Statins are effective cholesterol-lowering drugs. Lovastatin, one of the precursors of statins, is formed from dihydromonacolin L (DML), which is synthesized by lovastatin nonaketide synthase (LovB), with the assistance of a separate trans-acting enoyl reductase (LovC). A full DML synthesis comprises 8 polyketide synthetic cycles with about 35 steps. The assembling of the LovB–LovC complex, and the structural basis for the iterative and yet permutative functions of the megasynthase have remained a mystery. Here, we present the cryo-EM structures of the LovB–LovC complex at 3.60 Å and the core LovB at 2.91 Å resolution. The domain organization of LovB is an X-shaped face-to-face dimer containing eight connected domains. The binding of LovC laterally to the malonyl-acetyl transferase domain allows the completion of a L-shaped catalytic chamber consisting of six active domains. This architecture and the structural details of the megasynthase provide the basis for the processing of the intermediates by the individual catalytic domains. The detailed architectural model provides structural insights that may enable the re-engineering of the megasynthase for the generation of new statins.

This article provides a comprehensive overview of the structural enzymology of type I iPKSs, focusing on the individual domain structures, catalytic mechanisms and the representative programming activities that each domain contributes.

Nonribosomal peptide synthetases (NRPSs) are modular assembly-line megaenzymes that synthesize diverse metabolites with wide-ranging biological activities. The structural dynamics of synthetic elongation has remained unclear. Here, we present cryo-EM structures of PchE, an NRPS elongation module, in distinct conformations. The domain organization reveals a unique “H”-shaped head-to-tail dimeric architecture. The capture of both aryl and peptidyl carrier protein-tethered substrates and intermediates inside the heterocyclization domain and l-cysteinyl adenylate in the adenylation domain illustrates the catalytic and recognition residues. The multilevel structural transitions guided by the adenylation C-terminal subdomain in combination with the inserted epimerase and the conformational changes of the heterocyclization tunnel are controlled by two residues. Moreover, we visualized the direct structural dynamics of the full catalytic cycle from thiolation to epimerization. This study establishes the catalytic trajectory of PchE and sheds light on the rational re-engineering of domain-inserted dimeric NRPSs for the production of novel pharmaceutical agents.

Assembly-line polyketide synthases (PKSs) are molecular factories that produce diverse metabolites with wide-ranging biological activities. PKSs usually work by constructing and modifying the polyketide backbone successively. Here, we present the cryo-EM structure of CalA3, a chain release PKS module without an ACP domain, and its structures with amidation or hydrolysis products. The domain organization reveals a unique “∞”-shaped dimeric architecture with five connected domains. The catalytic region tightly contacts the structural region, resulting in two stabilized chambers with nearly perfect symmetry while the N-terminal docking domain is flexible. The structures of the ketosynthase (KS) domain illustrate how the conserved key residues that canonically catalyze C–C bond formation can be tweaked to mediate C–N bond formation, revealing the engineering adaptability of assembly-line polyketide synthases for the production of novel pharmaceutical agents.

Nonribosomal peptide synthetases (NRPS) are essential for the biosynthesis of therapeutically valuable molecules, including antibiotics, immunosuppressants, and anticancer agents. The assembly-line mechanism of NRPS offers significant potential for engineering novel natural products through reprogramming. However, the challenging purification of NRPS proteins has impeded the investigation of their assembly and catalytic mechanisms. In this study, we employed homologous recombination to insert a purification tag at the C-terminus of the NRPS gene within the chromosome. This genetic modification enabled efficient purification of NRPS proteins from the tagged mutant strain using a one-step affinity chromatography approach. Additionally, we discovered that MbtH-like proteins (MLPs) form stable complexes with all pyoverdine (PVD) NRPS subunits, allowing for the purification of the entire NRPS assembly line via tagged MLP. Negative stain electron microscopy analysis revealed that the purified PVD NRPS proteins exist as dynamically linear monomers. Our in-situ tag-based purification method enhances NRPS research in both biochemical and structural biology, providing a robust platform for further investigations into NRPS mechanisms and applications.