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Structural insights into two thiamine diphosphate-dependent enzymes and their synthetic applications in carbon–carbon linkage reactions

Nature Chemistry volume 17, pages 1107–1118 (2025)Cite this article

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Abstract

The α-hydroxy-β-keto acid synthases are thiamine diphosphate-dependent enzymes catalysing carbon–carbon linkage reactions in the biosynthesis of primary metabolites and various secondary metabolites. However, the substrate selectivity and catalytic stereoselectivity of α-hydroxy-β-keto acid synthases are poorly understood, greatly hindering their synthetic application in generating diverse carbon frameworks. We here report the discovery of two new α-hydroxy-β-keto acid synthases CsmA and BbmA, which show different substrate selectivities in catalysing carbon–carbon coupling reactions between two β-keto acids. Four crystal structures of CsmA or BbmA complexed with thiamine diphosphate and their substrates were determined, clearly revealing their structural bases of substrate selectivity and catalytic stereoselectivity. Substrate scope expansion enables us to generate 120 α-hydroxy-β-keto acids together with 240 NaBH4-reduction products. Furthermore, we applied CsmA and BbmA into enzymatic total synthesis, generating 36 γ-butyrolactone-containing furanolides. These results provide new structural insights into the catalyses of α-hydroxy-β-keto acid synthases and highlight their great potential in carboligation catalysis and synthetic applications.

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Fig. 1: The carbon–carbon linkage reactions catalysed by HKASs with two α-keto acids as the substrates.
Fig. 2: The CsmA-catalysed coupling reactions between different α-keto acids.
Fig. 3: The BbmA-catalysed coupling reactions between different α-keto acids.
Fig. 4: Crystal structures of CsmA–ThDP and BbmA–ThDP, and structure-based mutagenesis studies.
Fig. 5: Crystal structures of BbmA–ThDP–HPPA and BbmAG484F–ThDP–CBOA-covalent, and structure-based mutagenesis studies.
Fig. 6: The coupling reactions between different α-keto acids catalysed by CsmAG466F, BbmAL112S/W288T/G484F and BbmAG484F.
Fig. 7: The enzymatic total synthesis of furanolides using the characterized HKASs.

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Data availability

The structures of the CsmA–ThDP complex, BbmA–ThDP complex, BbmA–ThDP–HPPA complex and BbmAG484F–ThDP–CBOA-covalent complex have been deposited in the Protein Data Bank with accession numbers 8X3Z, 8X3Y, 8X3X and 8XOD, respectively. The gene sequences of csmA and csmB have been deposited in GenBank with accession numbers PP238510.1 and PP238511.1, respectively. Crystallographic data for small molecule structures have been deposited in the Cambridge Crystallographic Data Centre, under deposition numbers CCDC 2249922 (5b), CCDC 2249920 (6a), CCDC 2249926 (6b), CCDC 2297504 (13a), CCDC 2297502 (17b), CCDC 2253115 (40a), CCDC 2253125 (71b) and CCDC 2297497 (116a). Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/. DNA sequences, plasmids, strains, HPLC analyses of reactions, mass data, NMR data and spectra are available in the Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank F. Yin and H. Jia for X-ray diffraction tests, Q. Li and F. Liu for NMR data collection and J. Li and W. Ma for mass spectrometry data collection, from State Key Laboratory of Natural and Biomimetic Drugs (Peking University). We thank the National Center for Protein Sciences at Peking University for assistance with crystal screening. We thank the staff of the BL02U1/BL10U2/BL19U1 beam-lines of the National Facility for Protein Science in Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility for assistance during X-ray diffraction data collection. This research was supported by the National Key Research and Development Program of China (2023YFA0914102, M.M.) and the National Natural Science Foundation of China (92357305, M.M.) for experimental materials supply and data collection. This research was also supported by the National Key Research and Development Program of China (2023YFC3503900, M.M.) and the National Natural Science Foundation of China (82325046, M.M.; 82273829, M.M.; 22377004, D.Y.; 22107007, T.L.), and the China Postdoctoral Science Foundation (grant nos GZB20230046, T.L.; 2018M641123, G.W.).

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Author notes

  1. These authors contributed equally: Tan Liu, Guiyang Wang, Jiahui Yu, Mengyuan Li.

Authors and Affiliations

  1. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China

    Tan Liu, Guiyang Wang, Mengyuan Li, Jie Wang, Houhua Li, Min Ye, Donghui Yang & Ming Ma

  2. State Key Laboratory of Gene Function and Modulation Research, School of Life Sciences, Biomedical Pioneering Innovation Center (BIOPIC), Peking University, Beijing, China

    Jiahui Yu, Tianbo Peng & Xiao-dong Su

  3. Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University, Beijing, China

    Changtao Jiang

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  1. Tan Liu
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Contributions

M.M. conceived the project and designed the experiments. T.L., G.W., J.Y. and M.L. performed the experiments. T.P. carried out the MD simulations. J.W. helped with the chiral HPLC analysis. M.M., D.Y., X.d.S., C.J., M.Y. and H.L. analysed the experimental data. M.M., T.L., G.W., J.Y. and M.L. wrote the paper.

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Correspondence to Ming Ma.

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Extended data

Extended Data Fig. 1 HKAS-catalyzed α-keto acids coupling reactions in the biosynthesis of various natural products.

The red and blue moieties of the α-hydroxy-β-keto acids are derived from acyl donor and acceptor substrates, respectively.

Extended Data Fig. 2 Functional characterization of CsmA.

a, Acyloin-type natural products (1–3) isolated from C. sp. PKU-MA01392. b, HPLC analyses of CsmA-catalyzed reactions at different time points with IPA (S2) and HPPA (S3) as the substrates, and NaBH4 reduction of compound 1. The colours of the HPLC peaks corresponds to different substrates or products. c, The production of compounds 1, 4a, and 4b from CsmA catalysis.

Extended Data Fig. 3 Bioinformatic analysis of the putative HKASs.

a, Sequence alignment of ScyA and two candidate HKASs (CsmA and CsmB) from strain C. sp. PKU-MA01392. CsmA and CsmB share 26% and 24% sequence identities with ScyA, respectively. b, Phylogenetic analysis of CsmA, CsmB, and BbmA with known HKASs. CsmA, CsmB, and BbmA are shown in red, green, and blue, respectively. The coupling reaction types of these HKASs were labeled on the right of the enzyme names. MEGA 7.0 was used to construct the phylogenetic tree by maximum likelihood method. The PDB IDs or NCBI accession numbers of proteins are: 5FEM (ScAHAS), 6DEK(CaAHAS), 7EHE (ThAHAS), 6U9H (AtAHAS), 6LPI (EcAHAS), 1OZF (KpALS), 4RJJ (AlsS), WP_111325691.1 (ThZK0150), ALL53321.1 (NzsH), WP_012058936.1 (Cbei_2730), WP_012990202.1 (XclA), WP_063628719.1 (CybE) and WP_012408018.1 (ScyA) c, The encoding gene of CsmA and its neighboring genes. d, The encoding gene of CsmB and its neighboring genes that are responsible for branched-chain amino acid biosynthesis.

Extended Data Fig. 4 The conversion rates in the coupling reactions between IPA (S2) or HPPA (S3) and aliphatic α-keto acids catalyzed by CsmA, BbmA and the double mutants CsmAS110L/T278W, BbmAL112S/W288T.

(a)–(g), The total conversion rates of the two NaBH4-reduction products during the coupling reactions between IPA (S2) and PVA (S1, a), MOBA (S4, b), OPTA (S12, c), 3-MOVA (S13, d), OHA (S15, e), CPOA (S16, f), or CBOA (S17, g), respectively. (h)–(o), The total conversion rates of the two NaBH4-reduction products during the coupling reactions between HPPA (S3) and PVA (S1, h), MOBA (S4, i), OPTA (S12, j), 3-MOVA (S13, k), 4-MOVA (S14, l), OHA (S15, m), CPOA (S16, n), or CBOA (S17, o), respectively. All chromatograms are shown in the Supplementary Information (Supplementary Figs. 7 and 8). The conversion rates in percentile were determined from three original independent replicates of reactions (shown as dots) and the error bars were generated as mean values +/− SD (standard deviations) from the three independent replicates.

Source data

Extended Data Fig. 5 The conversion rates in type II coupling reactions catalyzed by CsmA, BbmA and the double mutants BbmAL112S/W288T.

(a)–(f), The total conversion rates of the two NaBH4 reduction products during the coupling reactions between IPA (S2) and HPPA (S3, a), PPA (S5, b), NOPA (S6, c), HMPPA (S7, d), OPBA (S8, e), or NPA (S9, f), respectively. g, The conversion rates in the homocoupling reactions of IPA (S2). (h)–(l), The total conversion rates of the two NaBH4-reduction products during the coupling reactions between HPPA (S3) and PPA (S5, h), NOPA (S6, i), HMPPA (S7, j), OPBA (S8, k), or NPA (S9, l), respectively. m, The conversion rates in the homocoupling reactions of HPPA (S3). All chromatograms are shown in the Supplementary Information (Supplementary Fig. 4). The conversion rates in percentile were determined from three original independent replicates of reactions (shown as dots) and the error bars were generated as mean values +/− SD (standard deviations) from the three independent replicates.

Source data

Extended Data Fig. 6 Structures of four HKASs in complex with ThDP cofactors.

a, The structure of CsmA-ThDP with two monomer molecules in one asymmetric unit. b, Amplified region of the ThDP (yellow stick) binding site in CsmA. c, Amplified region of the ADP (magenta stick) binding site in CsmA. d, The structure of BbmA-ThDP with two monomer molecules in one asymmetric unit. e, Amplified region of the ThDP (yellow stick) binding site in BbmA. f, Amplified region of the ADP (magenta stick) binding site in BbmA. g, The structure of AtAHAS with two monomer molecules in one asymmetric unit. h, Amplified region of the ThDP (yellow stick) binding site in AtAHAS. i, Amplified region of the FAD (cyan stick) binding site in AtAHAS. j, The structure of AlsS with two monomer molecules in one asymmetric unit. k, Amplified region of the ThDP (yellow stick) binding site in AlsS. l, Amplified region of the site (no FAD or ADP binding) in AlsS corresponding to the ADP binding site in CsmA and BbmA.

Extended Data Fig. 7 The MD simulations of acyl donor substrates in the active sites of CsmA and BbmA.

(a)–(d), The binding models of CsmA with acyl donors IPA (S2, a), HPPA (S3, b), OPBA (S8, c), or 4-MOVA (S14, d) from the MD simulations. (e)–(h), The binding models of BbmA with acyl donors OPTA (S12, e), 4-MOVA (S14, f), CBOA (S17, g), or IPA (S2, h) from the MD simulations. The ThDP are shown as yellow sticks. Hydrogen bond interactions are shown as yellow dashed lines, and hydrophobic interactions within 4 Å are shown as black dashed lines.

Extended Data Fig. 8 Crystal structures of BbmA-ThDP-HPPA and BbmAG484F-ThDP-CBOA-covalent, and structural comparisons of the active sites.

a, The structure of BbmA-ThDP-HPPA with two monomer molecules in one asymmetric unit. b, The structure of BbmAG484F-ThDP-CBOA-covalent shown with two monomer molecules. c, Structural comparison between the active sites of BbmA-ThDP (residues in cyan and ThDP in yellow) and BbmAG484F-ThDP-CBOA-covalent (residues in light pink and ThDP-CBOA-covalent in blue). d, Structural comparison between the active sites of BbmA-ThDP-HPPA (residues in magenta and ThDP in yellow) and BbmAG484F-ThDP-CBOA-covalent (residues in light pink and ThDP-CBOA-covalent in blue).

Extended Data Fig. 9 The α-keto acids coupling reactions catalyzed by BbmA and the mutants BbmAG484F, and sequence alignments of HKASs revealing the key residue for acyl acceptor selectivity.

a, LC-HRESIMS analysis of coupling reactions between IPA (S2) and individual aliphatic α-keto acids (S1, S4, or S12-S17) by BbmA and BbmAG484F. b, The key residues (highlighted with a red square) of known HKASs corresponding to G484 in BbmA by multiple sequence alignments.

Extended Data Fig. 10 The MD simulations of ThDP-acyl donor-Breslow intermediates and acyl acceptors in the active sites of CsmA or BbmA.

a, Model I: the initial binding of BbmA (cyan) with HPPA (green) and ThDP-CBOA-Breslow intermediate (blue) allowing an Si face attack. b, Model II: the initial binding of BbmA (cyan) with HPPA (orange) and ThDP-CBOA-Breslow intermediate (blue) allowing an Re face attack. c, Changes in the dihedral angle between the O-Cα-Cβ face and Cα-Cβ-Ha face during the 200 ns MD simulations for Model I and Model II. d, A representative MD snapshot at 110 ns for Model II, in which HPPA are shown as grey sticks. e, The MD simulations of CsmA (wheat) with HPPA (green) and ThDP-IPA-Breslow intermediate (blue) after 200 ns. f, The MD simulations of CsmA (wheat) with HPPA (green) and ThDP-OPBA-Breslow intermediate (blue) after 200 ns. g, The MD simulations of BbmA (cyan) with IPA (green) and ThDP-CBOA-Breslow intermediate (blue) after 200 ns. h, The MD simulations of BbmA (cyan) with HPPA (green) and ThDP-4-MOVA-Breslow intermediate (blue) after 200 ns.

Supplementary information

Supplementary Information

Supplementary Figs. 1–101 and Tables 1–8, including DNA sequences, plasmids, strains, HPLC analyses of reactions, mass data, NMR data and spectra.

Reporting Summary

Supplementary Data 1

Crystallographic data for compound 5b; CCDC reference 2249922.

Supplementary Data 2

Crystallographic data for compound 6a; CCDC reference 2249920.

Supplementary Data 3

Crystallographic data for compound 6b; CCDC reference 2249926.

Supplementary Data 4

Crystallographic data for compound 13a; CCDC reference 2297504.

Supplementary Data 5

Crystallographic data for compound 17b; CCDC reference 2297502.

Supplementary Data 6

Crystallographic data for compound 40a; CCDC reference 2253115.

Supplementary Data 7

Crystallographic data for compound 71b; CCDC reference 2253125.

Supplementary Data 8

Crystallographic data for compound 116a; CCDC reference 2297497.

Source data

Source Data Fig. 4

Original conversion rate source data.

Source Data Fig. 5

Original conversion rate source data.

Source Data Extended Data Fig. 4

Original conversion rate source data.

Source Data Extended Data Fig. 5

Original conversion rate source data.

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Liu, T., Wang, G., Yu, J. et al. Structural insights into two thiamine diphosphate-dependent enzymes and their synthetic applications in carbon–carbon linkage reactions. Nat. Chem. 17, 1107–1118 (2025). https://doi.org/10.1038/s41557-025-01822-y

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