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Structure-function analysis of exotoxin A proteins with mutations at histidine 426
M J Wick1, J M Cook, B H Iglewski
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, New York 14642.
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.
Insights
Histidine-426 in Pseudomonas aeruginosa exotoxin A is crucial for its ADP-ribosyltransferase and NAD-glycohydrolase activity. Substituting this residue significantly reduces enzymatic function, indicating a key structural role in the catalytic site.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Pseudomonas aeruginosa exotoxin A is a potent toxin.
- Previous studies indicated that substituting Tyrosine for Histidine-426 reduces ADP-ribosyltransferase activity.
Purpose of the Study:
- To investigate the precise role of Histidine-426 in the enzymatic activity of Pseudomonas aeruginosa exotoxin A.
- To determine if Histidine-426 has a catalytic function or a structural role in the enzyme's active site.
Main Methods:
- Oligonucleotide-directed mutagenesis was used to create mutant exotoxin A proteins with substitutions at position 426 (Ala, Glu, Gly, Lys, Pro).
- ADP-ribosyltransferase activity was measured using 34,000-Da carboxy-terminal exotoxin A peptides (H426n peptides).
- NAD-glycohydrolase activity was measured for full-size wild-type and mutant exotoxin A proteins (Ala-426, Tyr-426).
Main Results:
- Mutant H426n peptides showed ADP-ribosyltransferase activity ranging from 0.002% to 28% of wild-type levels.
- Exotoxin A mutants with Ala-426 or Tyr-426 exhibited less than 1% of wild-type NAD-glycohydrolase activity.
- Wild-type and mutant full-size proteins displayed conformational differences, evidenced by enzymatic activation and tryptic digestion patterns.
Conclusions:
- Histidine-426 is essential for the full expression of Pseudomonas aeruginosa exotoxin A's ADP-ribosyltransferase activity.
- Histidine-426 appears to play a critical structural role in domain III, orienting active-site residues within the catalytic cleft.
- The findings suggest His-426 is vital for maintaining the correct molecular architecture of the enzyme's active site.