Galactose oxidase of Dactylium dendroides. Gene cloning and sequence analysis

Abstract

The gaoA gene, encoding the secreted copper-containing enzyme galactose oxidase, has been isolated from the Deuteromycete fungus Dactylium dendroides. Degenerate oligonucleotide primers were designed from amino acid sequence data for use in the polymerase chain reaction. A 1.4-kilobase DNA fragment amplified from genomic DNA was used to screen a genomic library constructed in ZAP. A strongly hybridizing clone was rescued as a pBluescript derivative, pGAO9, by in vivo excision. The sequence of 3466 nucleotides of pGAO9 insert DNA was determined by progressively designing sequencing primers. The translation product of the single long open reading frame matches the available galactose oxidase peptide sequence data, which represents 42% of the residues in the protein. The mature enzyme has 639 residues, which have been assigned to a 1.7-A electron density map (Ito, N., Phillips, S. E. V., Stevens, C., Ogel, Z. B., McPherson, M. J., Keen, J. N., Yadav, K. D. S., and Knowles, P. F. (1991) Nature 350, 87-90). The gene lacks introns and encodes an mRNA of approximately 2.5 kilobases with three transcription initiation start points at least 324 nucleotides upstream of the translation start site. Multiple ATG codons are present between the transcription initiation region and the start of the mature protein; two in-frame ATGs could encode the initiating Met residue to give proteins with 89 or 41 residue N-terminal leader peptides. The shorter potential leader has N-terminal features characteristic of a secretion signal sequence and may also contain a pro-sequence processed by an enzyme specific for a monobasic (arginine) cleavage site, as proposed for other fungal genes. The codon bias of gaoA is characteristic of other filamentous fungal genes. No significant homologies exist between galactose oxidase and other protein sequences available in data bases.