L-Aspartate oxidase from Escherichia coli - I. Characterization of coenzyme binding and product inhibition

Michele Mortarino, Armando Negri, Gabriella Tedeschi, Tatjana Simonic, Stefano Duga, Hans Gunther Gassen, Severino Ronchi

Research output: Contribution to journalArticlepeer-review


This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of non-covalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant (K(d)) equal to 1.4 μM. The enzyme binds FAD by a simple second-order process with K(d) 0.67 μM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme.

Original languageEnglish
Pages (from-to)418-426
Number of pages9
JournalEuropean Journal of Biochemistry
Issue number2
Publication statusPublished - 1996


  • Binding
  • FAD
  • Inhibition
  • L-aspartate oxidase
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biochemistry


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