Probing the mega-synthases involved in primary and secondary metabolism

The carrier protein (CP) transports the growing fatty acid chain between enzyme domains of fatty acid synthase (FAS), polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) in primary and secondary metabolism. Because these mega-synthases operate upon CP-bound substrates, CP must stabilize and transport the growing chain. The transient nature of CP-enzyme interactions imposes a major obstacle to gaining high-resolution structural information about biosynthesis directed by the mega-synthases, and a new strategy is required to properly study protein-protein interactions. In this work, we describe the application of mechanism-based probes that allow site-selective covalent crosslinking of CP to downstream enzymes, as well as non-covalent probes that closely mimic the natural polyketide substrates. Several high resolution crystal structures of FAS and PKS complexes will be presented, in which ACP exhibits different conformations that represent snapshots of ACP in action. The 4’-phosphopantetheine (PPant) group of ACP first binds an arginine-rich groove of its partner enzyme, followed by an ACP helical conformational change that locks the ACP and its partner enzyme in place. Residues at the interface of ACP and its partner are further identified and validated by solution-phase NMR techniques, including HSQC perturbation and RDC measurements. These not only support the interpretation of crystal structures but also provide a dynamic animation of ACP in action. Mutations of both ACP and its partner enzymes generate both knock-out and enhanced ACP-partner interactions. Here the foundation is laid for defining the dynamic action of carrier protein activity in primary and secondary metabolism.