Chain elongation and cyclization in type III PKS DpgA

Area of Science:

• Biochemistry
• Molecular Biology
• Organic Chemistry

Background:

• Type III polyketide synthases (PKS) are crucial for synthesizing diverse natural products.
• The bacterial PKS DpgA catalyzes the formation of dihydroxyphenylacetyl-CoA (DPA-CoA).
• Previous models for DpgA-catalyzed chain elongation and cyclization remain incomplete.

Purpose of the Study:

• To elucidate the precise mechanism of chain elongation and cyclization in DpgA-catalyzed reactions.
• To investigate the roles of proposed labile intermediates, di- and tri-ketidyl-CoA (DK-CoA and TK-CoA).
• To determine the contribution of malonyl-CoA (MA-CoA) and other precursors in DPA-CoA synthesis.

Main Methods:

• Chemical synthesis of proposed labile intermediates (DK-CoA and TK-CoA).
• Enzymatic assays using DpgABD with synthesized intermediates and [(13)C(3)]malonyl-CoA (MA-CoA).
• Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) analysis of (13)C-enriched DPA-CoA products.

Main Results:

• Synthesized DK-CoA and TK-CoA were confirmed as intermediates in DpgA reactions.
• Partially (13)C-enriched DPA-CoA was formed using these intermediates and MA-CoA.
• NMR and MS data revealed a novel elongation mechanism: two DK-CoA molecules, or one DK-CoA and one acetoacetyl-CoA (AA-CoA), form DPA-CoA, not two AA-CoA molecules.
• Polyketidyl-CoA acts as both starter and extender, while polyketone-CoA without a terminal carboxyl group acts solely as an extender.

Conclusions:

• The study reveals a non-canonical chain elongation mechanism for DpgA, differing from previous assumptions.
• The terminal carboxyl group on ketidyl-CoA precursors is essential for the cyclization step, which likely occurs on the CoA molecule.
• These findings provide critical insights into the intricate biochemical pathways of type III PKS.