Modular polyketide synthases (PKSs) are biosynthetic assembly lines that construct structurally diverse natural products with wide-ranging applications in medicine and agriculture. Various mechanisms contribute to structural diversification during PKS-mediated chain assembly, including dehydratase (DH) domain-mediated elimination of water from R and S-configured 3-hydroxy-thioesters to introduce E- and Z-configured carbon–carbon double bonds, respectively. Here we report the discovery of a DH domain variant that catalyzes the sequential elimination of two molecules of water from a (3R, 5S)-3,5-dihydroxy thioester during polyketide chain assembly, introducing a conjugated E,Z-diene into various modular PKS products. We show that the reaction proceeds via a (2E, 5S)-2-enoyl-5-hydroxy-thioester intermediate and involves an additional universally conserved histidine residue that is absent from the active site of most conventional DH domains. These findings expand the diverse range of chemistries mediated by DH-like domains in modular PKSs, highlighting the catalytic versatility of the double hotdog fold.
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The minimum dataset necessary to interpret, verify and extend the research is provided in the main paper, extended data and supplementary information. The corresponding raw data (Figs. 2–5, Extended Data Figs. 1 and 2, Supplementary Table 1 and Supplementary Figs. 4 and 5), which were processed via standard deconvolution (intact protein mass spectrometry) and extracted ion chromatogram (LC–MS analyses) procedures, are available upon written request to the corresponding author.
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This research was supported by the Biotechnology and Biological Sciences Research Council (grant nos. BB/L021692/1, BB/K002341/1 and BB/M017982/1 to G.L.C.). M.J. and D.G. were supported by a Biotechnology and Biological Sciences Research Council Discovery Fellowship (grant no. BB/R012121/1) and the EPSRC MOAC Doctoral Training Centre (grant no. EP/F500378/1), respectively. M.R.-C. was supported by a mobility grant from the Government of Andorra (grant no. AM025-AND-2015). C.H. was supported by a PhD studentship from the University of Warwick. G.L.C. was the recipient of a Wolfson Research Merit Award from the Royal Society (grant no. WM130033).
D.M.R. is an employee and shareholder of Erebagen Ltd. G.L.C. is a Non-Executive Director, shareholder and paid consultant of Erebagen Ltd. The other authors declare no competing interests.
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Extended Data Fig. 1 Activity of the DH-like domain from module 13 of the gladiolin PKS towards 3S, 5S and 3S, 5R-configured stereoisomers of the natural substrate.
a, Deconvoluted mass spectrum of the DH-ACP di-domain following conversion of pantetheine thioester 12 to the corresponding CoA thioester and loading onto the apo-ACP domain. Negligible dehydration is observed. b, Deconvoluted mass spectrum of the DH-ACP di-domain following conversion of pantetheine thioester 13 to the corresponding CoA thioester and loading onto the apo-ACP domain. While a small quantity of mono-dehydrated product is observed, the major product is the holo-di-domain, which likely results from offloading of the substrate by δ-lactonization. The stereochemistry of the putative 2-enoyl-5-hydroxy and 2, 4-dienoyl thioesters resulting from single and double dehydration of the ACP thioesters derived from 12 and 13 are tentatively assigned based on established stereochemical relationships for other PKS DH domains (that is, R-configured hydroxyl groups result in E-configured alkenes and S-configured hydroxyl groups result in Z-configured alkenes). These stereochemical assignments could not be experimentally confirmed due to the very small quantities of singly and doubly dehydrated species produced.
Extended Data Fig. 2 Activity of the DH-like domain from module 13 of the gladiolin PKS towards the 3R, 5R-configured stereoisomer of the natural substrate.
a, Deconvoluted mass spectrum of the DH-ACP di-domain following conversion of pantetheine thioester 14 to the corresponding CoA thioester and loading onto the apo-ACP domain, showing the formation of singly and doubly dehydrated products. b, Extracted ion chromatograms (EICs) at m/z = 247.09 ± 0.02 from LC-MS comparisons of the cysteamine adducts of the 2, 4-dienoyl thioester resulting from double dehydration of the (3R, 5R)-3,5-dihydroxyacyl-ACP thioester by the DH-like domain (top) with synthetic standards of (2E, 4Z) (middle) and (2E, 4E)-2,4-dienoyl (bottom) panthetheine thioesters (10 and 11, respectively). c, Deconvoluted mass spectrum of the DH-ACP di-domain following conversion of pantetheine thioester 11 to the corresponding coenzyme A thioester and loading onto the apo-ACP domain, showing the formation of primarily a singly hydrated species. d, Deconvoluted mass spectrum of the DH-ACP di-domain following conversion of thioester 16 to the corresponding coenzyme A thioester and subsequent loading onto the apo-ACP domain, showing the formation of primarily the 2, 4-dienoyl thioester. All experiments were performed in triplicate and representative data are shown. e, Proposed mechanism for conversion of the (3R, 5R)-3,5-dihydroxyacyl-ACP thioester derived from 14 to the corresponding (2E, 4E)-2,4-dienoyl thioester by the DH-like domain. Hydrogen bonding of the C-3 and C-5 hydroxyl groups to Asp200 orients the substrate for E, E-diene formation.
Extended Data Fig. 3 Comparison of a model of the DH-like domain from module 13 of the gladiolin PKS with the structure of the DH-like domain from module 11 of the difficidin PKS and conservation of twin active site His residues in putative diene-forming DH-like domains from trans-AT PKSs.
a, Overlay of the AlphaFold model of the DH-like domain from the gladiolin PKS with the X-ray crystal structure of the DH-like domain from the difficidin PKS, highlighting the near identical positioning of the Asp and two His active site residues. b, Regions from a sequence alignment encompassing the two His residues observed in the active sites of the DH-like domains from the gladiolin and difficidin PKSs (H23/25 and H158/155, respectively), highlighting that they are universally conserved in other putative diene-forming DH-like domains from trans-AT PKSs. The labelling of DH-like domains corresponds to the subunit names and module numbers illustrated in Supplementary Fig. 1 (for example, Gbn_M13 is the DH-like domain from module 13 of GbnD5 subunit of the gladiolin PKS).
a, Dehydration of (3R)-3-hydroxyacyl thioesters yields the corresponding (2E)-2-enoyl thioester. b, Dehydration of (3S)-3-hydroxyacyl thioesters yields the Z-configured product. These reactions proceed via an E1CB mechanism involving a conserved active site His residue, which acts a general base / acid, and involve a net syn-elimination of the C-3 hydroxyl group and Hb. Hydrogen bonding of the C-3 hydroxyl group to a conserved active site Asp residue orients it in a gauche conformation with respect to Hb.
R1, R2 and R3 are residues corresponding to H44, Y168 and D206, respectively, of the DH domain from the EryAII subunit of the 6-DEB PKS (labelled EryDH1 in the figure). The functional group(s) introduced by each type of domain in trans-AT PKSs are shown to the left of the alignments. DH domains are labelled according to the order they appear in the PKS (that is, going from the N to C-terminus the first DH domain is labelled DH1, the second DH2 and so forth).
Neighbor-joining tree constructed from the sequences of DH-like domains from characterized trans-AT and cis-AT PKSs. Colored clades correspond to different catalytic functions in trans-AT PKSs, as follows: blue - 2-enoyl thioester synthases (DHe domains); red - 2, 4-dienoyl thioester synthases (DHd domains); green - 2-enoyl thioester isomerases (DHi domains); orange – tetrahydropyran/tetrahydrofuran synthases, (DHt domains). R1, R2 and R3 are the three conserved active site residues in the diene forming DH-like domains from trans-AT PKSs and the corresponding residues in other types of DH-like domain (see Extended Data Fig. 5). The structurally characterized DH-like domain from module 11 of the difficidin PKS (labelled DifDH8) and DH-like domain from module 13 of the gladiolin PKS (labelled GbnDH5) studied here are marked with asterisks, and examples of DH-like domains where the configuration of the diene in the final product differs from the predicted 2E, 4Z-configuration of the 2,4-dienoyl thioester intermediate are marked with black squares. An X between the DH and ACP domains indicates the presence of one or more other catalytic domains. DH domains are labelled according to the order they appear in the PKS (that is, going from the N to C-terminus, the first DH domain is labelled DH1, the second DH2 and so forth).
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Hobson, C., Jenner, M., Jian, X. et al. Diene incorporation by a dehydratase domain variant in modular polyketide synthases. Nat Chem Biol 18, 1410–1416 (2022). https://doi.org/10.1038/s41589-022-01127-y