Chemical Modification of 1-Aminocyclopropane Carboxylic Acid (ACC) Oxidase: Cysteine Mutational Analysis, Characterization, and Bioconjugation with a Nitroxide Spin Label

  • Sybille Tachon
  • Eugénie Fournier
  • Christophe Decroos
  • Pascal Mansuelle
  • Emilien Etienne
  • Marc Maresca
  • Marlène Martinho
  • Valérie BelleEmail author
  • Thierry Tron
  • Ariane Jalila SimaanEmail author
Original paper


1-Aminocyclopropane carboxylic acid oxidase (ACCO) catalyzes the last step of ethylene biosynthesis in plants. Although some sets of structures have been described, there are remaining questions on the active conformation of ACCO and in particular, on the conformation and potential flexibility of the C-terminal part of the enzyme. Several techniques based on the introduction of a probe through chemical modification of amino acid residues have been developed for determining the conformation and dynamics of proteins. Cysteine residues are recognized as convenient targets for selective chemical modification of proteins, thanks to their relatively low abundance in protein sequences and to their well-mastered chemical reactivity. ACCOs have generally 3 or 4 cysteine residues in their sequences. By a combination of approaches including directed mutagenesis, activity screening on cell extracts, biophysical and biochemical characterization of purified enzymes, we evaluated the effect of native cysteine replacement and that of insertion of cysteines on the C-terminal part in tomato ACCO. Moreover, we have chosen to use paramagnetic labels targeting cysteine residues to monitor potential conformational changes by electron paramagnetic resonance (EPR). Given the level of conservation of the cysteines in ACCO from different plants, this work provides an essential basis for the use of cysteine as probe-anchoring residues.


ACC oxidase Ethylene Iron Cysteine Mutagenesis Protein chemical modification Nitroxide EPR 



This work was supported by A*MIDEX project (No. ANR-11-IDEX-0001-02) funded by the « Ivestissements d’Avenir» French Government program, managed by the French National Research Agency (ANR). The authors are also grateful to the EPR facilities available at the national EPR network (IR CNRS 3443) and the Aix-Marseille Université EPR center.

Supplementary material

12033_2019_191_MOESM1_ESM.docx (866 kb)
Supplementary material 1 (DOCX 860 kb)


  1. 1.
    Bleecker, A. B., & Kende, H. (2000). Ethylene: a gaseous signal molecule in plants. Annual Review of Cell and Developmental Biology, 16(1), 1–18. Scholar
  2. 2.
    Dilley, D. R., Wang, Z., Kadirjan-Kalbach, D. K., Ververidis, F., Beaudry, R., & Padmanabhan, K. (2013). 1-Aminocyclopropane-1-carboxylic acid oxidase reaction mechanism and putative post-translational activities of the ACCO protein. AoB Plants, 5, plt031. Scholar
  3. 3.
    Simaan, A. J., & Réglier, M. (2015). ACC oxidase and ethylene biosynthesis in plants. In R. P. Hausinger & C. J. Schofield (Eds.), 2-Oxoglutarate-dependent oxygenases (Vol. 3, p. 425). Cambridge: Royal Society of Chemistry.CrossRefGoogle Scholar
  4. 4.
    Kal, S., & Que, L. (2017). Dioxygen activation by nonheme iron enzymes with the 2-His-1-carboxylate facial triad that generate high-valent oxoiron oxidants. JBIC Journal of Biological Inorganic Chemistry, 22(2–3), 339–365. Scholar
  5. 5.
    Murphy, L. J., Robertson, K. N., Harroun, S. G., Brosseau, C. L., Werner-Zwanziger, U., Moilanen, J., et al. (2014). A simple complex on the verge of breakdown: Isolation of the elusive cyanoformate ion. Science, 344(6179), 75–78. Scholar
  6. 6.
    Barlow, J. N., Zhang, Z., John, P., Baldwin, J. E., & Schofield, C. J. (1997). Inactivation of 1-aminocyclopropane-1-carboxylate oxidase involves oxidative modifications. Biochemistry, 36(12), 3563–3569. Scholar
  7. 7.
    Mantri, M., Zhang, Z., McDonough, M. A., & Schofield, C. J. (2012). Autocatalysed oxidative modifications to 2-oxoglutarate dependent oxygenases. The FEBS journal, 279(9), 1563–1575. Scholar
  8. 8.
    Zhang, Z., Ren, J.-S., Clifton, I. J., & Schofield, C. J. (2004). Crystal Structure and mechanistic implications of 1-aminocyclopropane-1-carboxylic acid oxidase—The ethylene-forming enzyme. Chemistry & Biology, 11(10), 1383–1394. Scholar
  9. 9.
    Sun, X., Li, Y., He, W., Ji, C., Xia, P., Wang, Y., et al. (2017). Pyrazinamide and derivatives block ethylene biosynthesis by inhibiting ACC oxidase. Nature Communications, 8, 15758–15814. Scholar
  10. 10.
    Yoo, A., Seo, Y. S., Jung, J.-W., Sung, S.-K., Kim, W. T., Lee, W., et al. (2006). Lys296 and Arg299 residues in the C-terminus of MD-ACO1 are essential for a 1-aminocyclopropane-1-carboxylate oxidase enzyme activity. Journal of Structural Biology, 156(3), 407–420. Scholar
  11. 11.
    Brisson, L., Bakkali-Taheri, N., Giorgi, M., Fadel, A., Kaizer, J., Réglier, M., et al. (2012). 1-Aminocyclopropane-1-carboxylic acid oxidase: insight into cofactor binding from experimental and theoretical studies. JBIC Journal of Biological Inorganic Chemistry, 17(6), 939–949. Scholar
  12. 12.
    Mizukami, S. (2011). Development of molecular imaging tools to investigate protein functions by chemical probe design. Chemical & Pharmaceutical Bulletin, 59(12), 1435–1446. Scholar
  13. 13.
    Hubbell, W. L., Cafiso, D. S., & Altenbach, C. (2000). Identifying conformational changes with site-directed spin labeling. Nature Structural Biology, 7(9), 735–739. Scholar
  14. 14.
    Markwick, P. R. L., Malliavin, T., & Nilges, M. (2008). Structural biology by NMR: Structure, dynamics, and interactions. PLoS Computational Biology, 4(9), e1000168–e1000177. Scholar
  15. 15.
    Albani, J. R. (2011). Structure and dynamics of macromolecules: Absorption and fluorescence studies.
  16. 16.
    Danielson, M. A., & Falke, J. J. (1996). Use of 19F NMR to probe protein structure and conformational changes. Annual Review of Biophysics and Biomolecular Structure, 25(1), 163–195. Scholar
  17. 17.
    Krall, N., da Cruz, F. P., Boutureira, O., & Bernardes, G. J. L. (2015). Site-selective protein-modification chemistry for basic biology and drug development. Nature Chemistry, 13(2), 168. Scholar
  18. 18.
    Mizukami, S., Hori, Y., & Kikuchi, K. (2013). Small-molecule-based protein-labeling technology in live cell studies: Probe-design concepts and applications. Accounts of Chemical Research, 47(1), 247–256. Scholar
  19. 19.
    Martinho, M., Fournier, E., LeBreton, N., Mileo, E., & Belle, V. (2018). Nitroxide spin labels: fabulous spy spins for biostructural EPR applications. In M. Martinho (Ed.), Electron paramagnetic resonance (Vol. 26, pp. 66–88). Cambridge: Royal Society of Chemistry. Scholar
  20. 20.
    Haugland, M. M., Lovett, J. E., & Anderson, E. A. (2017). Advances in the synthesis of nitroxide radicals for use in biomolecule spin labelling. Chemical Society Reviews. Scholar
  21. 21.
    Hubbell, W. L., López, C. J., Altenbach, C., & Yang, Z. (2013). Technological advances in site-directed spin labeling of proteins. Current Opinion in Structural Biology, 23(5), 725–733. Scholar
  22. 22.
    Mchaourab, H. S., Steed, P. R., & Kazmier, K. (2011). Toward the fourth dimension of membrane protein structure: Insight into dynamics from spin-labeling EPR spectroscopy. Structure, 19(11), 1549–1561. Scholar
  23. 23.
    Bordignon, E., & Bleicken, S. (2017). New limits of sensitivity of site-directed spin labeling electron paramagnetic resonance for membrane proteins. Biomembranes. Scholar
  24. 24.
    Boutureira, O., & Bernardes, G. J. L. (2015). Advances in chemical protein modification. Chemical Reviews, 115(5), 2174–2195. Scholar
  25. 25.
    Spicer, C. D., & Davis, B. G. (2014). Selective chemical protein modification. Nature Communications, 5, 4740. Scholar
  26. 26.
    Zheng, M., Zheng, L., Zhang, P., Li, J., & Zhang, Y. (2015). Development of bioorthogonal reactions and their applications in bioconjugation. Molecules, 20(2), 3190–3205. Scholar
  27. 27.
    Huang, Y., & Liu, L. (2016). Protein modification: Standing out from the crowd. Nature Chemistry, 8(2), 101–102. Scholar
  28. 28.
    Chalker, J. M., Bernardes, G. J. L., Lin, Y. A., & Davis, B. G. (2009). Chemical modification of proteins at cysteine: Opportunities in chemistry and biology. Chemistry An Asian Journal, 4(5), 630–640. Scholar
  29. 29.
    Hamilton, A. J., Bouzayen, M., & Grierson, D. (1991). Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast. Proceedings of the National Academy of Sciences, 88(16), 7434–7437. Scholar
  30. 30.
    Grierson, D., Hamilton, A. J., & Lycett, G. W. (2012). The life and times of ACC oxidase, alias TOM13. Molecular Biology Reports, 40(4), 3021–3022. Scholar
  31. 31.
    Zhang, Z., Schofield, C. J., Baldwin, J. E., Thomas, P., & John, P. (1995). Expression, purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from tomato in Escherichia coli. Biochemical Journal, 307(1), 77–85. Scholar
  32. 32.
    Bakkali-Taheri El, N., Tachon, S., Orio, M., Bertaina, S., Martinho, M., Robert, V., et al. (2017). Characterization of Cu(II)-reconstituted ACC Oxidase using experimental and theoretical approaches. Archives of Biochemistry and Biophysics, 623–624, 31–41. Scholar
  33. 33.
    Kelly, S. M., Jess, T. J., & Price, N. C. (2005). How to study proteins by circular dichroism. Biochimica et Biophysica Acta (BBA), 1751(2), 119–139. Scholar
  34. 34.
    Stoll, S., & Schweiger, A. (2006). EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. Journal of magnetic resonance (San Diego, Calif.: 1997), 178(1), 42–55. Scholar
  35. 35.
    Etienne, E., Le Breton, N., Martinho, M., Mileo, E., & Belle, V. (2017). SimLabel: A graphical user interface to simulate continuous wave EPR spectra from site-directed spin labeling experiments. Magnetic Resonance in Chemistry, 55(8), 714–719. Scholar
  36. 36.
    Clifton, I. J., McDonough, M. A., Ehrismann, D., Kershaw, N. J., Granatino, N., & Schofield, C. J. (2006). Structural studies on 2-oxoglutarate oxygenases and related double-stranded β-helix fold proteins. Journal of Inorganic Biochemistry, 100(4), 644–669. Scholar
  37. 37.
    Dupille, E., Rombaldi, C., Lelièvre, J. M., Cleyet-Marel, J. C., Pech, J. C., & Latché, A. (1993). Purification, properties and partial amino-acid sequence of 1-aminocyclopropane-1-carboxylic acid oxidase from apple fruits. Planta, 190(1), 65–70.CrossRefGoogle Scholar
  38. 38.
    Brunhuber, N. M. W., Mort, J. L., Christoffersen, R. E., & Reich, N. O. (2000). Steady-state kinetic mechanism of recombinant avocado ACC oxidase: Initial velocity and inhibitor studies. Biochemistry, 39(35), 10730–10738. Scholar
  39. 39.
    Thrower, J. S., Blalock, R., & Klinman, J. P. (2001). Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase. Biochemistry, 40(32), 9717–9724. Scholar
  40. 40.
    Dilley, D. R., Kadyrzhanova, D. K., & Wang, Z. (2001). Mechanism of carbon dioxide activation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase. Acta Horticulturae, 553, 143–144. Scholar
  41. 41.
    Dunning Hotopp, J. C., Auchtung, T. A., Hogan, D. A., & Hausinger, R. P. (2003). Intrinsic tryptophan fluorescence as a probe of metal and alpha-ketoglutarate binding to TfdA, a mononuclear non-heme iron dioxygenase. Journal of Inorganic Biochemistry, 93(1–2), 66–70. Scholar
  42. 42.
    Straganz, G. D., Egger, S., Aquino, G., D’Auria, S., & Nidetzky, B. (2006). Exploring the cupin-type metal-coordinating signature of acetylacetone dioxygenase Dke1 with site-directed mutagenesis: Catalytic reaction profile and Fe2+ binding stability of Glu-69→Gln mutant. Journal of Molecular Catalysis. B, Enzymatic, 39(1–4), 171–178. Scholar
  43. 43.
    Guo, Z., Cascio, D., Hideg, K., & Hubbell, W. L. (2008). Structural determinants of nitroxide motion in spin-labeled proteins: Solvent-exposed sites in helix B of T4 lysozyme. Protein Science, 17(2), 228–239. Scholar
  44. 44.
    Guo, Z., Cascio, D., Hideg, K., Kálái, T., & Hubbell, W. L. (2007). Structural determinants of nitroxide motion in spin-labeled proteins: Tertiary contact and solvent-inaccessible sites in helix G of T4 lysozyme. Protein Science, 16(6), 1069–1086. Scholar
  45. 45.
    Reginsson, G. W., & Schiemann, O. (2011). Pulsed electron–electron double resonance: Beyond nanometre distance measurements on biomacromolecules. Biochemical Journal, 434(3), 353–363. Scholar
  46. 46.
    Casey, T. M., & Fanucci, G. E. (2015). Spin labeling and double electron-electron resonance (DEER) to deconstruct conformational ensembles of HIV protease. In T. M. Casey (Ed.), Electron paramagnetic resonance investigations of biological systems by using spin labels, spin probes, and intrinsic metal ions, part B (Vol. 564, pp. 153–187). New York: Elsevier. Scholar
  47. 47.
    Fournier, E., Tachon, S., Belle, V., Simaan, A.J., Martinho, M., unpublished.Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.CNRSAix Marseille UnivMarseilleFrance
  2. 2.BIP, CNRSAix Marseille UnivMarseilleFrance
  3. 3.Plate-forme Protéomique, Marseille Protéomique (MaP), IBiSA Labeled, FR3479 Institut de Microbiologie de la Méditerranée, CNRSAix Marseille UnivMarseilleFrance

Personalised recommendations