Molecular cloning of the L-amino-acid oxidase gene from Neurospora crassa.

Abstract

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.