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

Abstract

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 þ , 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 þ substrate adopts a conformation remarkably different from that of the NAD þ analogue, bTAD, 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.

The 1.8 Å Cholix Toxin Crystal Structure in Complex with NAD+ and Evidence for a New Kinetic Model

“…3, e and f), wild type full-length toxin apparently has some activity, although at levels more than 15-fold lower than the corresponding catalytic fragment, cholix c . Activity was virtually abolished for the E581A and E574A/E581A mutants (29,300-and 12,600-fold, respectively), in part due to weaker NAD ϩ substrate binding, impaired productive substrate binding, and an inability to stabilize the transition state complex (11,14). Activity in these mutants is so low that accurate measurements are challenging.…”

“…A proposed overall cholix-NAD ϩ -eEF2 Michaelis complex is shown in Fig. 2c based on our previous x-ray structure of ExoA with eEF2 and NAD ϩ (13,14). A close-up of the cholix-NAD ϩ -eEF2 interface within the proposed complex is shown in Fig.…”

“…The DT group reaction has been extensively studied (13,14) and involves scission of the glycosidic bond (C-N) between nicotinamide and its conjugated ribose as well as transfer of the ADP-ribose group to a nucleophilic residue, diphthamide, on eEF2. Specifically, loop 1 flexibility allows this loop to form a solvent cover to exclude water and help stabilize the transition state during the reaction.…”

Pseudomonas aeruginosa exotoxin T induces potent cytotoxicity against a variety of murine and human cancer cell lines

“…Although these new recombinant immunotoxins have shown high potency in killing tumour cells with high specificity, they too share the limitation of targeting a single cellular substrate, eEF-2 (Jørgensen et al, 2008). In fact, mutations in eEF-2 have been shown to emerge easily, conferring resistance to these toxins in cancer cells (Foley et al, 1995;Jørgensen et al, 2008;Wei et al, 2012). & Barbieri, 2003;Garrity-Ryan et al, 2004), which play important roles in survival, proliferation, metastasis and angiogenesis in cancer.…”

“…For example, Pseudomonas exotoxin A and diphtheria toxin are the most common bacterial toxins that have been or are currently under clinical evaluation against a variety of haematologic malignancies and solid tumours with promising results (reviewed by Becker & Benhar, 2012). Although these new recombinant immunotoxins have shown high potency in killing tumour cells with high specificity, they too share the limitation of targeting a single cellular substrate, eEF-2 (Jørgensen et al, 2008). In fact, mutations in eEF-2 have been shown to emerge easily, conferring resistance to these toxins in cancer cells (Foley et al, 1995;Jørgensen et al, 2008;Wei et al, 2012).…”