L-aspartate oxidase is present in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3: characteristics and role in the de novo biosynthesis of nicotinamide adenine dinucleotide proposed by genome sequencing.
A gene encoding the L-aspartate oxidase homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. We succeeded in expressing the encoding gene in Escherichia coli and purified the product to homogeneity. Characterization of the protein revealed that it is the most thermostable L-aspartate oxidase detected so far. In addition to the oxidase activity, the enzyme catalyzed L-aspartate dehydrogenation in the presence of an artificial electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, and ferricyanide. L-Aspartate oxidase is known to function as the first enzyme in the de novo NAD biosynthetic pathway in prokaryotes. By a similarity search in public databases, the genes that encode the homologue of all other enzymes involved in the pathway were identified in the P. horikoshii OT-3 genome. This suggests that P. horikoshii OT-3 may use the de novo NAD biosynthetic pathway under anaerobic conditions.
Sakuraba
Haruhiko
H
Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, Tokushima 770-8506, Japan.
Satomura
Takenori
T
Kawakami
Ryushi
R
Yamamoto
Sanae
S
Kawarabayasi
Yutaka
Y
Kikuchi
Hisasi
H
Ohshima
Toshihisa
T
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D029968:Escherichia coli Proteins; D020745:Genome, Archaeal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000096:biosynthesis*; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D009243:NAD; D000594:Amino Acid Oxidoreductases; C031955:L-aspartate oxidase, E coli