Mapping the catalytic conformations of an assembly-line polyketide synthase module by single-particle cryo-EM

Assembly-line polyketide synthases, such as the 6-deoxyerythronolide B synthase (DEBS), are large multienzyme systems prized for their ability to produce specific and complex polyketide products. By transferring protein-tethered substrates across multiple active sites in a defined linear sequence, these enzymes facilitate programmed small molecule syntheses that could theoretically be harnessed to access countless polyketide product structures. Using single particle cryogenic electron microscopy (cryo-EM) to study DEBS module 1, we present the structural basis for inter-polypeptide substrate channelling as well as a model for structural dynamics during its catalytic cycle. Multiple near-atomic resolution structures (3.2–4.1 Å) reveal key domain-domain interfaces and an unexpected module asymmetry. We present an additional structure of DEBS module 1 in its product-bound state (4.3 Å) to rationalize a recently described “turnstile” mechanism that underlies vectorial biosynthesis by these assembly lines. Finally, we report on site- and domain-specific crosslinking to validate the observed structures as well as to unlock previously unknown conformations by single particle cryo-EM.