyltransferase reaction suggests that it is not the hydrogen bond-forming ability of His, but rather the acid-base properties which may be important for catalysis. The aromatic nature of the His imidazole ring seem less important since substitution of a Phe residue at this position does not maintain function. Although the data presented in this study is not sufficient to propose a detailed catalytic mechanism, we speculate that the acid-base properties of His may contribute to the interaction with the incoming EF-2 diphthamide residue, stabilize the transition state intermediate, and facilitate the transfer of the ADP-ribose moiety to the diphthamide site.

The functional differences between His of ETA and His of DT have encouraged us to search for additional active site residues which may be critical for ADP-ribosyltransferase activity. However, an inspection of the active site structure of ETA failed to indicate potential residues which might serve a function homolgous to the His of DT (data not shown).


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI123429-06 (to D. R. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Microbiology, The Ohio State University, 484 West 12th Ave., Columbus, OH 43210-1292; Tel.: 614-292-3761; Fax: 614-292-8120.

(^1)
The abbreviations used are: ETA, P. aeruginosa exotoxin A; DT, diphtheria toxin; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; EF-2, elongation factor 2; mAb, monoclonal antibody; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NAD, nicotinamide adenine dinucleotide; PBS, phosphate-buffered saline; PCR, polymerase chain reaction.

(^2)
D. J. Wozniak, X.-Y. Han, and D. R. Galloway (1994) submitted for publication.

(^3)
X.-Y. Han, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. Richard Swenson for a critical review of the manuscript and for helpful discussions.


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