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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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Evolutionary Selection on Barrier Activity: Bar1 Is an Aspartyl Protease with Novel Substrate Specificity
Stephen K Jones1, Starlynn C Clarke2, Charles S Craik2
1Department of Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island, USA.
Mbio
|November 26, 2015
Summary
Candida albicans Bar1 protease specifically cleaves α pheromones, regulating fungal mating. Its unique substrate specificity evolved alongside its target, offering biotechnological potential.
Area of Science:
- Mycology
- Biochemistry
- Evolutionary Biology
Background:
- Fungal species use peptide-based pheromones for sexual reproduction coordination.
- Extracellular proteases, like Bar1, antagonize pheromone signaling in fungi.
- Candida albicans Bar1 is an aspartyl protease that inactivates α pheromone.
Purpose of the Study:
- To investigate the properties and function of Candida albicans Bar1.
- To determine Bar1's substrate specificity and its role in regulating pheromone signaling.
- To explore the evolutionary relationship between Bar1 and its pheromone target.
Main Methods:
- Purification of Candida albicans Bar1.
- Enzymatic assays to determine pheromone cleavage activity and specificity.
- Genetic analysis of the BAR1 gene in C. albicans.
Main Results:
- Bar1 preferentially cleaves the endogenous α pheromone over those from related species.
- Proteolytic activity depends on C-terminal residues, not those at the scissile bond.
- Bar1 regulates both intraspecies and interspecies pheromone signaling.
- Bar1 exhibits near-neutral pH optimum and pepstatin A resistance.
Conclusions:
- Bar1's unusual substrate specificity is a result of coevolution with the α pheromone receptor Ste2.
- Bar1's function in regulating pheromone signaling has implications for fungal mating pathways.
- The unique properties of Bar1 suggest potential applications in biotechnology.
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