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

The cDNA library of the Japanese lacquer tree (Rhus vernicifera) was constructed by the reverse transcription of mRNA. A cDNA encoding laccase was amplified by PCR using primers based on the N-terminal amino acid sequences of the purified laccase and its peptide fragment formed by digestions with chymotrypsin and trypsin, and subcloned. The laccase cDNA clone contained a single, large open reading frame of 1599 nucleotides,encoding a protein of 533 amino acids with a calculated molecular mass of 58981Da. The lacquer laccase was found to have 42 to 62% identity with other plant laccases and 20 to 24% identity with microorganism laccases at the deduved amino acid level. Differing from microotganism laccases the lacquer laccase utilizes a Met residue in addition to one Cys and two His residues to constrict the type 1 Cu site. The secondary structure of the lacquer laccase was predicted to mainly consist of the β-structure(28.7%) and loop and random structures(67,0%) . The α-helix content was predicted to be only 4.3%. The location of these secondary structures was assumed to be very similar to those of ascorbate oxidase and fungal laccase, the crystal structures of which have been determined.