The structure of full-length AFPK supports the ACP linker in a role that regulates iterative polyketide and fatty acid assembly

Abstract

We present the full structures of two animal fatty acid synthase (FAS)-like polyketide synthases (AFPKs), PKS1 and PKS2 from Elysia chlorotica. Unlike the related FAS enzymes that use malonate to produce reduced lipids, EcPKS1 and EcPKS2 accept methylmalonyl-CoA to produce oxidized polypropionate products. When incubated with inhibitory malonyl-CoA (MC), the resulting EcPKS2(MC) structure revealed MC bound to the acyltransferase active site and the phosphopantetheinylated acyl carrier protein (ACP-pPant) bound to the ketosynthase (KS) active site. Remarkably, the entire linker from one modifying region to the ACP was visible in full-length EcPKS2(MC), revealing an asymmetric mega-enzyme structure. Mutations disrupting the affinity between the ACP linker and modifying domains altered substrate selectivity and active site selection, despite an expectation that the interactions are transient and that the wild-type linker alternates between the observed ordered and disordered conformations during the competing catalytic cycles of the homodimeric protein. A second structure, EcPKS2(AC), was acylated both on the KS catalytic cysteine and on the ACP-pPant. The ACP was docked at the dehydratase site, revealing further interactions between ACP and these FAS-like enzymes. The results suggest an unexpected role for the ACP linker in the control of substrate and product selectivity across the AFPK/FAS clade in animals and highlight ACP interfaces and mega-enzyme dynamics over the course of the catalytic cycle.