Active site mutations of Pseudomonas aeruginosa exotoxin A. Analysis of the His440 residue.

Pseudomonas aeruginosa exotoxin A (ETA) is a member of the family of bacterial ADP-ribosylating toxins which use NAD+ as the ADP-ribose donor. By analogy to diphtheria and pertussis toxins, the His440 residue of ETA has been proposed to be one of the critical residues within the active site of the toxin. In this study the role of the His440 residue was explored through site-directed mutagenesis which resulted in the production of ETA proteins containing Ala, Asn, and Phe substitutions at the 440 position. The His440-substituted ETA proteins were purified and analyzed. All substitutions at the 440 site displayed severely reduced ADP-ribosylation activity (> 1000-fold). However, NAD glycohydrolase activity remained intact and in the case of ETAH440N actually increased 10-fold. NAD+ binding is not affected by substitutions at the 440 site as indicated by similar Km values for the ETA variants tested. Conformational integrity of the mutant toxins appears to be largely unaffected as assessed by analysis with a conformation-sensitive monoclonal antibody as well as sensitivity to proteinase digestion. In view of the location of His440 residue within or close to the proposed NAD(+)-binding site, these results suggest that His440 may be a catalytic residue involved in the transfer of the ADP-ribose moiety to the EF-2 substrate.