Pseudomonas aeruginosa exotoxin A: effects of mutating tyrosine-470 and tyrosine-481 to phenylalanine

Abstract

Directed mutagenesis was used to probe the functions of Tyr-470 and Tyr-481 of Pseudomonas aeruginosa exotoxin A (ETA) with respect to cytotoxicity, ADP-ribosylation of elongation factor 2 (EF-2), and NAD-glycohydrolase activity. Both of these residues lie in the active site cleft, close to Glu-553, a residue believed to play a direct role in catalysis of ADP-ribosylation of EF-2. Substitution of Tyr-470 with Phe caused no change in any of these activities, thus eliminating the possibility that the phenolic hydroxyl group of Tyr-470 might be directly involved in catalysis. Mutation of Tyr-481 to Phe caused an approximately 10-fold reduction in NAD:EF-2 ADP-ribosyltransferase activity and cytotoxicity but no change in NAD-glycohydrolase activity. The latter mutation did not alter the KM of NAD in the NAD-glycohydrolase reaction, which suggests that the phenolic hydroxyl of Tyr-481 does does not participate in NAD binding. We hypothesize that the phenolic hydroxyl of Try-481 may be involved in the interaction of the toxin with substrate EF-2.