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Enzymatic control of cycloadduct conformation ensures reversible 1,3-dipolar cycloaddition in a prFMN-dependent decarboxylase

Nature Chemistry volume 11, pages 1049–1057 (2019)Cite this article

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Abstract

The UbiD enzyme plays an important role in bacterial ubiquinone (coenzyme Q) biosynthesis. It belongs to a family of reversible decarboxylases that interconvert propenoic or aromatic acids with the corresponding alkenes or aromatic compounds using a prenylated flavin mononucleotide cofactor. This cofactor is suggested to support (de)carboxylation through a reversible 1,3-dipolar cycloaddition process. Here, we report an atomic-level description of the reaction of the UbiD-related ferulic acid decarboxylase with substituted propenoic and propiolic acids (data ranging from 1.01–1.39 Å). The enzyme is only able to couple (de)carboxylation of cinnamic acid-type compounds to reversible 1,3-dipolar cycloaddition, while the formation of dead-end prenylated flavin mononucleotide cycloadducts occurs with distinct propenoic and propiolic acids. The active site imposes considerable strain on covalent intermediates formed with cinnamic and phenylpropiolic acids. Strain reduction through mutagenesis negatively affects catalytic rates with cinnamic acid, indicating a direct link between enzyme-induced strain and catalysis that is supported by computational studies.

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Fig. 1: Proposed mechanism of catalysis by AnFdc.
Fig. 2: Crystal structures of AnFdc with propionic acid substrates.
Fig. 3: Crystal structures of AnFdc with propiolic acid substrate analogues.
Fig. 4: Role of strain in the Fdc reaction.
Fig. 5: Potential energy diagram of the Fdc reaction.

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Data availability

The data generated and analysed in this study, including the computational modelling data associated with all of the figures, are available from the corresponding authors on reasonable request. The diffraction data and corresponding atomic models have been deposited in the PDB under accession codes 6R3G, 6R3F, 6R3I, 6R2Z, 6R30, 6R3O, 6R2T, 6R2R, 6R3N, 6R3L, 6R3J, 6R2P, 6R34, 6R33 and 6R32.

References

  1. Toney, M. D. Controlling reaction specificity in pyridoxal phosphate enzymes. Biochim. Biophys. Acta Proteins Proteom. 1814, 1407–1418 (2011).

    Article  CAS  Google Scholar 

  2. Kluger, R. & Tittmann, K. Thiamin diphosphate catalysis: enzymic and nonenzymic covalent intermediates. Chem. Rev. 108, 1797–1833 (2008).

    Article  CAS  Google Scholar 

  3. Piano, V., Palfey, B. A. & Mattevi, A. Flavins as covalent catalysts: new mechanisms emerge. Trends Biochem. Sci. 42, 457–469 (2017).

    Article  CAS  Google Scholar 

  4. Marshall, S. A., Payne, K. A. P. & Leys, D. The UbiX-UbiD system: the biosynthesis and use of prenylated flavin (prFMN). Arch. Biochem. Biophys. 632, 209–221 (2017).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Jeon, B.-S. et al. Investigation of the mechanism of the SpnF-catalyzed [4+2]-cycloaddition reaction in the biosynthesis of spinosyn A. Proc. Natl Acad. Sci. USA 114, 10408–10413 (2017).

    Article  CAS  Google Scholar 

  7. Meldal, M. & Tornøe, C. W. Cu-catalyzed azide−alkyne cycloaddition. Chem. Rev. 108, 2952–3015 (2008).

    Article  CAS  Google Scholar 

  8. Jacobs, M. J., Schneider, G. & Blank, K. G. Mechanical reversibility of strain-promoted azide–alkyne cycloaddition reactions. Angew. Chem. Int. Ed. 55, 2899–2902 (2016).

    Article  CAS  Google Scholar 

  9. Khanal, A., Long, F., Cao, B., Shahbazian-Yassar, R. & Fang, S. Evidence of splitting 1,2,3-triazole into an alkyne and azide by low mechanical force in the presence of other covalent bonds. Chemistry 22, 9760–9767 (2016).

    Article  CAS  Google Scholar 

  10. Krupička, M., Dopieralski, P. & Marx, D. Unclicking the click: metal-assisted mechanochemical cycloreversion of triazoles Is possible. Angew. Chem. Int. Ed. 56, 7745–7749 (2017).

    Article  Google Scholar 

  11. Stauch, T. & Dreuw, A. Force-induced retro-click reaction of triazoles competes with adjacent single-bond rupture. Chem. Sci. 8, 5567–5575 (2017).

    Article  CAS  Google Scholar 

  12. Payne, K. A. et al. New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition. Nature 522, 497–501 (2015).

    Article  CAS  Google Scholar 

  13. Ferguson, K. L., Eschweiler, J. D., Ruotolo, B. T. & Marsh, E. N. G. Evidence for a 1,3-dipolar cyclo-addition mechanism in the decarboxylation of phenylacrylic acids catalyzed by ferulic acid decarboxylase. J. Am. Chem. Soc. 139, 10972–10975 (2017).

    Article  CAS  Google Scholar 

  14. Bailey, S. S. et al. The role of conserved residues in UbiD/Fdc decarboxylase in oxidative maturation, isomerisation and catalysis of prenylated flavin mononucleotide. J. Biol. Chem. 293, 2272–2287 (2018).

    Article  CAS  Google Scholar 

  15. Borden, W. T. Pyramidalized alkenes. Chem. Rev. 89, 1095–1109 (1989).

    Article  CAS  Google Scholar 

  16. Okrasa, K. et al. Structure and mechanism of an unusual malonate decarboxylase and related racemases. Chemistry 14, 6609–6613 (2008).

    Article  CAS  Google Scholar 

  17. Jez, J. M., Ferrer, J. L., Bowman, M. E., Dixon, R. A. & Noel, J. P. Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase. Biochemistry 39, 890–902 (2000).

    Article  CAS  Google Scholar 

  18. Blake, C. C. et al. Crystallographic studies of the activity of hen egg-white lysozyme. Proc. R. Soc. Lond. B Biol. Sci. 167, 378–388 (1967).

    Article  CAS  Google Scholar 

  19. Vocadlo, D. J., Davies, G. J., Laine, R. & Withers, S. G. Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature 412, 835–838 (2001).

    Article  CAS  Google Scholar 

  20. Anderson, V. E. Quantifying energetic contributions to ground state destabilization. Arch. Biochem. Biophys. 433, 27–33 (2005).

    Article  CAS  Google Scholar 

  21. Zhang, Y. & Schramm, V. L. Ground-state destabilization in orotate phosphoribosyltransferases by binding isotope effects. Biochemistry 50, 4813–4818 (2011).

    Article  CAS  Google Scholar 

  22. Lehwess-Litzmann, A. et al. Twisted Schiff base intermediates and substrate locale revise transaldolase mechanism. Nat. Chem. Biol. 7, 678–684 (2011).

    Article  CAS  Google Scholar 

  23. Milić, D. et al. Crystallographic snapshots of tyrosine phenol-lyase show that substrate strain plays a role in C–C bond cleavage. J. Am. Chem. Soc. 133, 16468–16476 (2011).

    Article  Google Scholar 

  24. Vladimirova, A. et al. Substrate distortion and the catalytic reaction mechanism of 5-carboxyvanillate decarboxylase. J. Am. Chem. Soc. 138, 826–836 (2016).

    Article  CAS  Google Scholar 

  25. Lüdtke, S. et al. Sub-ångström-resolution crystallography reveals physical distortions that enhance reactivity of a covalent enzymatic intermediate. Nat. Chem. 5, 762–767 (2013).

    Article  Google Scholar 

  26. Albery, W. J. & Knowles, J. R. Efficiency and evolution of enzyme catalysis. Angew. Chem. Int. Ed. 16, 285–293 (1977).

    Article  CAS  Google Scholar 

  27. Abu Laban, N., Selesi, D., Rattei, T., Tischler, P. & Meckenstock, R. U. Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron-reducing enrichment culture. Environ. Microbiol. 12, 2783–2796 (2010).

    CAS  PubMed  Google Scholar 

  28. Luo, F. et al. Metatranscriptome of an anaerobic benzene-degrading, nitrate-reducing enrichment culture reveals involvement of carboxylation in benzene ring activation. Appl. Environ. Microbiol. 80, 4095–4107 (2014).

    Article  Google Scholar 

  29. Baunach, M. & Hertweck, C. Natural 1,3-dipolar cycloadditions. Angew. Chem. Int. Ed. 54, 12550–12552 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  31. Grimme, S., Ehrlich, S. & Goerigk, L. Effect of the damping function in dispersion corrected density functional theory. J. Comp. Chem. 32, 1456–1465 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the grants BBSRC BB/K017802 and ERC pre-FAB 695013. We acknowledge assistance via use of the Manchester Protein Structure Facility and Diamond Light Source for access (proposal numbers MX12788 and MX17773), which contributed to the results presented here. We also acknowledge the assistance given by IT Services and the use of the Computational Shared Facility at The University of Manchester. D.L. is a Royal Society Wolfson Merit Award holder.

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Authors and Affiliations

  1. Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester, UK

    Samuel S. Bailey, Karl A. P. Payne, Annica Saaret, Stephen A. Marshall, Irina Gostimskaya, Iaroslav Kosov, Karl Fisher, Sam Hay & David Leys

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Contributions

S.S.B. cloned, expressed and purified AnFdc1 and various variants. S.S.B. determined all crystal structures, with guidance from D.L. K.A.P.P. generated various AnFdc1 variants and collected solution data with cinnamic acid. A.S. assisted with crystallization of the Int3crotonic state. S.A.M. assisted with AnFdc1 reconstitution. I.G. assisted with crystallization of FMN-substituted AnFdc1 and the Int3butynoic state (with help from I.K.). K.F. assisted with protein purification and solution data collection and analysis. S.H. performed all of the computational studies. All authors discussed the results and participated in writing the manuscript. D.L. initiated and directed the research.

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Correspondence to Sam Hay or David Leys.

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Supplementary Information

Supplementary Figs. 1–10 and Supplementary Table 1.

Calculations archive file

Density functional theory coordinates used in this study.

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Bailey, S.S., Payne, K.A.P., Saaret, A. et al. Enzymatic control of cycloadduct conformation ensures reversible 1,3-dipolar cycloaddition in a prFMN-dependent decarboxylase. Nat. Chem. 11, 1049–1057 (2019). https://doi.org/10.1038/s41557-019-0324-8

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