Mechanistic Analysis of Programmed Iteration by Module 5 of the Nocardiosis-Associated Polyketide (NOCAP) Synthase

Assembly line polyketide synthases (PKSs) possess multimodular architectures in which each module harbors the requisite protein domains to catalyze a single round of polyketide chain elongation and postelongation modifications. Exceptions to this paradigm are modules that catalyze multiple elongation cycles, a phenomenon referred to as "programmed iteration". The molecular mechanism that allows PKS modules to iterate remains poorly understood. For example, Module 5 of the nocardiosis-associated polyketide (NOCAP) synthase catalyzes three elongation cycles during the biosynthesis of its undecaketide product, although in the absence of downstream modules, it has been shown to catalyze five elongation cycles. To understand the context-dependent control of its iterative capacity, we combined <i>in vitro</i> analysis of purified Module 5 of the NOCAP synthase with <i>in vivo</i> studies in <i>Escherichia coli</i>. Our findings reveal that, while the ability to iterate is an inherent property of Module 5, protein-protein interactions with its downstream module (Module 6) are key determinants of the number of elongation cycles catalyzed by Module 5 within the context of the complete assembly line. We also show that the intrinsic ability of Module 5 to iterate can be strongly influenced by the identity of its substrate. Our findings highlight the potential of Module 5 of the NOCAP synthase to reveal fundamentally new insights into the mechanistic differences between iterative and assembly line PKSs.