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Terminal alkyne formation by a pyridoxal phosphate-dependent enzyme

Nature Chemical Biology volume 22, pages 77–86 (2026)Cite this article

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Abstract

Terminal alkyne-containing natural products can undergo the bio-orthogonal ‘click’ reaction of Cu(I)-catalyzed azide–alkyne cycloaddition. Recently, an enzymatic mechanism for terminal alkyne formation was discovered in the biosynthesis of l-β-ethynylserine where the pyridoxal phosphate-dependent enzyme BesB forms a rare terminal alkyne-containing amino acid, l-propargylglycine, from a vinyl halide precursor, 4-chloro-l-allylglycine. Here we present the 1.3-Å-resolution crystal structure of BesB with detailed mechanistic and computational studies. We demonstrate that BesB can reversibly catalyze the exchange of the halogen in various 4-halo-allyl-l-glycines, implying the existence of an allene intermediate, which we then also observe. Taken together, this work supports a mechanism whereby an allene is formed from deprotonation-driven halogen loss and the terminal alkyne is then formed by isomerization of the allene. Our work further expands our understanding of the catalytic repertoire of pyridoxal phosphate-dependent enzymes and will enable development of metal-free allene-forming and alkyne-forming biocatalysts.

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Fig. 1: Terminal alkyne biosynthetic pathways.
Fig. 2: Crystal structure of SAcBesB.
Fig. 3: SAcBesB-catalyzed in vitro halide exchange.
Fig. 4: Allenylglycine generated by SAcBesB.
Fig. 5: Computational investigations on the proposed mechanism for BesB-catalyzed terminal alkyne bond formation.
Fig. 6: Proposed mechanism for BesB-based biosynthesis of a terminal alkyne bond.

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Data availability

The data that support the findings of this work are available in the article and its Supplementary Information. The crystallographic data were deposited to the PDB under accession codes 9AUA and 9AUB. Data are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2021-02626 to K.S.R.), the National Institutes of Health (R01 GM134271 to M.C.Y.C.), the National Science Foundation (CLP-2204014 to M.C.Y.C.), the Spanish MICINN (PID2022-141676NB-I00 and TED2021-130173B-C42 projects and RYC2020-028628-I fellowship to M.G.-B.) and the Generalitat de Catalunya AGAUR (2021SGR00623 project to M.G.-B.). J.A.M. would like to thank the HHMI Gilliam Fellowship for support. C.C.-T. is supported by the Eric and Wendy Schmidt AI in Science Postdoctoral Fellowship, a Schmidt Futures program. Some of the computational resources used were funded by FEDER and Spanish MINECO through projects CTQ2014-54306-P, CTQ2015-69363-P and MPCUdG2016/096 and by the European Research Council under the European Union’s ERC-StG-2015 (grant agreement 679001). We are grateful to the Canadian Light Source for access to beamline CMCF-BM and to the SSRL, SLAC National Accelerator Laboratory, for access to beamline 9-2.

Author information

Author notes

  1. Jason B. Hedges

    Present address: Xilio Therapeutics, Waltham, MA, USA

  2. Jorge A. Marchand

    Present address: Department of Chemical Engineering, University of Washington, Seattle, WA, USA

  3. Carla Calvó-Tusell

    Present address: Department of Molecular Biology, University of California, San Diego, La Jolla, CA, USA

Authors and Affiliations

  1. Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada

    Jason B. Hedges, Zi-Wang Wei & Katherine S. Ryan

  2. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA

    Jorge A. Marchand, Douglas C. Millar & Michelle C. Y. Chang

  3. Institute of Computational Chemistry and Catalysis, and Department of Chemistry, University of Girona, Girona, Spain

    Carla Calvó-Tusell & Marc Garcia-Borràs

  4. Department of Chemistry, University of California at Berkeley, Berkeley, CA, USA

    Michelle C. Y. Chang

  5. Department of Chemistry, Princeton University, Princeton, NJ, USA

    Michelle C. Y. Chang

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Contributions

J.B.H. carried out the structural and biochemical studies on SAcBesB. J.A.M. and D.C.M. carried out the biochemical studies on SSpBesB and SCaBesB and synthesized compound 5. C.C.-T. carried out the computational studies. Z.-W.W. carried out the kinetic analysis. M.G.-B. directed the computational studies, M.C.Y.C. directed the biochemical studies and K.S.R. directed the structural and biochemical studies. J.B.H., J.A.M., C.C.-T., M.G.-B., M.C.Y.C. and K.S.R. prepared the paper with feedback from all authors.

Corresponding authors

Correspondence to Marc Garcia-Borràs, Michelle C. Y. Chang or Katherine S. Ryan.

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Nature Chemical Biology thanks Yang Hai, Francesco Marchesani, Riccardo Miggiano, Andrea Mozzarelli, Darrin York and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Biosynthetic routes to internal alkyne-containing natural products.

a) Terminal alkynes can be synthesized using non-heme diiron-dependent enzymes. Similar non-heme diiron-dependent enzymes also catalyzed the formation of internal alkyne moieties found in several polyacetylenic fatty acids. Crep16 and Crep1-like desaturases have been characterized in the biosynthesis of crepenynic acid7, falcarindiol8, and dihydromatricaria acid9. b) An additional pathway to form alkynes has been discovered for the biosynthesis of the 1,3-enyne-containing cyclohexanoids, which are generated by P450-dependent oxidases such as AtyI and BisI, in the biosynthesis of asperpentyn13 and biscognienyne B14, respectively.

Extended Data Fig. 2 Purification and activity data for SAcBesB.

a) Propargylglycine biosynthetic gene cluster in S. achromogenes. b) SDS-PAGE gel of purified SAcBesB. c) Gel filtration data for SAcBesB. Protein standards for alcohol dehydrogenase, bovine serum albumin, and carbonic anhydrase indicated. d) Compound 3 production by SAcBesB using 1 as a substrate. e) Compound 3 production by SAcBesB using 2 as a substrate. Reactions repeated in triplicate with representative results shown.

Source data

Extended Data Fig. 3 Kinetics of the SAcBesB reaction.

Steady state kinetic analysis of BesB with respect to its substrate 4-chloroallylglycine (1). Each chosen concentration (20 μM to 2 mM) of 1 was performed in triplicate. Data points were subjected to non-linear curve fitting to Michaelis-Menten equation to obtain the kinetic parameters kcat = 0.0228 ± 0.0003 s-1, KM = 47 ± 3 μM.

Extended Data Fig. 4 Identification of stable PLP-pyruvic acid (PLP-PA) adduct in SAcBesB purified from Rhodococcus.

a) Example of the orange crystals of SAcBesB. b) UV-Vis spectrum of a SAcBesB crystal, with an extended absorbance at ~ 460 nm, indicating the bound PLP-adduct also persists throughout crystallographic experiments; however, we note the spectra for this sample was not of high quality. c) Examples of possible PLP-adducts which could be bound in the crystals. d) HPLC trace of PLP released from SAcBesB by treatment with KOH. PLP and PLP-PA are chemical standards, and the PLP-PA standard includes residual PLP from the synthesis. e) LC-MS analysis of the PLP released from SAcBesB. Traces depict the extracted ion chromatograms at m/z 318, which correspond to the PLP-PA adduct. High-resolution mass spectrometry was also used to verify the chemical formula of this compound as C11H13NO8P ([M + H]+ observed: 318.0377, [M + H]+ expected: 318.0379). f) Refinement of PLP-PA adduct in the electron density present. Left shows the clear separation between the catalytic lysine and the electron density present. Middle panel shows PLP-PA fit to the electron density present. Right panel shows the density after refinement. 2Fo-Fc electron density is shown in blue, contoured at 1.0 σ. Fo-Fc electron density is shown in green and red, and contoured at 3.0 σ and -3.0 σ respectively. PLP-PA adduct created used Phenix eLBOW. g) Abbreviated chemical mechanism for formation of the PLP-PA adduct from 2-aminoacrylate.

Extended Data Fig. 5 Sequence logos for BesB a) and other members of PF01053b).

Residues shown in both sets of sequence logos are the residues shown in stick form in Fig. 2e-f. The left set of logos shows the conserved Tyr and Arg involved in binding the phosphate of PLP. The right logo demonstrates the conservation of phenylalanine in BesB, and the YGG sequence which is most common amongst other PF01053 enzymes, as well as the conservation of other PLP-binding residues between the two enzymes. Sequence logos created using WebLogo 374.

Extended Data Fig. 6 SCaBesB-catalyzed halogen exchange.

Halogen exchange results for a) 4-iodo-allylglycine and b) 4-fluoro-allylglycine, with 1 and 2 as substrates in HEPES buffer (pH 7.5).

Extended Data Fig. 7 Reversibility of the reverse reaction as demonstrated by solvent exchange in D2O with SSpBesB.

a) All four non-acidic protons of 3 exchanged with D2O solvent when SSpBesB is present (Ammonium Bicarbonate Buffer, pH = 7.8). b) The masses detected by LC/MS-QTOF for each exchange of 3 in D2O.

Extended Data Fig. 8 Orientation of the allenylamine (10) in WT enzyme and F231Y variant.

a) MD simulations show that the presence of Tyr231 induces a reorientation of the allene moiety in the active site of F231Y variant. The ∠(C1 – C2 – N1 – C3) dihedral angle measured along independent MD trajectories (3 replicas of 500 ns for each for both the WT and F231Y variant) describes the reorientation of the allene moiety in BesB active site. In the right panel, the representation of the normalized kernel density plot of the monitored dihedral angle along the MD simulations for allenylamine (10) (see the left figure) for WT enzyme (depicted in gray) and F231Y variant (depicted in blue) are shown. b) Representative snapshot corresponding to the most populated clusters extracted from Molecular Dynamics (MD) simulations describing the interactions between allenylimine (10), Arg61, Asp233 and Phe/Tyr231. As shown in the figure, the interaction between Asp233, Arg61 and PLP-phosphate is maintained in both the WT enzyme and F231Y variant. However, repositioning of the allene moiety with respect to Arg61 in F231Y prevents the deprotonation by PLP-phosphate group. The introduction of Tyr231 affects the geometric stability of the different species in the BesB active site and the orientation of the alkene/allene moiety with respect to possible deprotonation groups. While along MD simulation of ketimine (8) Tyr231 shows a similar conformation that the one adopted for Phe231 in the WT enzyme, along the simulations with intermediate-9 and allenylamine (10), residue Tyr231 samples different orientations and interacts directly with the PLP-phosphate group or other residues found in the active site (Asp233 and Asp305). This interaction causes a rearrangement of the species in the active site, as well as affects the stability of the different species. For the F231Y variant, the analysis suggests that the two deprotonation sources -Asp233 and the PLP-phosphate group- (that showed low deprotonation barriers in DFT calculations, see Supplementary Fig. 29) are not close to the Hd of the intermediates for an efficient deprotonation (see Supplementary Figs. 45, 46). These results may explain why the tyrosine at position 231 contributes to trap the allenic intermediate. See additional details in Supplementary Fig. 48.

Supplementary information

Supplementary Information

Supplementary Methods, Tables 1–5, Figs. 1–50 and references.

Reporting Summary

Supplementary Data 1

Cartesian coordinates.

Source data

Source Data Extended Data Fig. 2

The uncropped gel of purified BesB at different concentrations. The rightmost lanes show the gel presented in Extended Data Fig. 2b.

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Hedges, J.B., Marchand, J.A., Calvó-Tusell, C. et al. Terminal alkyne formation by a pyridoxal phosphate-dependent enzyme. Nat Chem Biol 22, 77–86 (2026). https://doi.org/10.1038/s41589-025-01954-9

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