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Assembly-line polyketide synthases, such as the 6-deoxyerythronolide B synthase (DEBS), are large enzyme factories prized for their ability to produce specific and complex polyketide products. By channeling 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. Additionally, using cryogenic electron microscopy to study DEBS module 1, we present a structural model describing this substrate-channeling phenomenon. Our 3.2- to 4.3-angstrom-resolution structures of the intact module reveal key domain-domain interfaces and highlight an unexpected module asymmetry. We also present the structure of amore » product-bound module that shines light on a recently described “turnstile” mechanism for transient gating of active sites along the assembly line.« less

Assembly line polyketide synthases (PKSs) are large multimodular enzymes responsible for the biosynthesis of diverse antibiotics in bacteria. Structural and mechanistic analysis of these megasynthases can benefit from the discovery of reagents that recognize individual domains or linkers in a site-specific manner. Monoclonal antibodies not only have proven themselves as premier tools in analogous applications but also have the added benefit of constraining the conformational flexibility of their targets in unpredictable but often useful ways. Here we have exploited a library based on the naïve human antibody repertoire to discover a F_ab (3A6) that recognizes the terminal thioesterase (TE) domainmore » of the 6-deoxyerythronolide B synthase with high specificity. Biochemical assays were used to verify that 3A6 binding does not inhibit enzyme turnover. The co-crystal structure of the TE–3A6 complex was determined at 2.45 Å resolution, resulting in atomic characterization of this protein–protein recognition mechanism. F_ab binding had minimal effects on the structural integrity of the TE. In turn, these insights were used to interrogate via small-angle X-ray scattering the solution-phase conformation of 3A6 complexed to a catalytically competent PKS module and bimodule. Furthermore, we have developed a high-affinity monoclonal antibody tool that recognizes the TE domain of the 6-deoxyerythronolide B synthase while maintaining its native function.« less

Catalytic modules of assembly-line polyketide synthases (PKSs) have previously been observed in two very different conformations—an “extended” architecture and an “arch-shaped” architecture—although the catalytic relevance of neither has been directly established. By the use of a fully human naïve antigen-binding fragment (F_ab) library, a high-affinity antibody was identified that bound to the extended conformation of a PKS module, as verified by X-ray crystallography and tandem size-exclusion chromatography–small-angle X-ray scattering (SEC–SAXS). Kinetic analysis proved that this antibody-stabilized module conformation was fully competent for catalysis of intermodular polyketide chain translocation as well as intramodular polyketide chain elongation and functional group modification ofmore » a growing polyketide chain. Furthermore, the extended conformation of a PKS module is fully competent for all of its essential catalytic functions.« less