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.
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 degrees 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-alpha-aminoadipic acid, d-aspartic acid-beta-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 degrees C are reported.
Negri
A
A
Istituto di Fisiologia Veterinaria e Biochimica e Centro Interuniversitario per lo Studio delle Macromolecole Informazionali, Università di Milan, Via Celoria 10, I-20133, Milan, Italy.
Tedeschi
G
G
Ceciliani
F
F
Ronchi
S
S
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification*; D000818:Animals; D001051:Apoenzymes / Q000096:biosynthesis; D002417:Cattle; D050804:D-Aspartate Oxidase; D004926:Escherichia coli / Q000378:metabolism; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology*; D015870:Gene Expression; D007668:Kidney / Q000201:enzymology*; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D001051:Apoenzymes; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D000594:Amino Acid Oxidoreductases; D050804:D-Aspartate Oxidase