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

Laccases are biotechnological relevant enzymes belonging to the group of polyphenol oxidases. Three-dimensional crystal structures analyses of fungal laccases showed 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 the cDNAs encoding the Ery3 and Ery4 laccases from Pleurotus eryngii. The Ery3 gene was functionally expressed in Saccharomyces cerevisiae, whereas the recombinant Ery4 protein did not show enzymatic activity. In order to explain this evidence, we investigated the relationship between the structure of the carboxyl-terminal extension and the enzymatic laccase activity. The tasks of the present study were to determine the biological role of laccase C-terminal and to validate a “protein engineering” approach for the production of recombinant laccases with novel biochemical properties. We produced several mutant genes derived from the ERY4 by: i) progressive 3’-terminal deletions, ii) point mutations, iii) ERY3/ERY4 chimeras. The obtained genes were expressed in S. cerevisiae and several recombinant active laccase isoforms were produced, each showing different biological behaviors. The correlations between the structural information deriving from the biochemical and bioinformatic analyses shed light on the role of laccase C-terminal region in determining laccase functions. The obtained data also indicated that the employed approach could represent an efficient method for laccase protein engineering. To our knowledge, this study has produced the first evidences of the involvement of the C-terminal tail in the inactivation/activation mechanism of a basidiomycete laccase.