Switching from blue to yellow : altering the spectral properties of a high redox potential laccase by directed evolution

• During directed evolution to functionally express the high redox potential laccase from the PM1 basidiomycete in Saccharomyces cerevisiae, the characteristic max. absorption at the T1 copper site (Abs610T1Cu) was quenched, switching the typical blue color of the enzyme to yellow. To det. the mol. basis of this color change, we characterized the original wild-​type laccase and its evolved mutant. Peptide printing and MALDI-​TOF anal. confirmed the absence of contaminating protein traces that could mask the Abs610T1Cu, while conservation of the redox potential at the T1 site was demonstrated by spectroelectrochem. redox titrns. Both wild-​type and evolved laccases were capable of oxidizing a broad range of substrates (ABTS, guaiacol, DMP, synapic acid) and they displayed similar catalytic efficiencies. The laccase mutant could only oxidize high redox potential dyes (Poly R-​478, Reactive Black 5, Azure B) in the presence of exogenous mediators, indicating that the yellow enzyme behaves like a blue laccase. The main consequence of over-​expressing the mutant laccase was the generation of a six-​residue N-​terminal acidic extension, which was assocd. with the failure of the STE13 protease in the Golgi compartment giving rise to alternative processing. Removal of the N-​terminal tail had a neg. effect on laccase stability, secretion and its kinetics, although the truncated mutant remained yellow. The results of CD spectra anal. suggested that polyproline helixes were formed during the directed evolution altering spectral properties. Moreover, introducing the A461T and S426N mutations in the T1 environment during the first cycles of lab. evolution appeared to mediate the alterations to Abs610T1Cu by affecting its coordinating sphere. This laccase mutant is a valuable departure point for further protein engineering towards different fates.

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