Structural Basis for Intermodular Communication in Assembly-Line Polyketide Biosynthesis

Assembly-line polyketide synthases are large multienzyme systems with considerable potential for genetic reprogramming. To investigate the mechanisms by which reactive biosynthetic intermediates are directionally channeled across a defined sequence of active sites in a naturally occurring assembly line, we employed a bifunctional reagent to crosslink transient domain-domain interfaces of the 6-deoxyerythronolide B synthase. Structural resolution of these crosslinked states by single-particle cryogenic electron microscopy (cryo-EM) together with statistical per-particle image analysis of cryo-EM data revealed ketosynthase – acyl carrier protein (KS-ACP) interactions that discriminate between intra- and inter-modular communication, while reinforcing the relevance of asymmetric conformations during the catalytic cycle. Our findings provide a new foundation for the structure-based design of hybrid polyketide synthases comprised of biosynthetic modules from different assembly lines.