Structural studies of transient and higher-order interactions in polyketide synthases Yves U. Tittes 1 , Hugo Muñoz-Hernandez 1 , Jamie R. Alley 2 , Dominik A. Herbst 1 , Callie R. Huitt-Roehl 2 , Jacob M. Kravetz 2 , Philip A. Storm 2 , Craig A. Townsend 2 and Timm Maier 1 1 Biozentrum, University of Basel, Switzerland, 2 Department of Chemistry, Johns Hopkins University, USA The multi-step biosynthesis of polyketides by polyketide synthases (PKSs) relies on the productive interplay of enzymes or enzymatic domains, often embedded as functional modules into large multifunctional proteins, with carrier proteins for substrate shuttling. Substantial progress has been made in understanding the architecture of individual domains and modules in PKSs, but studying transient interactions for substrate transfer in the context of multidomain proteins and visualising assemblies of PKS modules remain substantial challenges. We have analysed transient states of substrate transfer in an iterative non-reducing PKS based on specific crosslinking stabilisation of carrier protein interactions and cryo electron microscopy (cryoEM). We obtain snapshots of functional states that reveal intrinsic and state-specific interactions and dynamics within iterative non-reducing PKS. A single functional core is sufficient for polyketide biosynthesis by iterative PKS, but the most versatile families of modular PKS utilise assembly lines of connected functional modules. The organisation of modular PKS beyond a single module provides the framework for directed substrate transfer between modules, but experimental structural studies of higher-order PKS assemblies have been hindered by their considerable intrinsic flexibility. We have studied a modular PKS bimodule core fragment containing two substrate elongating condensing domains. CryoEM analysis of the bimodule core at multiple levels provides structural insights from high-resolution analysis of individual condensing domains to the arrangement of successive modules at intermediate resolution. Overall, our analyses emphasise the relevance of transient and dynamic interactions between domains in PKS and provide important information for mechanistic studies of PKS in vitro as well as for resolving the structural organisation of polyketide biosynthesis in vivo .
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© The Author(s), 2022
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