Polyketide mimetics yield structural and mechanistic insights into product template domain function in nonreducing...

Area of Science:

• Biochemistry and Molecular Biology
• Structural Biology
• Natural Product Biosynthesis

Background:

• Product template (PT) domains in fungal nonreducing polyketide synthases (NR-PKSs) control crucial aldol cyclizations.
• Understanding PT domain function is limited by the instability of poly-β-ketone intermediates.
• Existing knowledge of NR-PKS catalytic mechanisms relies on indirect evidence due to intermediate instability.

Purpose of the Study:

• To develop and apply novel chemical mimetics for crystallographic studies of PT domain mechanisms.
• To elucidate the regioselective control exerted by PT domains during polyketide biosynthesis.
• To provide empirical evidence for proposed mechanisms of PT domain-catalyzed cyclization.

Main Methods:

• Crystallographic analysis of the PksA PT domain from aflatoxin biosynthesis.
• Utilized "atom replacement" mimetics (isoxazole rings linked by thioethers) to mimic poly-β-ketone intermediates.
• Co-crystallization with a heptaketide mimetic tethered to a modified 4'-phosphopantetheine.
• Incorporated docking simulations and mutational experiments.

Main Results:

• Determined the 1.8-Å co-crystal structure of the PksA PT domain with a heptaketide mimetic.
• Structural data support a mechanism involving deprotonation at C4 by His1345 and activation of the C9 carbonyl by a water network.
• Demonstrated the role of the 4'-phosphate in delivering the mimetic and positioning it for catalysis.
• Characterized protein-substrate mimic interactions influencing orientation and stabilization.

Conclusions:

• The developed "atom replacement" mimetics provide a viable strategy for studying unstable polyketide intermediates.
• The structural insights validate proposed catalytic mechanisms for PT domains in NR-PKSs.
• The findings offer a generalizable view of mimetic interactions within NR-PKS active sites.