Targeted Mutations in a Trametes villosa Laccase

Abstract

Trametes villosa laccase was mutated on a tetrapep- tide segment near the type 1 site. The mutations F463M and F463L were at the position corresponding to the type 1 copper axial methionine (M517) ligand in Zuc- chini ascorbate oxidase. The mutations E460S and A461E were near the T1 copper site. The mutated Tram- etes laccases were expressed in an Aspergillus oryzae host and characterized. The E460S mutation failed to produce a transformant with meaningful expression. The F463L and A461E mutations did not significantly alter the molecular and enzymological properties of the laccase. In contrast, the F463M mutation resulted in a type 1 copper site with an EPR signal intermediate be- tween that of the wild type laccase and plastocyanin, an altered UV-visible spectrum, and a decreased redox po- tential (by 0.1 V). In oxidizing phenolic substrate, the mutation led to a more basic optimal pH as well as an increase in kcat and Km. These effects are attributed to a significant perturbation of the T1 copper center caused by the coordination of the axial methionine (M463) ligand.