Nature, doi:10.1038/nature12588

Polyketide synthase machinery has, to this point, exclusively used head-to-tail connectivity to attach incoming monomers to the ends of growing polyketide chains (solid arrow). A new study from Bretschneider et al. overturns this dogma, reporting that an acyl carrier protein (ACP)-bound monomer can attack the vinylogous position of an α,β-unsaturated chain, introducing a branch point (dashed arrows). The authors' previous study of the rhizoxin biosynthetic pathway indicated that the connectivity of the molecule could not be rationalized by the known protein modules in the assembly line. To elucidate the pathway, the authors focused on an unusual ketosynthase-branching didomain found in the RhiE enzyme. In vitro reconstitution of the didomain, its ACP partner and other requisite proteins, along with chemical synthesis of activated substrates, provided direct evidence that a Michael addition was indeed occurring. NMR characterization of enzyme-bound, labeled substrates confirmed that C-C bond formation precedes release of the chain from the ketosynthase (KS) domain via an intramolecular esterification reaction; by synthetically removing the hydroxyl group necessary for the esterification, the authors were able to observe the doubly-bound intermediate. Surprisingly, structural and mutational evidence suggested the ketosynthase performs all reaction steps, while the new branching domain does not play a catalytic role, leaving the mechanism of this intriguing reaction to be resolved.