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

Abstract

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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Accessions

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.

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Acknowledgements

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

Affiliations

  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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Contributions

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.

Extended data

Supplementary information

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  1. 1.

    Supplementary Data 1

    Cartesian coordinates of optimized DFT models.

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DOI

https://doi.org/10.1038/nature14560

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