LETTER RESEARCH
potential. In the Fdc1/UbiD enzyme family, the prFMNiminium form
supports reversible decarboxylation of a wide range of (aromatic)
substrates. Although the [312] reaction between azomethine
ylide dipoles and alkene dipolarophiles has been extensively used
in organic chemistry11,12, the mechanism proposed here would pre-
sent the first example, to our knowledge, of a biological [312] reac-
tion. Combined with the recent description of both natural25 and
artificial26 bona fide [412] cycloaddition catalysing enzymes, this
hints at more widespread use of pericyclic reaction chemistry in nat-
ure. As Fdc1/UbiD enzymes have evolved from an NADH-FMN
oxidoreductase module8,21, the distinct possibility exists that other
unrelated prFMN-dependent enzymes might have developed from
distinct flavin-binding modules. Such distinct prFMN-dependent
enzymes could make use of different aspects of the prFMN chemistry,
as occurs with other organic cofactors27. Artificial flavoenzymes con-
taining N5-alkylated flavins have been created that are capable of
H2O2-driven enantioselective sulfoxidations28, using the fact that
N5-alkylated flavins can form remarkably stable 4a-peroxyflavins
and are powerful oxidising catalysts29. It is possible similar enzymes
already exist in nature.
14. Desai, B. J. 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. Okrasa, K. et al. Structure-guided directed evolution of alkenyl and arylmalonate
decarboxylases. Angew. Chem. 48, 7691–7694 (2009).
16. Lupa, B., Lyon, D., Gibbs, M. D., Reeves, R. A. & Wiegel, J. Distribution of genes
encoding the microbial non-oxidative reversible hydroxyarylic acid
decarboxylases/phenol carboxylases. Genomics 86, 342–351 (2005).
17. Zhang, H. & Javor, G. T. Regulation of the isofunctional genes ubiD and ubiX of the
ubiquinone biosynthetic pathway of Escherichia coli. FEMS Microbiol. Lett. 223,
67–72 (2003).
18. Kopec, J., Schnell, R. & Schneider, G. Structure of PA4019, a putative aromatic acid
decarboxylase from Pseudomonas aeruginosa. Acta Crystallogr. F 67, 1184–1188
(2011).
19. Plumridge, A. etal. 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. Lin, F., Ferguson, K. L., Boyer, D. R., Lin, X. N. & Marsh, E. N. 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. Christendat, D. et al. Structural proteomics of an archaeon. Nature Struct. Biol. 7,
903–909 (2000).
22. Walsh, C. T. & Wencewicz, T. A. Flavoenzymes: versatile catalysts in biosynthetic
pathways. Nat. Prod. Rep. 30, 175–200 (2013).
23. Xu, S. 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. Prantz, K. & Mulzer, J. Synthetic applications of the carbonyl generating Grob
fragmentation. Chem. Rev. 110, 3741–3766 (2010).
25. Kim, H. J., Ruszczycky, M. W., Choi, S.-H., Lie, Y.-N. & Liu, H.-W. Enzyme-catalysed
[412] cycloaddition is a key step in the biosynthesis of spinosyn A. Nature 473,
109–112 (2011).
26. Preiswerk, N. 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. Richter, M. Functional diversity of organic molecule enzyme cofactors. Nat. Prod.
Rep. 30, 1324–1345 (2013).
Online Content Methods, along with any additional Extended Data display items
to these sections appear only in the online paper.
Received 8 December 2014; accepted 13 May 2015.
Published online 17 June 2015.
1. Aussel, L. et al. Biosynthesis and physiology of coenzyme Q in bacteria. Biochim.
Biophys. Acta 1837, 1004–1011 (2014).
28. de Gonzalo, G., Smit, C., Jin, J., Minnaard, A. J. & Fraaije, M. W. Turning a riboflavin-
binding protein into a self-sufficient monooxygenase by cofactor redesign. Chem.
Commun. 47, 11050–11052 (2011).
29. Imada, Y., Iida, H., Kitagawa, T. & Naota, T. Aerobic reduction of olefins by in situ
generation of diimide with synthetic flavin catalysts. Chemistry 17, 5908–5920
(2011).
2. Gulmezian, M., Hyman, K. R., Marbois, B. N., Clarke, C. F. & Javor, G. T. The role of
UbiX in Escherichia coli coenzyme Q biosynthesis. Arch. Biochem. Biophys. 467,
144–153 (2007).
3. Leppik, R. A., Young, I. G. & Gibson, F. Membrane-associated reactions in
ubiquinone biosynthesis in Escherichia coli. 3-octoprenyl-4-hydroxybenzoate
carboxy-lyase. Biochim. Biophys. Acta 436, 800–810 (1976).
4. Erb, T. J. Carboxylases in natural and synthetic microbial pathways. Appl. Environ.
Microbiol. 77, 8466–8477 (2011).
5. Boll, M., Loeffler, C., Morris, B. E. L. & Kung, J. W. Anaerobic degradation of
homocyclic aromatic compounds via arylcarboxyl-coenzyme A esters: organisms,
strategies and key enzymes. Environ. Microbiol. 16, 612–627 (2014).
6. Mukai, N., Masaki, K., Fujii, T., Kawamukai, M. & Iefuji, H. PAD1 and FDC1 are
essential for the decarboxylation of phenylacrylic acids in Saccharomyces
cerevisiae. J. Biosci. Bioeng. 109, 564–569 (2010).
7. Rangarajan, E. S. et al. Crystal structure of a dodecameric FMN-dependent UbiX-
like decarboxylate (Pad1) from Eschericia coli O157:H7. Protein Sci. 13,
3006–3016 (2004).
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 helpedtocontributetotheresultspresentedhere. WethankD. 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.
8. Jacewicz, A., Izumi, A., Brunner, K., Schnell, R. & Schneider, G. Structural insights
into the UbiD protein family from the crystal structure of PA0254 from
Pseudomonas aeruginosa. PLoS ONE 8, e63161 (2013).
9. Stratford, M. 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. White, M. D. et al. UbiX is a flavin prenyltransferase required for bacterial
(2015).
Author Contributions K.A.P.P. carried out molecular biology, biophysical and struc-
tural biology studies of A. niger Fdc1. B.K. carried out molecular biology experiments
underpinningbiophysicalandstructural biologystudies ofS. cerevisiaeFdc1performed
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.
11. Pellissier, H. Asymmetric 1,3-dipolar cycloadditions. Tetrahedron 63, 3235–3285
(2007).
Author Information Coordinates and structure factors have been deposited in the
4ZAA, 4ZAC and 4ZAD. Reprints and permissions information is available at
12. Ess, D. H. & Houk, K. N. Theory of 1,3-dipolar cycloadditions: Distortion/Interaction
and frontier molecular orbital models. J. Am. Chem. Soc. 130, 10187–10198
(2008).
13. Li, T., Huo, L., Pulley, C. & Liu, A. Decarboxylation mechanisms in biological system.
Bioorg. Chem. 43, 2–14 (2012).
0 0 M O N T H 2 0 1 5
| V O L 0 0 0 | N A T U R E | 5
G
2015 Macmillan Publishers Limited. All rights reserved