title: Purification and properties of D-aspartate oxidase from Cryptococcus humicolus UJ1.
abstract: 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.
authors.affiliation: Department of BioEngineering, Nagaoka University of Technology, Niigata, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Yamada
authors.forename: H
authors.initials: H
authors.lastname: Ujiie
authors.forename: Y
authors.initials: Y
authors.lastname: Kera
authors.forename: T
authors.initials: T
authors.lastname: Nakase
authors.forename: K
authors.initials: K
authors.lastname: Kitagawa
authors.forename: T
authors.initials: T
authors.lastname: Imasaka
authors.forename: K
authors.initials: K
authors.lastname: Arimoto
authors.forename: M
authors.initials: M
authors.lastname: Takahashi
authors.forename: Y
authors.initials: Y
authors.lastname: Matsumura
mesh_terms: 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
chemical_list: D000596:Amino Acids; D004791:Enzyme Inhibitors; D000594:Amino Acid Oxidoreductases; D050804:D-Aspartate Oxidase; C494102:DDO protein, human