Letter | Published:

New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition

Nature volume 522, pages 497501 (25 June 2015) | Download Citation


The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis1,2,3 or microbial biodegradation of aromatic compounds4,5,6, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear7,8,9. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad110. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor–cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry11,12, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.

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Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 4ZA4, 4ZA5, 4ZA7, 4ZA8, 4ZAB, 4ZA9, 4ZAA, 4ZAC and 4ZAD.


  1. 1.

    et al. Biosynthesis and physiology of coenzyme Q in bacteria. Biochim. Biophys. Acta 1837, 1004–1011 (2014)

  2. 2.

    , , , & The role of UbiX in Escherichia coli coenzyme Q biosynthesis. Arch. Biochem. Biophys. 467, 144–153 (2007)

  3. 3.

    , & Membrane-associated reactions in ubiquinone biosynthesis in Escherichia coli. 3-octoprenyl-4-hydroxybenzoate carboxy-lyase. Biochim. Biophys. Acta 436, 800–810 (1976)

  4. 4.

    Carboxylases in natural and synthetic microbial pathways. Appl. Environ. Microbiol. 77, 8466–8477 (2011)

  5. 5.

    , , & Anaerobic degradation of homocyclic aromatic compounds via arylcarboxyl-coenzyme A esters: organisms, strategies and key enzymes. Environ. Microbiol. 16, 612–627 (2014)

  6. 6.

    , , , & PAD1 and FDC1 are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae. J. Biosci. Bioeng. 109, 564–569 (2010)

  7. 7.

    et al. Crystal structure of a dodecameric FMN-dependent UbiX-like decarboxylate (Pad1) from Eschericia coli O157:H7. Protein Sci. 13, 3006–3016 (2004)

  8. 8.

    , , , & Structural insights into the UbiD protein family from the crystal structure of PA0254 from Pseudomonas aeruginosa. PLoS ONE 8, e63161 (2013)

  9. 9.

    et al. Mapping the structural requirements of inducers and substrates for decarboxylation of weak acid preservatives by the food spoilage mould Aspergillus niger. Int. J. Food Microbiol. 157, 375–383 (2012)

  10. 10.

    et al. UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis. Nature (2015)

  11. 11.

    Asymmetric 1,3-dipolar cycloadditions. Tetrahedron 63, 3235–3285 (2007)

  12. 12.

    & Theory of 1,3-dipolar cycloadditions: Distortion/Interaction and frontier molecular orbital models. J. Am. Chem. Soc. 130, 10187–10198 (2008)

  13. 13.

    , , & Decarboxylation mechanisms in biological system. Bioorg. Chem. 43, 2–14 (2012)

  14. 14.

    et al. Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution. Proc. Natl Acad. Sci. USA 111, 15066–15071 (2014)

  15. 15.

    et al. Structure-guided directed evolution of alkenyl and arylmalonate decarboxylases. Angew. Chem. 48, 7691–7694 (2009)

  16. 16.

    , , , & Distribution of genes encoding the microbial non-oxidative reversible hydroxyarylic acid decarboxylases/phenol carboxylases. Genomics 86, 342–351 (2005)

  17. 17.

    & Regulation of the isofunctional genes ubiD and ubiX of the ubiquinone biosynthetic pathway of Escherichia coli. FEMS Microbiol. Lett. 223, 67–72 (2003)

  18. 18.

    , & Structure of PA4019, a putative aromatic acid decarboxylase from Pseudomonas aeruginosa. Acta Crystallogr. F 67, 1184–1188 (2011)

  19. 19.

    et al. The decarboxylation of the weak-acid preservative, sorbic acids, is encoded by linked genes in Aspergillus spp. Fungal Genet. Biol. 47, 683–692 (2010)

  20. 20.

    , , , & Isofunctional enzymes Pad1 and UbiX catalyse formation of a novel cofactor required by ferulic acid decarboxylase and 4-hydroxy-3-polyprenylbenzoic acid decarboxylase. ACS Chem. Biol. 10, 1137–1144 (2015)

  21. 21.

    et al. Structural proteomics of an archaeon. Nature Struct. Biol. 7, 903–909 (2000)

  22. 22.

    & Flavoenzymes: versatile catalysts in biosynthetic pathways. Nat. Prod. Rep. 30, 175–200 (2013)

  23. 23.

    et al. Crystal structures of isoorotate decarboxylases reveal a novel catalytic mechanism of 5-carboxyl-uracil decarboxylation and shed light on the search for DNA decarboxylase. Cell Res. 23, 1296–1309 (2013)

  24. 24.

    & Synthetic applications of the carbonyl generating Grob fragmentation. Chem. Rev. 110, 3741–3766 (2010)

  25. 25.

    , , , & Enzyme-catalysed [4+2] cycloaddition is a key step in the biosynthesis of spinosyn A. Nature 473, 109–112 (2011)

  26. 26.

    et al. Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase. Proc. Natl Acad. Sci. USA 111, 8013–8018 (2014)

  27. 27.

    Functional diversity of organic molecule enzyme cofactors. Nat. Prod. Rep. 30, 1324–1345 (2013)

  28. 28.

    , , , & Turning a riboflavin-binding protein into a self-sufficient monooxygenase by cofactor redesign. Chem. Commun. 47, 11050–11052 (2011)

  29. 29.

    , , & Aerobic reduction of olefins by in situ generation of diimide with synthetic flavin catalysts. Chemistry 17, 5908–5920 (2011)

  30. 30.

    & Microbial l-phenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani. Biochem. J. 149, 65–72 (1975)

  31. 31.

    & Comparing HPLC and UV spectrophotometric analysis methods for determining the stability of sorbic acid in nonionic creams containing lactic acid. Drug Dev. Ind. Pharm. 26, 539–547 (2000)

  32. 32.

    et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

  33. 33.

    XDS. Acta Crystallogr. D 66, 125–132 (2010)

  34. 34.

    et al. Atomic-resolution structure of an N5 flavin adduct in d-arginine dehydrogenase. Biochemistry 50, 6292–6294 (2011)

  35. 35.

    et al. Gaussian 09 (Gaussian, Wallingford, CT, revision B.01, 2010)

  36. 36.

    & (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997)

  37. 37.

    , , , & Hybrid density functional theory for pi-stacking interactions: application to benzenes, pyridines, and DNA bases. J. Comput. Chem. 27, 491 (2006)

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The main part of this work was supported by BBSRC grants (BB/K017802/1 with Shell and BB/M/017702/1). Early studies were supported by EU grant FP-7 256808 to D.L. and N.S.S. S.H. is a BBSRC David Phillips research fellow. N.S.S. is an EPSRC Established Career Fellow and Royal Society Wolfson Award holder. We thank Diamond Light Source for access to MX beamlines (proposal number MX8997), which helped to contribute to the results presented here. We thank D. Procter (University of Manchester) for discussions. The authors acknowledge the assistance given by IT Services and the use of the Computational Shared Facility at The University of Manchester.

Author information


  1. Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK

    • Karl A. P. Payne
    • , Mark D. White
    • , Karl Fisher
    • , Basile Khara
    • , Samuel S. Bailey
    • , Nicholas J. W. Rattray
    • , Drupad K. Trivedi
    • , Royston Goodacre
    • , Rebecca Beveridge
    • , Perdita Barran
    • , Stephen E. J. Rigby
    • , Nigel S. Scrutton
    • , Sam Hay
    •  & David Leys
  2. Innovation/Biodomain, Shell International Exploration and Production, Westhollow Technology Center, 3333 Highway 6 South, Houston, Texas 77082-3101, USA

    • David Parker


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K.A.P.P. carried out molecular biology, biophysical and structural biology studies of A. niger Fdc1. B.K. carried out molecular biology experiments underpinning biophysical and structural biology studies of S. cerevisiae Fdc1 performed by M.D.W. K.F. and S.E.J.R. performed and analysed EPR experiments. S.H. performed DFT calculations. N.J.W.R., D.K.T. and R.G. undertook liquid chromatography–mass spectrometry of extracts and interpreted the data on substrate–product species. R.B. and P.B. performed native mass spectrometry. S.S.B. solved the C. dubliniensis Fdc1 structure. All authors discussed the results with N.S.S. and D.P. and all participated in writing the manuscript. D.L. initiated and directed this research.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to David Leys.

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    Supplementary Data 1

    Cartesian coordinates of optimized DFT models.

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