Enzyme reaction mechanisms of bacterial mono-ADP-ribosyltransferase family

Decreases in childhood immunization effectiveness and frequencies and in world health programs make it necessary to develop new antitoxin technologies. Targeting exotoxins with specific inhibitors is an attractive approach to treatments, since bacterial resistance is not expected to develop with antitoxins. The development of a comprehensive knowledge of the structure, function, and catalytic mechanism of the mART reaction characteristic of this large family of exotoxins will lead to the development of effective antitoxin agents.

The main objective of this research is to determine the molecular and structural events involved in the catalytic mechanism of the mono-ADP-ribosyltransferase (mART) family of enzymes (both novel and currently studies), most of which serve as virulence factors in a number of pathogenic bacteria.

The issue of antimicrobial resistance has become a growing concern, especially as we witness the ever increasing rates of in vitro resistance among previously susceptible organisms and the emergence of intrinsically resistant organisms as pathogens in immunocompromised hosts. In order to curtail the development and spread of antimicrobial resistance, it will require both the preservation of current antimicrobials through their appropriate use, as well as the discovery and development of new agents. A sound fundamental knowledge of the molecular mechanisms of virulence factors such as the bacterial exotoxins, many of which are necessary for causing disease in the host, is essential for the development of treatment strategies against the offending pathogens. This research is aimed at revealing the molecular mechanisms of the mART family of enzymes that function as virulence factors in a number of pathogenic bacteria. Undoubtedly, the new knowledge and insight accrued from this research program will provide the basis for the prevention and treatment of bacterial infections.

Pseudomonas aeruginosa is an important human pathogen that is particularly troublesome in the hospital environment. It is a serious concern for various immune-compromised individuals, whose immune systems are incapable of combating the arsenal of virulence factors produced by this organism. The most potent and fatal virulence factor is the ETA protein, which causes rapid cell death of host cells leading to significant necrosis of the invaded tissue. ETA is a mART enzyme that kills infected host eukaryotic cells by catalyzing the transfer of ADP-ribose from NAD⁺ to a highly conserved eukaryotic protein translation factor known as elongation factor-2 (eEF-2).

ETA belongs to a family of related bacterial toxins that can be classified according to their protein substrates into four groups:

1. elongation factor-2 ADP-ribosylating toxins
   (diphtheria, DT; and ETA);
2. heterotrimeric G-protein ADP-ribosylating toxins (cholera and pertussis toxin);
3. toxins that ADP-ribosylate small GTPases (clostridium botulinum C3 exoenzyme); and
4. ADP-ribosyltransferases with actin as a substrate (clostridrial ADP-ribosyltransferases and Clostrium perfringens toxin).

Interestingly, ETA is also catalytically related to the DNA repair enzyme family, poly-(ADP-ribose) polymerases (PARPs). The latter are involved in a variety of physiological events such as chromatin decondensation, DNA replication and repair, gene expression, malignant transformation, cellular differentiation and apoptosis.

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A proposed catalytic mechanism based on the recent x-ray structures and recent work in our group and others is shown in above figure. The kinetic and stereochemical data indicate that the reaction mechanism involves an SN1 nucleophilic substitution by which the diphthamide residue (a post-translationally modified His residue) of eEF2 attacks the anomeric carbon of the nicotinamide ribose forming a new glycosidic bond between ADP-ribose and diphthamide-eEF2. Glu 553 forms a critical H-bond with the 2'-OH group of the nicotinamide ribose forming an oxocarbenium ion that serves to enhance the catalytic rate by rendering the C1 carbon more susceptible to nucleophilic attack by the N3 of diphthamide. The acceptor for the ADP-ribose transfer reaction is, therefore, the diphthamide residue within eEF2 and ribosylation of the protein results in the inhibition of protein synthesis and cell death. Recent kinetic isotope effect (KIE) studies by Parikh and Schramm suggest that the presence of eEF2 in the Michaelis complex must also stabilize a ribooxacarbenium ion and/or activate the leaving group without adding nucleophilic participation. The proposed mechanism is called "nucleophilic displacement by electrophile migration." Furthermore, recent kinetic studies in the our lab indicate that the transfer of the ADP-ribose group from NAD⁺ to eEF2 proceeds through a random-order ternary complex mechanism where NAD⁺ binds first forming a binary complex that then associates with eEF2.

We will continue our efforts to elucidate the molecular mechanisms of this important class of toxins through a number of approaches:

1. site-directed mutagenesis of enzyme active sites
2. fluorescence spectroscopic analyses
3. rigorous steady state and stopped-flow kinetic analyses
4. inhibitor identification and application
5. X-ray structure determination

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