Structure determination, heterologous expression and directed evolution of fungal laccases

Laccases are blue multi-copper oxidases that catalyse the oxidation of a wide variety of substrates, including various phenolic compounds. Molecular oxygen is used as an electron acceptor and no separate co-factor is required. Laccases contain four coppers, which are distinguished by their spectroscopic and paramagnetic properties. The binding pocket for the organic substrate is situated near the mononuclear copper site, and the electrons are transferred to the trinuclear copper site, where the molecular oxygen is reduced. Laccases are widely distributed in fungi and plants, and they are involved mainly in lignin degradation and biosynthesis. Potential areas of commercial use include e.g. pulp bleaching, textile dye decolorization, bioglueing, detoxification and biosensors. Cost effective enzyme production and enhancement of the enzyme properties are the key issues in developing enzymatic oxidation technologies. Our aim is to study and improve the performance of fungal laccases by means of protein engineering, including also directed evolution methods. The three-dimensional structure of Melanocarpus albomyces laccase has been determined successfully in an intact four copper atom form at 2.4 resolution [Hakulinen et al. (2002), Nat. Str. Biol. 9, 601-605] The structure revealed an overall similar fold to other known laccase structures. The major differences were observed in the loops forming the substrate-binding site for the aromatic compounds. We have also produced the Melanocarpus enzyme in our fungal production host, Trichoderma reesei, with yields of several hundreds of mg/l, which is the highest expression level reported so far for laccases. For directed evolution purposes, a heterologous expression system has been set-up in Saccharomyces cerevisiae under the control of an inducible galactokinase (GAL1) promoter using the Phlebia radiata laccase gene. So far two different random mutant libraries of the P.radiata laccase gene have been created, where the mutagenesis has been targeted to the loop structures involved in the binding of the aromatic substrate. Some 3000 clones from the mutant banks have been screened using our HTS robotic system. Out of the 3000 clones, 15 interesting clones with improved activity on a non-phenolic ABTS as compared to activity on a phenolic DMP compound have been picked for further characterisation