Structure-function analysis of exotoxin A proteins with mutations at histidine 426

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.