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Analysis of heterocycle formation and stereochemical control by a non-ribosomal peptide synthetase condensation domain

Nature Synthesis (2026)Cite this article

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Abstract

Non-ribosomal peptide synthetases (NRPSs) produce diverse bioactive peptides, including antibiotics and antitumour agents, by integrating proteinogenic and non-proteinogenic amino acids alongside tailoring modifications, such as epimerization (E) and cyclization. Here we demonstrate that the tetrahydropyrimidine ring of pyoverdine is synthesized by the condensation (C) domain PvdL-C3, revealing cyclization capability of a canonical NRPS domain. Analyses of PvdL-E2C3A3 cross-module structures reveal a stable E-C didomain catalytic platform maintained by the donor communication-mediating domain. Through mutagenesis and evolutionary analyses, we identified key active site residues shaping the catalytic channel, controlling both efficiency and specificity. We further engineered PvdL-C3 to accept l-epimers, unveiling latent substrate flexibility. Ultimately, our work provides a unifying model where domain architecture and catalytic channel features collectively govern NRPS function, elucidating a cyclization mechanism and providing a structural blueprint for future enzyme engineering.

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Fig. 1: Module 3 of PvdL catalyses the tetrahydropyrimidine ring formation in pyoverdine.
Fig. 2: Cryo-EM structures of the cross-modular part of PvdL reveal a stable E2-C3 catalytic platform and its distinct conformational states.
Fig. 3: Structural snapshots of the PvdL-C3 domain in distinct binding states.
Fig. 4: Structure-guided mutations and catalytic mechanism of PvdL-C3.
Fig. 5: Phylogenetic, structural and mutational analysis of CcDab.
Fig. 6: Key residues controlling chirality in PvdL-C3.
Fig. 7: Structural basis for the reversal of stereoselectivity in the C₃-R3393A mutant.

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Data availability

All data that support the findings of this study are available within the tables and figures in Supplementary Information. The 3D cryo-EM maps generated in this study have been deposited in the Electron Microscopy Data Bank (https://www.emdataresource.org/) under accession numbers EMD-64387, EMD-62988, EMD-62989 and EMD-62990 (Supplementary Table 4). The atomic coordinates have been deposited in the Protein Data Bank (https://www.rcsb.org) under accession numbers 9UP2, 9LCO, 9LCP and 9LCQ. Cryo-EM maps are available via figshare at https://doi.org/10.6084/m9.figshare.30618215 (ref. 72). Source data are provided with this paper.

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Acknowledgements

We thank the National Facility for Protein Science in Shanghai (NFPS), Zhangjiang Laboratory, for collecting the cryo-EM images used in this study. The computations in this paper were run on the π 2.0 cluster supported by the Center for High Performance Computing at Shanghai Jiao Tong University. This work was financially supported by the National Key R&D Program of China (grants 2021YFA0910500 to J.L.), the National Science Foundation of China (grants 32270033 and 22577073 to Z.W., 32271302 to J.L., 32201035 to J.W., 32171252 to Z.D.), the China Postdoctoral Science Foundation (grants 2022T150412 and 2022M720085 to J.W.) and the Shanghai ‘Super Postdoctoral’ Incentive Program (grant 202112 to J.W.), Shanghai Pilot Program for Basic Research-Shanghai Jiao Tong University. We declare that the English grammar and flow of the text were polished by using Google’s Gemini large language models.

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  1. These authors contributed equally: Wei Cao, Jialiang Wang, Shyue Leh Chen.

Authors and Affiliations

  1. State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China

    Wei Cao, Jialiang Wang, Shyue Leh Chen, Dandan Li, Xiaozheng Wang, Zixin Deng, Jingdan Liang & Zhijun Wang

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Contributions

Z.W., W.C., J.W. and J.L. conceived of the study. W.C. and S.L.C. performed the biochemical reconstitution and mutagenesis experiments. W.C., J.W. and D.L. conducted the protein structural analysis and W.C. and X.W. performed the compound synthesis and analysis. Data curation was performed by W.C. and J.W. and formal analysis was conducted by Z.W., W.C., J.W. and J.L. The original draft of the paper was written by W.C. and Z.W. and all authors contributed to the review and editing. The project was supervised by J.L. (biochemical experiments) and Z.W. (biochemical and structural analysis experiments). Overall project administration was handled by Z.W. and Z.D., who also led the funding acquisition.

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Correspondence to Zhijun Wang.

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Supplementary information

Supplementary Information (download PDF )

Supplementary Methods, Tables 1–6 and Figs. 1–33.

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Supplementary Video 1 (download MP4 )

3D variability analysis (3DVA) of the A conformation dataset revealing an oscillation of approximately 20° of the E2 domain relative to the C3 structural domain.

Supplementary Video 2 (download MP4 )

3D variability analysis (3DVA) of the D-conformation dataset showing the oscillation of the E2 domain relative to the C3 domain, and the movement of PCP2 from the E2 active site to the C3 donor site.

Supplementary Video 3 (download MP4 )

3D variability analysis (3DVA) illustrating the conformational switch of the C3 domain between open and closed states, synchronized with the binding and dissociation of the PCP2 domain.

Supplementary Data 1 (download XLSX )

Source data for the chiral analysis bar chart presented in Supplementary Fig. 28.

Source data

Source Data Fig. 2 (download TXT )

Source data for the COMD mutation analysis bar chart (Fig. 2h) and the sequence conservation analysis (Fig. 2i).

Source Data Fig. 4 (download XLSX )

Source data for the relative production bar charts presented in Fig. 4b,d.

Source Data Fig. 5 (download TXT )

Source data for the phylogenetic tree analysis (Fig. 5a) and the enzymatic activity bar chart (Fig. 5c).

Source Data Fig. 6 (download TXT )

Source data for the multiple sequence alignment (Fig. 6a) and relative abundance bar chart (Fig. 6c).

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Cao, W., Wang, J., Chen, S.L. et al. Analysis of heterocycle formation and stereochemical control by a non-ribosomal peptide synthetase condensation domain. Nat. Synth (2026). https://doi.org/10.1038/s44160-026-01014-7

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