Modular polyketide synthase ketosynthases collaborate with upstream dehydratases to install double bonds

Area of Science:

• Biochemistry
• Molecular Biology
• Organic Chemistry

Background:

• Polyketide assembly lines are crucial for producing diverse natural products.
• Ketosynthases (KS) are key enzymes in these pathways, responsible for catalyzing carbon-carbon bond formation.
• The selection of specific intermediates by KS enzymes is critical for determining the final polyketide structure.

Purpose of the Study:

• To investigate the role of the VMYH motif in ketosynthase selectivity.
• To understand how this motif differentiates between α,β-unsaturated and D-β-hydroxy intermediates.
• To elucidate the mechanism of substrate selection in polyketide biosynthesis.

Main Methods:

• Site-directed mutagenesis was used to alter the VMYH motif in a model tetraketide synthase.
• Enzyme activity assays were performed to assess the selectivity of the modified ketosynthases.
• Analysis of reaction products was conducted to determine the preferred intermediates.

Main Results:

• The VMYH motif was identified as essential for ketosynthase selectivity.
• Alterations to the VMYH motif significantly decreased the enzyme's ability to select α,β-unsaturated intermediates.
• Replacing VMYH with the TNGQ motif (found in KS accepting D-β-hydroxy intermediates) further impaired selectivity for α,β-unsaturated substrates.

Conclusions:

• The VMYH motif plays a critical role in guiding ketosynthases to select specific intermediates during polyketide assembly.
• This motif acts as a gatekeeper, ensuring the correct substrate is chosen from a pool of possibilities.
• Understanding this selectivity mechanism provides insights into controlling polyketide biosynthesis for potential applications.