CRM66 (cross-reactive 66kDa protein) is an inactive mutant form of Pseudomonas aeruginosa exotoxin A that has been isolated from a mutant strain of P. aeruginosa derived from nitrosoguanidine-based mutagenesis. The mutation within this enzyme toxin was previously identified as H426Y and it was shown to possess significantly reduced enzymic activity. Furthermore, it was previously suggested that His-426 may directly participate in the catalytic mechanism of the exotoxin A enzyme and that it may also play an important role in the binding of the protein substrate of exotoxin A, a critical protein factor in eukaryotic protein translation known as elongation factor-2. In order to more thoroughly characterize the role of His-426 in the enzyme mechanism of exotoxin A, amino acid substitutions were made within helix 1 of the enzyme domain in the vicinity of the His-426 residue. Analysis of the site-directed mutagenesis results involving kinetic and protein structural integrity measurements revealed that His-426 H-bonds to Tyr-502 and that replacement of His-426 with polar substitutions leads to structural alterations of the enzyme's folded conformation. Furthermore, it was shown that His-426 is not important for the binding of either of the two substrates of exotoxin A, NAD+ or elongation factor-2. In summary, these data show that His-426 is not an active-site residue and that it is not important for substrate binding or orientation, but that it plays an important structural role in helping to maintain the folded conformation of the enzyme toxin. Therefore, the role of His-426 would seem to be to tether helix 1 to the main body of the enzyme, and mutations resulting in the disruption of this region of the enzyme result in a significantly impaired enzyme.
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November 2002
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Research Article|
November 01 2002
A re-evaluation of the role of histidine-426 within Pseudomonas aeruginosa exotoxin A
Tania M. ROBERTS;
Tania M. ROBERTS
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1
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A. Rod MERRILL
A. Rod MERRILL
1
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1
1To whom correspondence should be addressed (e-mail merrill@chembio.uoguelph.ca).
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Publisher: Portland Press Ltd
Received:
May 13 2002
Revision Received:
July 31 2002
Accepted:
August 02 2002
Accepted Manuscript online:
August 02 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 367 (3): 601–608.
Article history
Received:
May 13 2002
Revision Received:
July 31 2002
Accepted:
August 02 2002
Accepted Manuscript online:
August 02 2002
Citation
Tania M. ROBERTS, A. Rod MERRILL; A re-evaluation of the role of histidine-426 within Pseudomonas aeruginosa exotoxin A. Biochem J 1 November 2002; 367 (3): 601–608. doi: https://doi.org/10.1042/bj20020768
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