Molecular cloning of the L-amino-acid oxidase gene from Neurospora crassa.
The addition of D-phenylalanine to starved cultures of Neurospora crassa leads to de novo synthesis of L-amino-acid oxidase. Poly(A) RNA from D-phenylalanine-treated mycelium was therefore used to generate a cDNA library which was subsequently screened by hybrid-selected translation. A positive L-amino-acid oxidase clone served as a probe to isolate the complete gene from a genomic library of N. crassa. The nucleotide sequence obtained revealed an open reading frame coding for a protein of 695 amino acids. A comparison of the deduced primary structure with the partial amino-terminal sequence of the isolated enzyme showed that the protein is synthesized as a precursor. The proform exceeds the mature enzyme by 129 amino acids. The presence of a cluster of basic amino acid residues preceding Ala129 in the precursor suggests a post-translational modification brought about by limited proteolysis. N. crassa L-amino-acid oxidase shares a highly conserved region with many well-characterized flavoproteins that is known to constitute part of the flavin-adenine dinucleotide-binding site.
Niedermann
D M
DM
Biochemisches Institut, Universität Zürich, Switzerland.
Lerch
K
K
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D015723:Gene Library; D005800:Genes, Fungal; D050830:L-Amino Acid Oxidase; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D004271:DNA, Fungal; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase