Primary structure of a Japanese lacquer tree as a prototype enzyme of multicopper oxidases

The cDNA librare of the Japanease lacquwe tree (Rhus vernicifera) was constructed by the reverse transcription of mDNA. A cDNA oncoding laccase was amplified by PCR using primers based on the N-terminal amino acid sequences of the purified laccase and its peptide fragments formedby digestions with chrmotrypsin, and subclones. The laccase cDNA clone cntaines a single, large open reading frame of 1599 nucleotides, incoding a protein of 533 amino acids with a calculated molecular mass of 58 981 Da. The lacquer laccase was found to have 42 to 62% identity with other plant laccases and 20 to 24% identity with microorganism laccses at the deduced amono acid level. Differing from microooganism laccases the lacquer laccase utilizas a met residue in addition to one Cys and two His residues to construct the type 1 Cu site. The a-helix conttent was predicted to be only 4.3%. The location of these secondary structures was assumed to be very similar to those of asorbate oxidase and fungal laccase, the crystal structures of which have been determined.