title: Identification and active site analysis of the 1-aminocyclopropane-1-carboxylic acid oxidase catalysing the synthesis of ethylene in Agaricus bisporus.
abstract: BACKGROUND
  1-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.
  
  
  METHODS
  Recombinant 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.
  
  
  RESULTS
  Ab-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.
  
  
  CONCLUSIONS
  These 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Demei
authors.initials: D
authors.lastname: Meng
authors.forename: Lin
authors.initials: L
authors.lastname: Shen
authors.forename: Rui
authors.initials: R
authors.lastname: Yang
authors.forename: Xinhua
authors.initials: X
authors.lastname: Zhang
authors.forename: Jiping
authors.initials: J
authors.lastname: Sheng
mesh_terms: D000364:Agaricus / Q000201:enzymology* / Q000235:genetics; D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D005030:Ethylenes / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
chemical_list: D005030:Ethylenes; C036216:ethylene; D000594:Amino Acid Oxidoreductases; C059460:1-aminocyclopropane-1-carboxylic acid oxidase
keywords: 1-Aminocyclopropane-1-carboxylate oxidase (ACO); 1-aminocyclopropane-1-carboxylic acid; 3-(N-morpholino) propanesulphonic acid; 3D; ACC; ACC oxidase; ACC synthase; ACO; ACS; ANS; Ab-ACO; Active site; Agaricus bisporus (J.E. Lange) Imbach; Agaricus bisporus (J.E. Lange) Imbach ACC oxidase; CD; DTT; EFE; Ethylene; Ethylene forming enzyme; IBs; IPTG; K(m); MOPS; Michaelis constant; Mushroom; PCR; PMSF; S-adenosyl-L-methionine; SAM; SDS-PAGE; anthocyanidin synthase; circular dichroism; dithiothreitol; ethylene-forming enzyme; inclusion bodies; phenylmethanesulphonyl fluoride; polymerase chain reaction; sodium dodecyl sulphate polyacrylamide gel electrophoresis; three-dimensional; β-D-thiogalactoside