Expression of the E. coli nadB gene and characterization of the gene product L-aspartate oxidase.
Quinolinic acid is synthesized in E. coli by the enzymes L-aspartate oxidase and quinolinate synthase A, the genes of which are named nadB and nadA. In our previous work we cloned and characterized the two genes (Flachmann, R., Kunz, N., Seifert, J., Gütlich, M., Wientjes, F.J., Läufer, A. & Gassen, H.G. (1988) Eur. J. Biochem. 175, 221-228). Here we report on the expression of the nadB gene under control of the inducible left promoter of the bacteriophage lambda. The yield of the active gene product L-aspartate oxidase was enhanced up to 20% of the soluble cell protein. The enzyme was purified to homogeneity in a three-step procedure and the reading frame of the L-aspartate oxidase gene was confirmed by Edman degradation of five cyanogen bromide peptides. L-Aspartate oxidase shows no classical Michaelis-Menten behaviour but is subject to a substrate inactivation. The apparent Km values were different for substrate concentrations below and above 1mM and were determined to 0.5 mM and 4.1mM, respectively. The active form of the enzyme is a monomer of 60,284 Da and contains one molecule of FAD and nine cysteine residues, four of which built up two disulfide bonds. The isoelectric point of the protein was determined to be at pH 5.6. Chemical modifications of the enzyme showed that at least one tyrosine and one histidine residue are essential for enzyme activity. The coenzyme-binding domain is located in the amino-terminal part of the polypeptide chain as revealed by a sequence comparison to other dinucleotide binding enzymes. Furthermore, there is evidence for a relationship to fumarate reductase and succinate dehydrogenase of E. coli.
Seifert
J
J
Institut für Biochemie, Technische Hochschule Darmstadt.
Kunz
N
N
Flachmann
R
R
Läufer
A
A
Jany
K D
KD
Gassen
H G
HG
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic
D029968:Escherichia coli Proteins; D000594:Amino Acid Oxidoreductases; C031955:L-aspartate oxidase, E coli