The active site and substrate-binding mode of 1-aminocyclopropane-1-carboxylate oxidase determined by site-directed mutagenesis and comparative modelling studies

Biochem J. 2004 Jun 1;380(Pt 2):339-46. doi: 10.1042/BJ20031762.


The active site and substrate-binding mode of MD-ACO1 (Malus domestica Borkh. 1-aminocyclopropane-1-carboxylate oxidase) have been determined using site-directed mutagenesis and comparative modelling methods. The MD-ACO1 protein folds into a compact jelly-roll motif comprised of eight a-helices, 12 b-strands and several long loops. The active site is well defined as a wide cleft near the C-terminus. The co-substrate ascorbate is located in cofactor Fe2+-binding pocket, the so-called '2-His-1-carboxylate facial triad'. In addition, our results reveal that Arg244 and Ser246 are involved in generating the reaction product during enzyme catalysis. The structure agrees well with the biochemical and site-directed mutagenesis results. The three-dimensional structure together with the steady-state kinetics of both the wild-type and mutant MD-ACO1 proteins reveal how the substrate specificity of MD-ACO1 is involved in the catalytic mechanism, providing insights into understanding the fruit ripening process at atomic resolution.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Amino Acid Oxidoreductases / genetics*
  • Amino Acid Oxidoreductases / metabolism*
  • Amino Acid Sequence
  • Ascorbic Acid / metabolism
  • Binding Sites / genetics
  • Ferrous Compounds / metabolism
  • Kinetics
  • Malus / chemistry
  • Malus / enzymology
  • Models, Molecular
  • Models, Structural
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed / genetics*
  • Plant Proteins / genetics
  • Plant Proteins / metabolism
  • Protein Binding / genetics
  • Protein Binding / physiology
  • Protein Interaction Mapping
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity / genetics


  • Ferrous Compounds
  • Plant Proteins
  • Recombinant Proteins
  • Amino Acid Oxidoreductases
  • 1-aminocyclopropane-1-carboxylic acid oxidase
  • Ascorbic Acid