A mechanism-based fluorescence transfer assay for examining ketosynthase selectivity

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Polyketide synthases (PKS) are crucial for producing diverse natural products, including therapeutics.
• Enzyme specificity in PKS has limited the engineering of novel compounds.
• Developing new tools to understand PKS is vital for drug discovery.

Purpose of the Study:

• To develop a novel, mechanism-based fluorescence transfer assay for studying ketosynthase (KS) domains.
• To characterize the substrate specificity of the KS domain from 6-deoxyerythronolide synthase module 6.
• To demonstrate the assay's utility for both didomains and full PKS modules.

Main Methods:

• A fluorescence transfer assay was designed to monitor KS domain activity.
• The assay was applied to the KS domain of 6-deoxyerythronolide synthase module 6.
• Various thioester substrates, including α-branched and electronically diverse compounds, were tested.

Main Results:

• The developed assay provides a simple and distinct visible readout of KS activity.
• The KS domain showed a strong preference for longer, α-branched thioester substrates.
• Substrate electronic properties, modulated by p-substituted aromatic groups, were also assessed.

Conclusions:

• The new fluorescence assay is a valuable tool for polyketide biosynthesis and engineering.
• Understanding KS domain specificity is key to rationally designing PKS for novel therapeutics.
• This assay facilitates the study of enzyme mechanisms and substrate recognition in PKS.