Dehydratase-specific probes for fatty acid and polyketide synthases

Area of Science:

• Biochemistry
• Enzymology
• Chemical Biology

Background:

• Dehydratase (DH) enzymes are crucial components of fatty acid synthases (FASs) and polyketide synthases (PKSs).
• Existing probes, like 3-decynoyl-N-acetylcysteamine (3-decynoyl-NAC), exhibit nonspecific reactivity, limiting their utility for DH enzyme detection.
• There is a need for specific and reliable probes to study DH enzymes within complex biological systems.

Purpose of the Study:

• To design, synthesize, and validate a novel dehydratase (DH)-specific reactive probe.
• To develop a tool that facilitates the detection, enrichment, and identification of DH enzymes in FAS and PKS.
• To engineer a probe that overcomes the limitations of previous DH inhibitors.

Main Methods:

• Design and synthesis of a DH-specific probe featuring a sulfonyl 3-alkyne reactive warhead.
• Engineering the probe to prevent hydrolysis and nonenzymatic inactivation.
• Coupling the probe with a fluorescent tag for visualization and detection.
• Testing the probe against recombinant type I and type II FAS and PKS enzyme systems and whole proteomes.
• Conducting activity studies, including enzyme inactivation and antibiotic susceptibility assays.

Main Results:

• The developed sulfonyl 3-alkyne probe selectively targets DH enzymes.
• The probe demonstrates efficacy in detecting DH enzymes in both purified FAS/PKS systems and complex proteomes.
• Activity studies confirm the probe's mechanism-based inactivation of DH enzymes, such as FabA.
• The probe's design minimizes nonspecific reactivity, enhancing its specificity.

Conclusions:

• The novel DH-specific probe provides a powerful tool for the functional characterization and molecular identification of DH enzymes.
• This probe accelerates research in fatty acid and polyketide synthesis pathways.
• The sulfonyl 3-alkyne scaffold represents a promising strategy for developing selective enzyme-targeting probes.