Purification and properties of D-aspartate oxidase from Cryptococcus humicolus UJ1.
D-Aspartate oxidase (EC 1.4.3.1), which is highly specific to D-aspartate, was inducibly produced by a yeast strain which was isolated from soil and identified as Cryptococcus humicolus UJ1. The enzyme was purified to homogeneity as indicated on SDS-polyacrylamide gel electrophoresis. The molecular mass of the monomer subunit was determined to be 40 kDa. The native enzyme was suggested to be a homotetramer by its behavior on gel filtration. The enzyme was shown to be a flavoprotein by its absorption spectral properties, and the flavin was found to be tightly, but not covalently, bound FAD. The purified preparation had a specific activity of 76.1 mumol/min per mg protein with D-aspartate as substrate. Optimum pH was 7.5 and optimum temperature was around 35 degrees C. D-Glutamate was a very poor substrate for the enzyme. N-Methyl-D-aspartate was better than D-glutamate as substrate but markedly poorer than D-aspartate. Malonate was the most effective competitive inhibitor of the compounds tested. The N-terminal amino-acid sequence of the enzyme showed a significant homology with those of D-aspartate oxidases from beef kidney and Octopus vulgaris and those of D-amino-acid oxidases from various sources.
Yamada
R
R
Department of BioEngineering, Nagaoka University of Technology, Niigata, Japan.
Ujiie
H
H
Kera
Y
Y
Nakase
T
T
Kitagawa
K
K
Imasaka
T
T
Arimoto
K
K
Takahashi
M
M
Matsumura
Y
Y
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D016384:Consensus Sequence; D003454:Cryptococcus / Q000201:enzymology* / Q000254:growth & development; D050804:D-Aspartate Oxidase; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D049831:Octopodiformes; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity; D013552:Swine
D000596:Amino Acids; D004791:Enzyme Inhibitors; D000594:Amino Acid Oxidoreductases; D050804:D-Aspartate Oxidase; C494102:DDO protein, human