Characterization of a D-amino acid oxidase with high activity against cephalosporin C from the yeast Trigonopsis variabilis.
We describe an improved method to purify D-amino acid oxidase with activity towards cephalosporin C. The protein has a carbohydrate content of 1.3% and two molecules of non-covalently bound flavin cofactor per protein molecule. HPLC profiles and enzymatic analysis have indicated that the cofactor is FAD, even though fluorescence spectroscopy shows a slightly altered spectral profile in the 400-500 nm range compared to authentic FAD. N-terminal sequencing of the protein revealed a high level of similarity (56% identity in 25 amino acids) between the fungal and mammalian oxidase, and probably represents a "Rossman fold" with a beta-alpha-beta structure for the binding of the adenosyl moiety of the cofactor.
Szwajcer-Dey
E
E
Pure and Applied Biochemistry, University of Lund, Sweden.
Miller
J R
JR
Kovacevic
S
S
Mosbach
K
K
D000595:Amino Acid Sequence; D002511:Cephalosporins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003605:D-Amino-Acid Oxidase / Q000302:isolation & purification / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D003904:Mitosporic Fungi / Q000201:enzymology*; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence
D002511:Cephalosporins; D005182:Flavin-Adenine Dinucleotide; C025163:cephalosporin C; D003605:D-Amino-Acid Oxidase