Institution: | 1. Aix Marseille Univ, Centrale Marseille, CNRS, iSm2, Marseille, France
Aix Marseille Univ, CNRS, BIP, Marseille, France;2. Aix Marseille Univ, Centrale Marseille, CNRS, iSm2, Marseille, France;3. Manchester Institute of Biotechnology and Department of, Chemical Engineering and Analytical Science, The University of Manchester, 131 Princess Street, Manchester, M1 7DN UK;4. Aix Marseille Univ, CNRS, BIP, Marseille, France;5. CNRS, FR3479 Institut de Microbiologie de la Méditerranée, Plateforme Protéomique, Marseille Protéomique (MaP), IBiSA labeled, Aix Marseille Univ, Marseille, France |
Abstract: | 1-Aminocyclopropane-1-carboxylic oxidase (ACCO) is a non-heme iron(II)-containing enzyme involved in the biosynthesis of the phytohormone ethylene, which regulates fruit ripening and flowering in plants. The active conformation of ACCO, and in particular that of the C-terminal part, remains unclear and open and closed conformations have been proposed. In this work, a combined experimental and computational study to understand the conformation and dynamics of the C-terminal part is reported. Site-directed spin-labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy was used. Mutagenesis experiments were performed to generate active enzymes bearing two paramagnetic labels (nitroxide radicals) anchored on cysteine residues, one in the main core and one in the C-terminal part. Inter-spin distance distributions were measured by pulsed EPR spectroscopy and compared with the results of molecular dynamics simulations. The results reveal the existence of a flexibility of the C-terminal part. This flexibility generates several conformations of the C-terminal part of ACCO that correspond neither to the existing crystal structures nor to the modelled structures. This highly dynamic region of ACCO raises questions on its exact function during enzymatic activity. |