Identification and active site analysis of the 1-aminocyclopropane-1-carboxylic acid oxidase catalysing the synthesis of ethylene in Agaricus bisporus |
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Authors: | Demei Meng Lin Shen Rui Yang Xinhua Zhang Jiping Sheng |
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Affiliation: | 1. College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China;2. School of Agricultural Economics and Rural Development, Renmin University of China, Beijing 100872, China;3. School of Agriculture and Food Engineering, Shandong University of Technology, Zibo 255049, Shandong, China |
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Abstract: | Background1-Aminocyclopropane-1-carboxylate oxidase (ACO) is a key enzyme that catalyses the final step in the biosynthesis of the plant hormone ethylene. Recently, the first ACO homologue gene was isolated in Agaricus bisporus, whereas information concerning the nature of the ethylene-forming activity of this mushroom ACO is currently lacking.MethodsRecombinant ACO from A. bisporus (Ab-ACO) was purified and characterised for the first time. Molecular modelling combined with site-directed mutagenesis and kinetic and spectral analysis were used to investigate the property of Ab-ACO.ResultsAb-ACO has eight amino acid residues that are conserved in the Fe (II) ascorbate family of dioxygenases, including four catalytic residues in the active site, but Ab-ACO lacks a key residue, S289. In comparison to plant ACOs, Ab-ACO requires ACC and Fe (II) but does not require ascorbate. In addition, Ab-ACO had relatively low activity and was completely dependent on bicarbonate, which could be ascribed to the replacement of S289 by G289. Moreover, the ferrous ion could induce a change in the tertiary, but not the secondary, structure of Ab-ACO.ConclusionsThese results provide crucial experimental support for the ability of Ab-ACO to catalyse ethylene formation in a similar manner to that of plant ACOs, but there are differences between the biochemical and catalytic characteristics of Ab-ACO and plant ACOs.General significanceThis work enhances the understanding of the ethylene biosynthesis pathways in fungi and could promote profound physiological research of the role of ethylene in the regulation of mushroom growth and development. |
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Keywords: | ACC, 1-aminocyclopropane-1-carboxylic acid ACO, ACC oxidase Ab-ACO, Agaricus bisporus (J.E. Lange) Imbach ACC oxidase SAM, S-adenosyl-L-methionine ACS, ACC synthase EFE, ethylene-forming enzyme PCR, polymerase chain reaction IPTG, β-D-thiogalactoside MOPS, 3-(N-morpholino) propanesulphonic acid DTT, dithiothreitol PMSF, phenylmethanesulphonyl fluoride SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis IBs, inclusion bodies 3D, three-dimensional CD, circular dichroism ANS, anthocyanidin synthase Km, Michaelis constant |
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