Characterization of the enzymatic component of Clostridium perfringens iota-toxin

The iota(a) component (i(a)) of Clostridium perfringens ADP ribosylates nonmuscle beta/gamma actin and skeletal muscle alpha-actin. Replacement of Arg-295 in i(a) with alanine led to a complete loss of NAD(+)-glycohydrolase (NADase) and ADP-ribosyltransferase (ARTase); that of the residue with lysine caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them. Substitution of alanine for Glu-378 and Glu-380 caused a complete loss of NADase and ARTase. However, exchange of Glu-378 to aspartic acid or glutamine resulted in little effect on NADase activity but a drastic reduction in ARTase activity (<0.1% of the wild-type activity). Exchange of Glu-380 to aspartic acid caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them; that of the residue to glutamine caused a complete loss of ARTase activity. Replacement of Ser-338 with alanine resulted in 0.7 to 2.3% wild-type activities, and that of Ser-340 and Thr-339 caused a reduction in these activities of 5 to 30% wild-type activities. The kinetic analysis showed that Arg-295 and Ser-338 also play an important role in the binding of NAD(+) to i(a), that Arg-295, Glu-380, and Ser-338 play a crucial role in the catalytic rate of NADase activity, and that these three amino acid residues and Glu-378 are essential for ARTase activity. The effect of amino acid replacement in i(a) on ARTase activity was similar to that on lethal and cytotoxic activities, suggesting that lethal and cytotoxic activities in i(a) are dependent on ARTase activity.     

Clostridium perfringens iota toxin. Mapping of the Ia domain involved in docking with Ib and cellular internalization

Clostridium perfringens iota toxin consists of two unlinked proteins. The binding component (Ib) is required to internalize into cells an enzymatic component (Ia) that ADP-ribosylates G-actin. To characterize the Ia domain that interacts with Ib, fusion proteins were constructed between the C. botulinum C3 enzyme, which ADP-ribosylates Rho, and various truncated versions of Ia. These chimeric molecules retained the wild type ADP-ribosyltransferase activity specific for Rho and were recognized by antibodies against C3 enzyme and Ia. Internalization of each chimera into Vero cells was assessed by measuring the disorganization of the actin cytoskeleton and intracellular ADP-ribosylation of Rho. Fusion proteins containing C3 linked to the C terminus of Ia were transported most efficiently into cells like wild type Ia in an Ib-dependent manner that was blocked by bafilomycin A1. The minimal Ia fragment that promoted translocation of Ia-C3 chimeras into cells consisted of 128 central residues (129-257). These findings revealed that iota toxin is a suitable system for mediating the entry of heterologous proteins such as C3 into cells.     

Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia.     

Structure-function analysis of the Rho-ADP-ribosylating exoenzyme C3stau2 from Staphylococcus aureus

Exoenzyme C3stau2 from Staphylococcus aureus is a new member of the family of C3-like ADP-ribosyltransferases that ADP-ribosylates RhoA, -B, and -C. Additionally, it modifies RhoE and Rnd3. Here we report on studies of the structure-function relationship of recombinant C3stau2 by site-directed mutagenesis. Exchange of Glu(180) with leucine caused a complete loss of both ADP-ribosyltransferase and NAD glycohydrolase activity. By contrast, exchange of the glutamine residue two positions upstream (Gln(178)) with lysine blocked ADP-ribosyltransferase activity without major changes in NAD glycohydrolase activity. NAD and substrate binding of this mutant protein was comparable to that of the recombinant wild type. Exchange of amino acid Tyr(175), which is part of the recently described "ADP-ribosylating toxin turn-turn" (ARTT) motif [Han, S., Arvai, A. S., Clancy, S. B., and Tainer, J. A. (2001) J. Mol.Biol. 305, 95-107], with alanine, lysine, or threonine caused a loss of or a decrease in ADP-ribosyltransferase activity but an increase in NAD glycohydrolase activity. Recombinant C3stau2 Tyr175Ala and Tyr175Lys were not precipitated by matrix-bound Rho, supporting a role of Tyr(175) in protein substrate recognition. Exchange of Arg(48) and/or Arg(85) resulted in a 100-fold reduced transferase activity, while the recombinant C3stau2 double mutant R48K/R85K was totally inactive. The data indicate that amino acid residues Arg(48), Arg(85), Tyr(175), Gln(178), and Glu(180) are essential for ADP-ribosyltransferase activity of recombinant C3stau2 and support the role of the ARTT motif in substrate recognition of RhoA by C3-like ADP-ribosyltransferases.     

Site-directed mutagenesis of active site residues of phosphite dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the unusual oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH. PTDH shares significant amino acid sequence similarity with D-hydroxy acid dehydrogenases (DHs), including strongly conserved catalytic residues His292, Glu266, and Arg237. Site-directed mutagenesis studies corroborate the essential role of His292 as all mutants of this residue were completely inactive. Histidine-selective inactivation studies with diethyl pyrocarbonate provide further evidence regarding the importance of His292. This residue is most likely the active site base that deprotonates the water nucleophile. Kinetic analysis of mutants in which Arg237 was changed to Leu, Lys, His, and Gln revealed that Arg237 is involved in substrate binding. These results agree with the typical role of this residue in D-hydroxy acid DHs. However, Glu266 does not play the typical role of increasing the pK(a) of His292 to enhance substrate binding and catalysis as the Glu266Gln mutant displayed an increased k(cat) and unchanged pH-rate profile compared to those of wild-type PTDH. The role of Glu266 is likely the positioning of His292 and Arg237 with which it forms hydrogen bonds in a homology model. Homology modeling suggests that Lys76 may also be involved in substrate binding, and this postulate is supported by mutagenesis studies. All mutants of Lys76 display reduced activity with large effects on the K(m) for phosphite, and Lys76Cys could be chemically rescued by alkylation with 2-bromoethylamine. Whereas a positively charged residue is absolutely essential for activity at the position of Arg237, Lys76 mutants that lacked a positively charged side chain still had activity, indicating that it is less important for binding and catalysis. These results highlight the versatility of nature's catalytic scaffolds, as a common framework with modest changes allows PTDH to catalyze its unusual nucleophilic displacement reaction and d-hydroxy acid DHs to oxidize alcohols to ketones.     

Functional comparison of the NAD binding cleft of ADP-ribosylating toxins

Although a common core structure forms the active site of ADP-ribosylating (ADPRT) toxins, the limited-sequence homology within this region suggests that different mechanisms are being used by toxins to perform their shared function. To explain differences in their mechanisms of NAD binding and hydrolysis, the functional interrelationship of residues predicted to perform similar functions in the beta3-strand of the NAD binding cleft of different ADPRT toxins was compared. Replacing Tyr54 in the A-subunit of diphtheria toxin (DTA) with a serine, its functional homologue in cholera toxin (CT), resulted in the loss of catalytic function but not NAD binding. The catalytic role of the aromatic portion of Tyr54 in the ADPRT reaction was confirmed by the ability of a Tyr54-to-phenylalanine DTA mutant to retain ADPRT activity. In reciprocal studies, positioning a tyrosine in the beta3-strand of the A1-subunit of CT (CTA1) caused both loss of function and altered structure. The restricted flexibility of the CTA1 active site relative to function became evident upon the loss of ADPRT activity when a conservative Val60-to-leucine mutation was performed. We conclude from our studies that DT and CT maintain a similar mechanism of NAD binding but differ in their mechanisms of NAD hydrolysis. The aromatic moiety at position 54 in DT is integral to NAD hydrolysis, while NAD hydrolysis in CT appears highly dependent on the precise positioning of specific residues within the beta3-strand of the active-site cleft.     

Homology modeling and molecular dynamics studies of a novel C3-like ADP-ribosyltransferase

The novel C3-like ADP-ribosyltransferase is produced by a Staphylococcus aureus strain that especially ADP-ribosylates RhoE/Rnd3 subtype proteins, and its three-dimensional (3D) structure has not known. In order to understand the catalytic mechanism, the 3D structure of the protein is built by using homology modeling based on the known crystal structure of exoenzyme C3 from Clostridium botulinum (1G24). Then the model structure is further refined by energy minimization and molecular dynamics methods. The putative nicotinamide adenine dinucleotide (NAD(+))-binding pocket of exoenzyme C3(Stau) is determined by Binding-Site Search module. The NAD(+)-enzyme complex is developed by molecular dynamics simulation and the key residues involved in the combination of enzyme binding to the ligand-NAD(+) are determined, which is helpful to guide the experimental realization and the new mutant designs as well. Our results indicated that the key binding-site residues of Arg48, Glu180, Ser138, Asn134, Arg85, and Gln179 play an important role in the catalysis of exoenzyme C3(Stau), which is in consistent with experimental observation.     

The nature and character of the transition state for the ADP-ribosyltransferase reaction

Exotoxin A (ExoA) from Pseudomonas aeruginosa is an important virulence factor that belongs to a class of exotoxins that are secreted by pathogenic bacteria which cause human diseases such as cholera, diphtheria, pneumonia and whooping cough. We present the first crystal structures, to our knowledge, of ExoA in complex with elongation factor 2 (eEF2) and intact NAD(+), which indicate a direct role of two active-site loops in ExoA during the catalytic cycle. One loop moves to form a solvent cover for the active site of the enzyme and reaches towards the target residue (diphthamide) in eEF2 forming an important hydrogen bond. The NAD(+) substrate adopts a conformation remarkably different from that of the NAD(+) analogue, betaTAD, observed in previous structures, and fails to trigger any loop movements. Mutational studies of the two loops in the toxin identify several residues important for catalytic activity, in particular Glu 546 and Arg 551, clearly supporting the new complex structures. On the basis of these data, we propose a transition-state model for the toxin-catalysed reaction.     

Computer modelling of the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of NAD binding

Five ADP-ribosylating bacterial toxins, pertussis toxin, cholera toxin, diphtheria toxin, Escherichia LT toxin and Pseudomonas exotoxin A, show significant homology in selected segments of their sequence. Site-directed mutagenesis and chemical modification of residues within these regions cause loss of catalytic activity and of NAD binding. On the basis of these results and of molecular modelling based on the three-dimensional structure of exotoxin A, the geometry of an NAD binding site common to all the toxins is deduced and described in the paper. For diphtheria toxin, sequence similarity with exotoxin A is such that its preliminary structure can be computed by molecular modelling, whereas for the other toxins similarity appears to be restricted to the NAD binding site. Moreover, an analysis of molecular fitting of the NAD molecule into its binding cavity suggests a new model for the conformation of the bound NAD that better accounts for all available experimental information.     

Rho-specific Bacillus cereus ADP-ribosyltransferase C3cer cloning and characterization

C3-like ADP-ribosyltransferases represent an expanding family of related exoenzymes, which are produced by Clostridia and various Staphylococcus aureus strains. Here we report on the cloning and biochemical characterization of an ADP-ribosyltransferase from Bacillus cereus strain 2339. The transferase encompasses 219 amino acids; it has a predicted mass of 25.2 kDa and a theoretical isoelectric point of 9.3. To indicate the relationship to the family of C3-like ADP-ribosyltransferases, we termed the enzyme C3cer. The amino acid sequence of C3cer is 30 to 40% identical to other C3-like exoenzymes. By site-directed mutagenesis, Arg(59), Arg(97), Tyr(151), Arg(155), Thr(178), Tyr(180), Gln(183), and Glu(185) of recombinant C3cer were identified as pivotal residues of enzyme activity and/or protein substrate recognition. Precipitation experiments with immobilized RhoA revealed that C3cerTyr(180), which is located in the so-called "ADP-ribosylating toxin turn-turn" (ARTT) motif, plays a major role in the recognition of RhoA. Like other C3-like exoenzymes, C3cer ADP-ribosylates preferentially RhoA and RhoB and to a much lesser extent RhoC. Because the cellular accessibility of recombinant C3cer is low, a fusion toxin (C2IN-C3cer), consisting of the N-terminal 225 amino acid residues of the enzyme component of C2 toxin from Clostridium botulinum and C3cer was used to study the cytotoxic effects of the transferase. This fusion toxin caused rounding up of Vero cells comparable to the effects of Rho-inactivating toxins.     

Structural and enzymatic analysis of soybean beta-amylase mutants with increased pH optimum

Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted. These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.     

The involvement of Arg265 of mouse ribonucleotide reductase R2 protein in proton transfer and catalysis

Ribonucleotide reductase class I enzymes consist of two non-identical subunits, R1 and R2, the latter containing a diiron carboxylate center and a stable tyrosyl radical (Tyr*), both essential for catalysis. Catalysis is known to involve highly conserved amino acid residues covering a range of approximately 35 A and a concerted mechanism involving long range electron transfer, probably coupled to proton transfer. A number of residues involved in electron transfer in both the R1 and R2 proteins have been identified, but no direct model has been presented regarding the proton transfer side of the process. Arg265 is conserved in all known sequences of class Ia R2. In this study we have used site-directed mutagenesis to gain insight into the role of this residue, which lies close to the catalytically essential Asp266 and Trp103. Mutants to Arg265 included replacement by Ala, Glu, Gln, and Tyr. All mutants of Arg265 were found to have no or low catalytic activity with the exception of Arg265 to Glu, which shows approximately 40% of the activity of native R2. We also found that the Arg mutants were capable of stable tyrosyl radical generation, with similar kinetics of radical formation and R1 binding as native R2. Our results, supported by molecular modeling, strongly suggest that Arg265 is involved in the proton-coupled electron transfer pathway and may act as a proton mediator during catalysis.     

Roles of Glu 349 and Asp 352 in membrane insertion and translocation by diphtheria toxin.

Acidic conditions within the endosomal lumen induce the T domain of receptor-bound diphtheria toxin (DT) to insert into the endosomal membrane and mediate translocation of the toxin's catalytic domain to the cytosol. A conformational rearrangement in the toxin occurring near pH5 allows a buried apolar helical hairpin of the native T domain (helices TH8 and TH9) to undergo membrane insertion. If the inserted hairpin spans the bilayer, as hypothesized, then the two acidic residues within the TL5 interhelical loop, Glu 349 and Asp 352, should become exposed at the neutral cytosolic face of the membrane and reionize. To investigate the roles of these residues in toxin action, we characterized mutant toxins in which one or both acidic residues had been replaced with nonionizable ones. Each of two double mutants examined showed a several-fold reduction in cytotoxicity in 24-h Vero cell assays (sixfold for E349A + D352A and fourfold for E349Q + D352N), whereas the individual E349Q and D352N mutations caused smaller reductions in toxicity. The single and double mutations also attenuated the toxin's ability to permeabilize Vero cells to Rb+ at low pH and decreased channel formation by the toxin in artificial planar bilayers. Neither of the double mutations affected the pH-dependence profile of the toxin's conformational rearrangement in solution, as measured by binding of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 6-sulfonate. The results demonstrate that, although there is no absolute requirement for an acidic residue within the TL5 loop for toxicity, Glu 349 and Asp 352 do significantly enhance the biological activity of the protein. The data are consistent with a model in which ionization of these residues at the cytosolic face of the endosomal membrane stabilizes the TH8/TH9 hairpin in a transmembrane configuration, thereby facilitating channel formation and translocation of the toxin's catalytic chain.     

Identification of domain-domain docking sites within Clostridium symbiosum pyruvate phosphate dikinase by amino acid replacement

Potential domain-domain docking residues, identified from the x-ray structure of the Clostridium symbiosum apoPPDK, were replaced by site-directed mutagenesis. The steady-state and transient kinetic properties of the mutant enzymes were determined as a way of evaluating docking efficiency. PPDK mutants, in which one of two stringently conserved docking residues located on the N-terminal domain (Arg(219) and Glu(271)) was substituted, displayed largely unimpeded catalysis of the phosphoenolpyruvate partial reaction at the C-terminal domain, but significantly impaired catalysis (>10(4)) of the ATP pyrophosphorylation of His(455) at the N-terminal domain. In contrast, alanine mutants of two potential docking residues located on the N-terminal domain (Ser(262) and Lys(149)), which are not conserved among the PPDKs, exhibited essentially normal catalytic turnover. Arg(219) and Glu(271) were thus proposed to play an important role in guiding the central domain and, hence, the catalytic His(455) into position for catalysis. Substitution of central domain residues Glu(434)/Glu(437) and Thr(453), the respective docking partners of Arg(219) and Glu(271), resulted in mutants impaired in catalysis at the ATP active site. The x-ray crystal structure of the apo-T453A PPDK mutant was determined to test for possible misalignment of residues at the N-terminal domain-central domain interface that might result from loss of the Thr(453)-Glu(271) binding interaction. With the exception of the mutation site, the structure of T453A PPDK was found to be identical to that of the wild-type enzyme. It is hypothesized that the two Glu(271) interfacial binding sites that remain in the T453A PPDK mutant, Thr(453) backbone NH and Met(452) backbone NH, are sufficient to stabilize the native conformation as observed in the crystalline state but may be less effective in populating the reactive conformation in solution.     

Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design

Homology modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase (PTDH) as the principal determinants of nicotinamide cofactor (NAD(+) and NADP(+)) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both separately and in combination resulted in PTDH mutants with relaxed cofactor specificity. All three mutants exhibited significantly better catalytic efficiency for both cofactors, with the best kinetic parameters displayed by the double mutant, which had a 3.6-fold higher catalytic efficiency for NAD(+) and a 1000-fold higher efficiency for NADP(+). The cofactor specificity was changed from 100-fold in favor of NAD(+) for the wild-type enzyme to 3-fold in favor of NADP(+) for the double mutant. Isoelectric focusing of the proteins in a nondenaturing gel showed that the replacement with more basic residues indeed changed the effective pI of the protein. HPLC analysis of the enzymatic products of the double mutant verified that the reaction proceeded to completion using either substrate and produced only the corresponding reduced cofactor and phosphate. Thermal inactivation studies showed that the double mutant was protected from thermal inactivation by both cofactors, while the wild-type enzyme was protected by only NAD(+). The combined results provide clear evidence that Glu175 and Ala176 are both critical for nicotinamide cofactor specificity. The rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.     

Transcytosis of iota-toxin across polarized CaCo-2 cells

Iota-toxin from Clostridium perfringens type E is a binary toxin consisting of two independent proteins, an enzymatic Ia and binding Ib component. Ia catalyses ADP-ribosylation of actin monomers, thus disrupting the actin cytoskeleton. In this report, we show that Ia plus Ib applied apically or basolaterally induce a rapid decrease in the transepithelial resistance (TER) of CaCo-2 cell monolayers and disorganization of actin filaments as well as the tight and adherens junctions. Ib alone, on the apical or basolateral side, slowly decreased the TER without affecting the actin cytoskeleton, possibly via pore formation. Interestingly, the two iota-toxin components inoculated separately on each cell surface induced cytopathic effects and a TER decrease. Anti-Ib sera, raised against the whole molecule or the Ia docking domain and applied to the opposite cell side versus Ib, neutralized the TER decrease. In addition, radioactive Ib incubated in the basolateral compartment was detected on the apical side by selective cell surface biotinylation. This argues for a transcytotic routing of Ib to mediate internalization of Ia from the opposite cell surface. Bafilomycin A1 also prevented the cytopathic effects of Ia and Ib applied separately to each cell side, possibly by blocking translocation of Ia into the cytosol and/or the intracellular transport of Ib. Ib is either routed into the cell independently of Ia, trans-cytosed and permanently exposed on the opposite cell surface or continuously recycled between an endosomal compartment and the cell surface.     

DNA polymerase beta: contributions of template-positioning and dNTP triphosphate-binding residues to catalysis and fidelity

The specific catalytic roles of two groups of DNA polymerase beta active site residues identified from crystal structures were investigated: residues possibly involved in DNA template positioning (Lys280, Asn294, and Glu295) and residues possibly involved in binding the triphosphate moiety of the incoming dNTP (Arg149, Ser180, Arg183, and Ser188). Eight site-specific mutants were constructed: K280A, N294A, N294Q, E295A, R149A, S180A, R183A, and S188A. Two-dimensional NMR analysis was employed to show that the global conformation of the mutants has not been perturbed significantly. Pre-steady-state kinetic analyses with single-nucleotide gapped DNA substrates were then performed to obtain the rate of catalysis at saturating dNTP (k(pol)), the apparent dissociation constant for dNTP (K(d)), catalytic efficiency k(pol)/K(d), and fidelity. Of the three template-positioning residues, Asn294 and Glu295 (but not Lys280) contribute significantly to k(pol). Taken together with other data, the results suggest that these two residues help to stabilize the transition state during catalysis even though they interact with the DNA template backbone rather than directly with the incoming dNTP or the opposite base on the template. Furthermore, the fidelity increases by up to 19-fold for N294Q due to differential k(pol) effects between correct and incorrect nucleotides. Of the four potential triphosphate-binding residues, Ser180 and Arg183 contribute significantly to k(pol) while the effects of R149A are relatively small and are primarily on K(d), and Ser188 appears to play a minimal role in the catalysis by Pol beta. These results identify several residues important for catalysis and quantitate the contributions of each of those residues. The functional data are discussed in relation to the prediction on the basis of available crystal structures.     

Immunochemical analysis of Pseudomonas aeruginosa exotoxin A. Analysis of the His426 determinant.

This study describes a combined immunochemical and genetic approach defining a site on Pseudomonas aeruginosa exotoxin A (ETA) which is critical to the ADP-ribosyltransferase (ADPRT) activity of the toxin. The sequential epitope of a monoclonal antibody (TO-1) which binds to domain III (residues 405-613), containing the ADPRT activity of ETA, has been defined using a series of synthetic peptides. This epitope spans residues 422-432 which composes the major alpha-helical segment of domain III and includes His426 which has previously been shown to be essential for ADPRT activity (Wozniak, D.J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8880-8884). The critical His426 residue which projects into a major cleft becomes exposed when the ETA protein is in an ADPRT-active configuration. Since the TC-1 mAb does not block the binding of NAD+, it is possible that the alpha-helix site containing the TC-1 epitope and the His426 residue is associated with the interaction between ETA and its elongation factor 2 substrate.     

An enzyme-linked immunosorbent assay for the association of the catalytic domain of diphthamide-specific ribosyltransferases to eukaryotic elongation factor-2.

The X-ray structure of the catalytic domain of Pseudomonas aeruginosa exotoxin A (PE24) has recently been solved to high resolution, facilitating studies on the interaction of PE24 with its target substrate, eukaryotic elongation factor-2 (eEF-2). PE24 exhibits mono-ADP-ribosyltransferase (ADPRT) activity in a mechanism that has been proposed to feature a nucleophilic attack by the diphthamide residue (nucleophile) of eEF-2 on the C-1 of the nicotinamide ribose of NAD(+). The interaction of wheat germ eEF-2 with PE24 was studied by employing an enzyme-linked immunosorbent assay (ELISA), devised to assess protein-protein interactions. It was shown that the proteins associate with each other only in the presence of the enzyme's nucleotide substrate, NAD(+), and exhibit a dose-dependent association that is saturable. The apparent dissociation constant (K(d)) for this protein-protein interaction is 50 nM and is salt-dependent. The association is maximal at low ionic strength and is progressively weaker at higher salt concentrations, which corroborates previous findings on the salt dependence of ADPRT activity for this toxin. This finding suggests that the sensitivity of ADPRT activity toward high salt resides in the interaction between the catalytic domain of the toxin and eEF-2. A major product of the glycohydrolase activity of PE24, nicotinamide, inhibits the binding between PE24 and eEF-2 with an ID(50) of 20 microM. The naturally occurring, noncatalytic mutant of PE24, H426Y, did not bind eEF-2 in the ELISA, verifying that His 426 is located at the center of the eEF-2 binding site within ETA.