CHARACTERIZATION AND STRUCTURAL-ANALYSIS OF THE LACCASE-I GENE FROM THE NEWLY ISOLATED LIGNINOLYTIC BASIDIOMYCETE PM1 (CECT 2971)

We have isolated and characterized the cDNA and genomic DNA coding for a phenoloxidase, laccase I, previously purified from culture supernat ant of the newly isolated ligninolytic basidiomycete PM1 (CECT 2971). A cDNA library from basidiomycete PM1 was constructed, and laccase-enc oding cDNAs were identified by screening with antiserum raised against the purified enzyme. The lac1 gene coding for the laccase was identif ied in a partial genomic library by using the isolated cDNA as a probe . Nucleotide sequence determination of the full-length cDNA revealed a n open reading frame of 1,551 bp encoding a polypeptide of 517 amino a cid residues with a putative signal peptide of 21 amino acid residues. Ten small introns interrupted the genomic DNA. A single 1.8-kb transc ript mRNA was detected by Northern (RNA) blot analysis, and its 5' end maps to a position 51 bp upstream from the site of initiation of prot ein synthesis. Eukaryotic regulatory sequences, CAAT and TATA, were ob served in the 5' flanking region, which also contains sequences simila r to those of copper-regulated proteins. Comparative analysis of the p redicted amino acid sequence showed that basidiomycete PM1 laccase I h ad great similarity to the laccases from Coriolus versicolor, Coriolus hirsutus, and Phlebia radiata.