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 noncovalently 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 (Kd) equal to 1.4 microM. The enzyme binds FAD by a simple second-order process with Kd 0.67 microM. 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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Oxidoreductases / chemistry*
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Amino Acid Oxidoreductases / isolation & purification
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Amino Acid Oxidoreductases / metabolism*
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Amino Acid Sequence
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Bacillus subtilis / enzymology
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Base Sequence
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Binding Sites
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Conserved Sequence
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Escherichia coli / enzymology*
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Escherichia coli / genetics
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Escherichia coli Proteins
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Flavin-Adenine Dinucleotide / metabolism*
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Genes, Bacterial
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Isoenzymes / chemistry
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Isoenzymes / isolation & purification
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Isoenzymes / metabolism
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Kinetics
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Oligodeoxyribonucleotides
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Point Mutation
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Sequence Homology, Amino Acid
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Spectrophotometry
Substances
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Escherichia coli Proteins
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Isoenzymes
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Oligodeoxyribonucleotides
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Recombinant Proteins
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Flavin-Adenine Dinucleotide
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Amino Acid Oxidoreductases
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L-aspartate oxidase, E coli