title: Structure-function analysis of exotoxin A proteins with mutations at histidine 426.
abstract: Substitution of Tyr for His-426 of Pseudomonas aeruginosa exotoxin A results in a mutant protein with reduced ADP-ribosyltransferase activity (M. J. Wick and B. H. Iglewski, J. Bacteriol. 170:5385-5388, 1988). To investigate the role of His-426 in enzymatic activity, oligonucleotide-directed mutagenesis was used to construct mutant proteins encoding Ala, Glu, Gly, Lys, or Pro at position 426. The effect of these amino acid substitutions on ADP-ribosyltransferase activity was analyzed in 34,000-Da carboxy-terminal exotoxin A peptides (H426n peptides). ADP-ribosyltransferase activity of the H426n peptides fell within a range between 0.002 and 28% of wild-type levels of activity, suggesting that His-426 is required for full expression of enzymatic activity of exotoxin A. To investigate a possible catalytic function of His-426, the abilities of full-size (66,000-Da) wild-type exotoxin A and mutant proteins encoding either Ala-426 or Tyr-426 to hydrolyze NAD were compared by measuring NAD-glycohydrolase activity. This analysis revealed that exotoxin A encoding either Ala-426 or Tyr-426 expressed less than 1% of wild-type levels of NAD-glycohydrolase activity. Several criteria, including differential enzymatic activation properties and unique tryptic digestion patterns, revealed that the wild-type and mutant full-size proteins exhibit conformational differences. Our data suggest that His-426 plays a critical structural role in establishing the molecular architecture of the catalytic site in domain III and is important in orienting active-site residues in the cleft.
authors.affiliation: Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, New York 14642.
authors.forename: M J
authors.initials: MJ
authors.lastname: Wick
authors.forename: J M
authors.initials: JM
authors.lastname: Cook
authors.forename: B H
authors.initials: BH
authors.lastname: Iglewski
mesh_terms: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins / Q000032:analysis*; D001483:Base Sequence; D005098:Exotoxins / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D006639:Histidine; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D006639:Histidine; D009243:NAD; D011065:Poly(ADP-ribose) Polymerases; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A