State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and School of Life Science & Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
National Facility for Protein Science in Shanghai, Chinese Academy of Sciences, Shanghai, 201204, China.
Nat Commun. 2022 Feb 1;13(1):592. doi: 10.1038/s41467-022-28284-x.
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.
非核糖体肽合成酶（NRPSs）是模块化的装配线巨型酶，能够合成具有广泛生物活性的各种代谢物。其合成延伸的结构动力学仍不清楚。在这里，我们展示了 PchE（一种 NRPS 延伸模块）在不同构象下的冷冻电镜结构。结构域组织揭示了独特的“H”形头对头二聚体架构。在杂环化结构域内捕获芳基和肽酰载体蛋白连接的底物和中间体，以及在腺苷酸化结构域内捕获 L-半胱氨酸腺苷酸，说明了催化和识别残基。由腺苷酸化 C 末端亚结构域引导的多层次结构转变，结合插入的差向异构酶和杂环化隧道的构象变化，由两个残基控制。此外，我们还可视化了从硫醇化到差向异构化的完整催化循环的直接结构动力学。本研究确立了 PchE 的催化轨迹，并为新型药物制剂的生产提供了对插入结构域的二聚 NRPS 进行合理重新设计的思路。
