Molecular approach to study the structure-function of a Pleurotus eryngii laccase isoform.

Laccases are biotechnologically interesting enzymes belonging to the polyphenol oxidases family. They are widely distributed throughout the phylogenetic scale from bacteria to mammals. In fungi the analysis of three-dimensional crystal structures of laccases indicate that ascomycete laccases are processed at their C-termini, at a conserved cleavage site, resulting in the proteolytic removal of C-terminal residues. We have isolated and cloned in expression vectors the cDNAs encoding two laccase isoforms (Ery3 and Ery4) from the basidiomycete Pleurotus eryngii. The Ery3 enzyme expressed in Saccharomyces cerevisiae, is functional, whereas the recombinant Ery4 protein does not show enzymatic activity. In order to explain this evidence, we investigated the relationship between the structure of the C-terminal extension and laccase enzymatic activity. The tasks of the present study were to determine the biological role of laccase C-terminal, and to validate a "molecular engineering" approach for the production of recombinant laccases with novel biochemical properties. Genetically engineered mutant genes were produced from Ery4 by: i) progressive 3'-terminal deletions, ii) point mutations, iii) Ery3/Ery4 chimeras. The mutant genes were expressed in S. cerevisiae and active recombinant laccase isoforms were produced, exhibiting each a different biological behaviour. The correlations between the structural information deriving from both biochemical and bioinformatic analyses shed light on the role of Cterminal region in determining laccase functions. The obtained data also indicated that our approach could represent an efficient method for laccase genetic engineering. To our knowledge, this study has produced the first evidences obtained by biotechnological approach of the involvement of the C-terminal tail in the inactivation/activation process of a basidiomycete laccase.