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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

Abstract

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.

Keywords

ACC oxidase Ethylene Iron Cysteine Mutagenesis Protein chemical modification Nitroxide EPR 

Notes

Funding

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)

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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

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