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 journalArticle

33 Citations (Scopus)

Abstract

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
Volume239
Issue number2
Publication statusPublished - 1996

Fingerprint

L-aspartate oxidase
Flavin-Adenine Dinucleotide
Coenzymes
Escherichia coli
Flavoproteins
Mutagenesis
Holoenzymes
Enzymes
Site-Directed Mutagenesis
Aspartic Acid
Assays
Monomers
Binding Sites
Oxidation
Proteins

Keywords

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

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mortarino, M., Negri, A., Tedeschi, G., Simonic, T., Duga, S., Gassen, H. G., & Ronchi, S. (1996). L-Aspartate oxidase from Escherichia coli - I. Characterization of coenzyme binding and product inhibition. European Journal of Biochemistry, 239(2), 418-426.

L-Aspartate oxidase from Escherichia coli - I. Characterization of coenzyme binding and product inhibition. / Mortarino, Michele; Negri, Armando; Tedeschi, Gabriella; Simonic, Tatjana; Duga, Stefano; Gassen, Hans Gunther; Ronchi, Severino.

In: European Journal of Biochemistry, Vol. 239, No. 2, 1996, p. 418-426.

Research output: Contribution to journalArticle

Mortarino, M, Negri, A, Tedeschi, G, Simonic, T, Duga, S, Gassen, HG & Ronchi, S 1996, 'L-Aspartate oxidase from Escherichia coli - I. Characterization of coenzyme binding and product inhibition', European Journal of Biochemistry, vol. 239, no. 2, pp. 418-426.
Mortarino, Michele ; Negri, Armando ; Tedeschi, Gabriella ; Simonic, Tatjana ; Duga, Stefano ; Gassen, Hans Gunther ; Ronchi, Severino. / L-Aspartate oxidase from Escherichia coli - I. Characterization of coenzyme binding and product inhibition. In: European Journal of Biochemistry. 1996 ; Vol. 239, No. 2. pp. 418-426.
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