Polyketide synthases (PKSs) represent large multifunctional enzymes involved in the biosynthesis of numerous small molecules that arise from their structural diversity. A thorough understanding of PKS catalytic mechanisms is a vital prerequisite to rationally design novel antibiotics and pharmacologically relevant small molecules. Assembly line PKSs are a specific type of PKS composed of various catalytic domains that mediate a conserved set of chemical transformations on a growing polyketide chain. The biosynthetic machinery of an assembly line PKS minimally comprises of a ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) domain. Some PKS modules include the AT as an integral domain of a multifunctional polypeptide (cis-AT PKS), while other PKS modules utilize a standalone AT (trans-AT PKS). Typically, each module catalyzes a single elongation and modification step before the growing polyketide chain is either translocated onto the KS domain of the following module or off-loaded from the assembly line by a thioesterase/thioester reductase domain. However, not all PKSs abide to this collinear feature, such as the nocardiosis-associated polyketide (NOCAP) synthase which harbors a trans-AT (Module 5) that catalyzes three successive elongation and modification cycles on the nascent polyketide chain. Such a phenomenon known as module iteration or “stuttering”, arises when an elongated and modified polyketide product is translocated from the ACP domain of Module 5 back to its own KS domain. In this talk, we aim to report the origins and molecular mechanisms of module iteration (“split-and-stuttering”) in the context of Module 5 of the NOCAP synthase.
