title: Biochemical analysis of CRM 66. A nonfunctional Pseudomonas aeruginosa exotoxin A.
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.
authors.affiliation: Department of Microbiology, Ohio State University, Columbus 43210-1292.
authors.forename: D R
authors.initials: DR
authors.lastname: Galloway
authors.forename: R C
authors.initials: RC
authors.lastname: Hedstrom
authors.forename: J L
authors.initials: JL
authors.lastname: McGowan
authors.forename: S P
authors.initials: SP
authors.lastname: Kessler
authors.forename: D J
authors.initials: DJ
authors.lastname: Wozniak
mesh_terms: D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins; D001428:Bacterial Vaccines / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D002352:Carrier Proteins; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005098:Exotoxins / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D006639:Histidine / Q000378:metabolism; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism / Q000633:toxicity*; D011487:Protein Conformation; D054406:Pseudomonas Vaccines; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000378:metabolism; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000032:analysis; D014443:Tyrosine / Q000378:metabolism; D017778:Vaccines, Combined; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D002352:Carrier Proteins; D005098:Exotoxins; D006639:Histidine; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D011065:Poly(ADP-ribose) Polymerases; D054406:Pseudomonas Vaccines; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D014443:Tyrosine; D017778:Vaccines, Combined; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C027553:Pseudomonas exotoxin binding protein, mouse; C039987:polyvalent pseudomonas vaccine