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Purification of beef kidney d-aspartate oxidase overexpressed in Escherichia coli and characterization of its redox potentials and oxidative activity towards agonists and antagonists of excitatory amino acid receptors
- Source :
- Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1431:212-222
- Publication Year :
- 1999
- Publisher :
- Elsevier BV, 1999.
-
Abstract
- The flavoenzyme d -aspartate oxidase from beef kidney (DASPO, EC 1.4.3.1) has been overexpressed in Escherichia coli. A purification procedure, faster than the one used for the enzyme from the natural source (bDASPO), has been set up yielding about 2 mg of pure recombinant protein (rDASPO) per each gram of wet E. coli paste. rDASPO has been shown to possess the same general biochemical properties of bDASPO, except that the former contains only FAD, while the latter is a mixture of two forms, one active containing FAD and one inactive containing 6-OH-FAD (9–20% depending on the preparation). This results in a slightly higher specific activity (about 15%) for rDASPO compared to bDASPO and in facilitated procedures for apoprotein preparation and reconstitution. Redox potentials of −97 mV and −157 mV were determined for free and l -(+)-tartrate complexed DASPO, respectively, in 0.1 M KPi, pH 7.0, 25°C. The large positive shift in the redox potential of the coenzyme compared to free FAD (−207 mV) is in agreement with similar results obtained with other flavooxidases. rDASPO has been used to assess a possible oxidative activity of the enzyme towards a number of compounds used as agonists or antagonists of neurotransmitters, including d -aspartatic acid, d -glutamic acid, N-methyl- d -aspartic acid, d,l -cysteic acid, d -homocysteic acid, d,l -2-amino-3-phosphonopropanoic acid, d -α-aminoadipic acid, d -aspartic acid-β-hydroxamate, glycyl- d -aspartic acid and cis-2,3-piperidine dicarboxylic acid. Kinetic parameters for each substrate in 50 mM KPi, pH 7.4, 25°C are reported.
- Subjects :
- D-aspartate oxidase
D-Aspartate Oxidase
Stereochemistry
Biophysics
Gene Expression
Cysteic acid
Kidney
Biochemistry
Cofactor
Substrate Specificity
chemistry.chemical_compound
Apoenzymes
Structural Biology
Aspartic acid
Escherichia coli
Excitatory Amino Acid Agonists
Animals
Molecular Biology
chemistry.chemical_classification
biology
Glutamic acid
Homocysteic acid
Amino acid
Dicarboxylic acid
chemistry
biology.protein
Cattle
Amino Acid Oxidoreductases
Excitatory Amino Acid Antagonists
Oxidation-Reduction
Subjects
Details
- ISSN :
- 01674838
- Volume :
- 1431
- Database :
- OpenAIRE
- Journal :
- Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
- Accession number :
- edsair.doi.dedup.....d567cb65209b2fe3fd252fc63327c29d
- Full Text :
- https://doi.org/10.1016/s0167-4838(99)00027-8