Biochemical Analysis of CRM 66. A Nonfunctional Pseudomonas Aeruginosa Exotoxin A

J Biol Chem. 1989 Sep 5;264(25):14869-73.

Abstract

Pseudomonas aeruginosa exotoxin A (ETA) is an ADP-ribosyltransferase which inactivates protein synthesis by covalently attaching the ADP-ribose portion of NAD+ onto eucaryotic elongation factor 2 (EF-2). A direct biochemical comparison has been made between ETA and a nonenzymatically active mutant toxin (CRM 66) using highly purified preparations of each protein. The loss of ADP-ribosyltransferase activity and subsequent cytotoxicity have been correlated with the presence of a tyrosine residue in place of a histidine at position 426 in CRM 66. In the native conformation, CRM 66 demonstrated a limited ability (by a factor or at least 100,000) to modify EF-2 covalently and lacked in vitro and in vivo cytotoxicity, yet CRM 66 appeared to be normal with respect to NAD+ binding. Upon activation with urea and dithiothreitol, CRM 66 lost ADP-ribosyltransferase activity entirely yet CRM 66 retained the ability to bind NAD+. Replacement of Tyr-426 with histidine in CRM 66 completely restored cytotoxicity and ADP-ribosyltransferase activity. These results support previous findings from this laboratory (Wozniak, D. J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8880-8884) which suggest that the His-426 residue of ETA is not involved in NAD+ binding but appears to be associated with the interaction between ETA and EF-2.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ADP Ribose Transferases*
  • Animals
  • Bacterial Toxins*
  • Bacterial Vaccines / isolation & purification*
  • Bacterial Vaccines / metabolism
  • Bacterial Vaccines / toxicity
  • Carrier Proteins*
  • Cell Line
  • Cricetinae
  • Cricetulus
  • Exotoxins / isolation & purification*
  • Exotoxins / metabolism
  • Exotoxins / toxicity
  • Histidine / metabolism
  • Peptide Elongation Factor 2
  • Peptide Elongation Factors / metabolism
  • Poly(ADP-ribose) Polymerases / metabolism
  • Poly(ADP-ribose) Polymerases / toxicity*
  • Protein Conformation
  • Pseudomonas Vaccines
  • Pseudomonas aeruginosa / enzymology*
  • Pseudomonas aeruginosa / metabolism
  • Receptors, Cell Surface*
  • Receptors, Cholinergic / analysis
  • Tyrosine / metabolism
  • Virulence Factors*

Substances

  • Bacterial Toxins
  • Bacterial Vaccines
  • Carrier Proteins
  • Exotoxins
  • Peptide Elongation Factor 2
  • Peptide Elongation Factors
  • Pseudomonas Vaccines
  • Pseudomonas exotoxin binding protein, mouse
  • Receptors, Cell Surface
  • Receptors, Cholinergic
  • Virulence Factors
  • polyvalent pseudomonas vaccine
  • Tyrosine
  • Histidine
  • ADP Ribose Transferases
  • Poly(ADP-ribose) Polymerases
  • toxA protein, Pseudomonas aeruginosa