Understanding Substrate Selectivity of Phoslactomycin Polyketide Synthase by Using Reconstituted in Vitro Systems

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Synthesis

Background:

• Polyketide synthases (PKSs) are crucial enzymes for synthesizing diverse natural products.
• Understanding the substrate specificity of PKS modules is key to elucidating their biosynthetic mechanisms.

Purpose of the Study:

• To establish an in vitro model using the phoslactomycin (Pn) PKS to investigate the site-specific incorporation of different extender units.
• To determine the substrate specificity of individual Pn PKS modules, particularly concerning malonyl-CoA and ethylmalonyl-CoA.

Main Methods:

• Constructed Pn PKS systems by combining up to six modules with varying termination sites for controlled release of polyketide chains.
• Performed competitive assays using up to seven different extender units to challenge the specificity of Pn PKS modules in vitro.

Main Results:

• Malonyl-CoA modules within the Pn PKS exclusively incorporated their natural substrate.
• The ethylmalonyl-CoA module (PnC) demonstrated tolerance for various α-substituted derivatives while discriminating against malonyl-CoA.
• The ratio of transacylation to hydrolysis in PnC was identified as a critical factor controlling extender unit incorporation.

Conclusions:

• The study elucidates the molecular basis for site-specific selectivity and promiscuity in Pn PKS.
• The findings provide insights into how PKS enzymes achieve precise incorporation of diverse building blocks during polyketide biosynthesis.