Abstract
The functional diversification of biosynthetic enzymes underlies the chemical richness of natural products, yet how primary metabolic enzymes evolve to acquire specialized functions in secondary metabolism remains elusive. Here, we report a tripartite enzyme complex from oral Streptococcus species—comprising 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS), acetyl-CoA acetyltransferase (ACAT), and a DUF35 protein—that catalyzes an unusual Friedel–Crafts C-acetylation on a pyrrolidine-2,4-dione scaffold, completing the biosynthesis of the antibiotic reutericyclin A. Cryo-electron microscopy of the S. macacae-derived thiolase complex (SmaATase) reveals a conserved architecture resembling the archaeal HMGS/ACAT/DUF35 complex involved in the mevalonate pathway, yet with key catalytic residues rewired to reprogram substrate specificity. Biochemical characterization, molecular modeling, and evolutionary analysis confirmed that the ancestral activity of HMG-CoA synthesis has been lost, while the complex has been repurposed to mediate Friedel–Crafts C-acylation of small molecule acceptors. These findings reveal a rare example of thiolase complex neofunctionalization, shedding light on an underexplored trajectory in enzyme evolution and offering a template for engineering C–C bond-forming catalysts in synthetic biology.
Data availability
Data supporting the findings of this work are available within the paper and its Supplementary Information files. The cryo-EM structures and corresponding density maps are available in the Protein Data Bank under accession codes 9VBO (MucA4B2C2) and 9VBT (MucA4B4C4), and in the Electron Microscopy Data Bank database under accession codes EMD-64929 (MucA4B2C2) and EMD-64933 (MucA4B4C4). The crystal structures of PpATase (5MG5) and the archaeal HMGS/ACAT/DUF35 complex (6ESQ) were used in this study for structural comparison. The protein mass spectrometry data have been deposited to the ProteomeXchange Consortium via the iProX72,73 partner repository with the dataset identifier PXD072906. MD simulation files, including the initial and final coordinates along with the parameter files, are deposited in the Figshare repository https://doi.org/10.6084/m9.figshare.30646505. Source data are provided with this paper.
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Acknowledgements
This work has been supported by the National Natural Science Foundation of China (32401633 to G.L., 82173719 to X.T., and 32322039 to X.P.), Guangdong Basic and Applied Basic Research Foundation (2023A1515111192 to G.L. and 2024A1515010922 to X.T.), Guangdong S&T Program (2024B1111160007 to X.T.), Shenzhen Science and Technology Program (KJZD20240903101104007 to X.T.), Shenzhen Bay Laboratory Startup Fund (21230051 to X.T. and QH28001 to K.K.H.), and the Major Program of Shenzhen Bay Laboratory (C1012523006 and S211101001 to X.T.). The authors are grateful to T. Zheng and E. Li from the Multi-Omics Mass Spectrometry Core of Shenzhen Bay Laboratory for the help with IMS-TOF mass measurements, H.Yin and J.Zheng from Shenzhen Medical Academy of Research and Translation for the acquisition of Orbitrap mass spectrometry data, and K.Wang from Refeyn Co., Ltd. for the assistance with MP measurements. We thank L. Dai (Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences) for providing S. salivarius DA547 and S. oralis DA1241.
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X.T. and G.L. conceived and designed the project. G.L., R.S., A.W., and C.P. conducted biochemical experiments, metabolic profiling, and antibacterial activity assays. Z.S. (Zilin Shen) and Z.L. acquired the cryo-EM data and performed structural modeling and refinement under the supervision of X.P., G.W. performed bioinformatic analyses and genome mining under the supervision of Y.-X.L., and F.L. carried out native mass spectrometry experiments under the supervision of K.K.H. Z.S. (Zhuanglin Shen) performed molecular docking and molecular dynamic simulations. C.M. executed chemical synthesis and compound isolation. G.L. and X.T. prepared the manuscript with input from all authors. X.T. supervised the project.
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Liao, G., Sun, R., Shen, Z. et al. Evolutionary repurposing of a metabolic thiolase complex enables antibiotic biosynthesis. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68910-6
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DOI: https://doi.org/10.1038/s41467-026-68910-6
