Cloning and expression in Escherichia coli of the D-aspartate oxidase gene from the yeast Cryptococcus humicola and characterization of the recombinant enzyme.
The D-aspartate oxidase (DDO) from the yeast Cryptococcus humicola UJ1 (ChDDO) is highly specific to D-aspartate. The gene encoding ChDDO was cloned and expressed in Escherichia coli. Sequence analysis of the ChDDO gene showed that an open reading frame of 1,110 bp interrupted by two introns encodes a protein of 370 amino acids. The deduced amino acid sequence showed an FAD-binding motif and a peroxisomal targeting signal 1 in the N-terminal region and at the C-terminus, respectively, and also the presence of certain catalytically important amino acid residues corresponding to those catalytically important in D-amino acid oxidase (DAO). The sequence exhibited only a moderate identity to human (27.4%) and bovine (28.0%) DDOs, and a rather higher identity to yeast and fungal DAOs (30.4-33.2%). Similarly, phylogenetic analysis showed that ChDDO is more closely related to yeast and fungal DAOs than to mammalian DDOs. The gene expression was regulated at the transcriptional level and specifically induced by the presence of D-aspartate as the sole nitrogen source. ChDDO was expressed in an active form in E. coli to an approximately 5-fold greater extent than in yeast. The purified recombinant enzyme was identical to the native enzyme in physicochemical and catalytic properties.
Takahashi
Shouji
S
Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188.
Takahashi
Toshiyuki
T
Kera
Yoshio
Y
Matsunaga
Ryuji
R
Shibuya
Hiroo
H
Yamada
Ryo-hei
RH
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000494:pharmacology; D000645:Ammonium Sulfate / Q000737:chemistry; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002847:Chromatography, Agarose; D003001:Cloning, Molecular; D003454:Cryptococcus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D003605:D-Amino-Acid Oxidase / Q000235:genetics; D050804:D-Aspartate Oxidase; D026603:D-Aspartic Acid / Q000494:pharmacology; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D000596:Amino Acids; D004271:DNA, Fungal; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D026603:D-Aspartic Acid; D000594:Amino Acid Oxidoreductases; D050804:D-Aspartate Oxidase; D003605:D-Amino-Acid Oxidase; D000645:Ammonium Sulfate