Structure and expression of cDNA for D-amino acid oxidase active against cephalosporin C from Fusarium solani.
D-Amino acid oxidase (DAO) was extracted and purified from cultured mycelia of Fusarium solani M-0718 (FERM P-2688). The enzyme was able to oxidatively deaminate cephalosporin C to 7-beta-(5-carboxy-5-oxopentanamido)cephalosporanic acid. Ninety-eight amino acid residues of the F. solani DAO were determined by sequence analysis of 9 peptides derived from Acromobacter protease I digests of the protein. Complementary DNAs encoding F. solani DAO were isolated from the F. solani cDNA library by hybridization with synthetic oligonucleotide probes corresponding to the partial amino acid sequences. Analysis of the nucleotide sequences of the clones revealed a 1,186-nucleotide sequence with a 5'-terminal untranslated region of 41 nucleotides, an open reading frame of 1,083 nucleotides that encoded 361 amino acids, and a 3'-terminal untranslated region of 62 nucleotides. The amino acid sequence of F. solani DAO had 25% homology to that of porcine kidney DAO [EC 1.4.3.3] and 37% homology to that of Trigonopsis variabilis DAO. The constructed plasmid overproduced F. solani DAO in Escherichia coli. The recombinant DAO had almost the same molecular activity as the native DAO against cephalosporin C.
Isogai
T
T
Exploratory Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Ibaraki.
Ono
H
H
Ishitani
Y
Y
Kojo
H
H
Ueda
Y
Y
Kohsaka
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002511:Cephalosporins / Q000378:metabolism*; D003605:D-Amino-Acid Oxidase / Q000096:biosynthesis / Q000235:genetics*; D004271:DNA, Fungal / Q000096:biosynthesis* / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005670:Fusarium / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016366:Open Reading Frames; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D002511:Cephalosporins; D004271:DNA, Fungal; D015345:Oligonucleotide Probes; C025163:cephalosporin C; D003605:D-Amino-Acid Oxidase