Trapping of a polyketide synthase module after C-C bond formation reveals transient acyl carrier domain interactions.
Modular polyketide synthases (PKSs) are giant assembly lines producing an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)-bound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the inter-domain interactions after C-C bond formation in a chain-branching module of the rhizoxin PKS. Mechanism-based crosslinking of an engineered module was achieved using a synthetic surrogate that serves as a Michael acceptor. The crosslinked protein allowed to identify an asymmetric state of the dimeric protein complex upon C-C bond formation by cryo-electron microscopy (cryo-EM). The possible existence of two ACP binding sites, one of them a potential.