Abstract
Trametes villosa laccase was mutated on a tetrapeptide 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 Zucchini ascorbate oxidase. The mutations E460S and A461E were near the T1 copper site. The mutated Trametes 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 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 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.
Highlights
Trametes villosa laccase was mutated on a tetrapeptide 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 Zucchini ascorbate oxidase
Crystal structure analyses, and spectroscopy indicate that all multicopper oxidases contain at least one type 1 (T1)1 copper center, one type 2 (T2) copper center, and one type 3 (T3) copper center [3,4,5,6,7]
Summary
(Received for publication, January 12, 1999, and in revised form, February 24, 1999). The mutations F463M and F463L were at the position corresponding to the type 1 copper axial methionine (M517) ligand in Zucchini ascorbate oxidase. The typical T1 site, such as that found in the blue copper protein plastocyanin (Pc) and the multi-copper protein Zucchini ascorbate oxidase (zAO), contains two histidines (His), a cysteine (Cys) that forms a short S-Cu bond, and a methionine (Met) that forms a long S-Cu bond. These four ligands bind T1 copper in a distorted tetrahedral coordination geometry (Fig. 1, A and B). Underlined letters represent the mutated residues of this study. *, ligand to T3 copper; ‡, ligand to T1 copper
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