β-Branching in the biosynthesis of bongkrekic acid: a complex affair

Bongkrekic acid is a potent respiratory toxin which inhibits the mitochondrial ATP/ADP carrier protein. The polyketide synthase that biosynthesises bongkrekic acid recruits a discrete cassette of β-branching enzymes (BonF-BonI) to install two distinct β-branches: an <i>endo</i>-β-methyl branch in module 1, and a carboxymethyl β-branch in module 11. Both β-branches contribute to specific interactions with bongkrekic acid's biological target. However, a critical component of the β-branching cassette, the donor acyl carrier protein (ACP_D), has not been identified in previous studies. Furthermore for the module 11 carboxymethyl β-branch to be retained, conversion to an <i>endo</i>-β-methyl branch <i>via</i> the enoyl-coenzyme A hydratase (ECH), BonI, must be avoided. The mechanistic basis for these divergent β-branching pathways is poorly understood, both in the bongkrekic acid biosynthetic pathway and more generally where it arises in polyketide biosynthesis. Here, we confirm the roles of BonF-BonI by reconstituting β-branching in modules 1 and 11 <i>in vitro</i> and uncover the previously unannotated ACP_D, BonN, to complete the β-branching cassette. We further demonstrate promiscuous BonI interactions with both module 1 and 11 ACPs that confounds simple ACP selectivity arguments for carboxymethyl β-branch <i>versus endo</i>-β-methyl branch installation, suggesting that this is instead regulated by a complex interplay between substrate and kinetic control.