Structural basis for intermodular communication in assembly-line polyketide biosynthesis

Assembly-line polyketide synthases (PKSs) are modular multi-enzyme systems with considerable potential for genetic reprogramming. Understanding how they selectively transport biosynthetic intermediates along a defined sequence of active sites could be harnessed to rationally alter PKS product structures. Here, to investigate functional interactions between PKS catalytic and substrate acyl carrier protein (ACP) domains, we employed a bifunctional reagent to crosslink transient domain–domain interfaces of a prototypical assembly line, the 6-deoxyerythronolide B synthase, and resolved their structures by single-particle cryogenic electron microscopy (cryo-EM). Together with statistical per-particle image analysis of cryo-EM data, we uncovered interactions between ketosynthase (KS) and ACP domains that discriminate between intra-modular and inter-modular communication while reinforcing the relevance of conformational asymmetry during the catalytic cycle. Our findings provide a foundation for the structure-based design of hybrid PKSs comprising biosynthetic modules from different naturally occurring assembly lines.. Authors: Cogan, Dillon P. [Univ. of Southern California, Los Angeles, CA (United States)] (ORCID:0000000345632397); Soohoo, Alexander M. [Stanford Univ., CA (United States)] (ORCID:0009000684685212); Chen, Muyuan [SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL); Stanford Univ., CA (United States)]; Liu, Yan [SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL); Stanford Univ., CA (United States)] (ORCID:0000000156838695); Brodsky, Krystal L. [Stanford Univ., CA (United States)]. DOE Contract: AC02-76SF00515. Subjects: 59 BASIC BIOLOGICAL SCIENCES; biosynthesis; chemical tools; enzyme mechanisms; structural biology