Related Experiment Videos
Replacement of histidine 340 with alanine inactivates the group A Streptococcus extracellular cysteine protease
S Gubba1, V Cipriano, J M Musser
1Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, Texas 77030, USA.
Abstract:
Streptococcus pyogenes expresses a highly conserved extracellular cysteine protease that is a virulence factor for invasive disease, including soft tissue infection. Site-directed mutagenesis was used to generate a His340Ala recombinant mutant protein that was made as a stable 40-kDa zymogen by Escherichia coli. Purified His340Ala protein was proteolytically inactive when bovine casein and human fibronectin were used as substrates. Wild-type 28-kDa streptococcal protease purified from S. pyogenes processed the 40-kDa mutant zymogen to a 28-kDa mature form, a result suggesting that the derivative protein retained structural integrity. The data are consistent with the hypothesis that His340 is an enzyme active site residue, an idea confirmed by recent solution of the zymogen crystal structure (T. F. Kagawa, J. C. Cooney, H. M. Baker, S. McSweeney, M. Liu, S. Gubba, J. M. Musser, and E. N. Baker, Proc. Natl. Acad. Sci. USA 97:2235-2240, 2000). The data provide additional insight into structure-function relationships in this S. pyogenes virulence factor.
Insights
Histidine 340 is crucial for the activity of Streptococcus pyogenes extracellular protease, a key virulence factor. Mutating this residue inactivated the protease, confirming its role in invasive disease.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Streptococcus pyogenes possesses a conserved extracellular cysteine protease.
- This protease is a significant virulence factor in invasive diseases, such as soft tissue infections.
Purpose of the Study:
- To investigate the role of Histidine 340 (His340) in the enzymatic activity of the Streptococcus pyogenes extracellular protease.
- To confirm His340 as a critical active site residue.
Main Methods:
- Site-directed mutagenesis was employed to create a His340Ala mutant protein.
- The mutant protein was expressed as a zymogen in Escherichia coli and purified.
- Proteolytic activity was assessed using casein and fibronectin substrates.
- Processing of the mutant zymogen by wild-type protease was analyzed.
Main Results:
- The His340Ala mutant protein was proteolytically inactive against tested substrates.
- Wild-type protease successfully processed the mutant zymogen into a mature form, indicating retained structural integrity.
- These findings support His340 as an essential active site residue.
Conclusions:
- Histidine 340 is a critical residue within the active site of the Streptococcus pyogenes extracellular cysteine protease.
- The study provides further insight into the structure-function relationship of this important virulence factor.