Targeted mutations in a Trametes villosa laccase - Axial perturbations of the T1 copper

Trametes villosa laccase was mutated on a tetrapeptide segment near the typ e 1 site. The mutations F463M and F463L were at the position corresponding to the type 1 copper axial methionine (M517) ligand in Zucchini ascorbate o xidase, The mutations E460S and A461E were near the T1 copper site. The mut ated Trametes laccases were expressed in an Aspergillus oryzae host and cha racterized. The E460S mutation failed to produce a transformant with meanin gful 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 interme diate between that of the wild type laccase and plastocyanin, an altered UV -visible spectrum, and a decreased redox potential (by 0.1 V). In oxidizing phenolic substrate, the mutation led to a more basic optimal pH as well as an increase in h(cat) and K-m. These effects are attributed to a significa nt perturbation of the T1 copper center caused by the coordination of the a xial methionine (M463) ligand.