Purification and characterization of a secreted laccase of Gaeumannomyces graminis var. tritici

We purified a secreted fungal laccase from filtrates of Gaeumannomyces gram inis var. tritici cultures induced with copper and xylidine, The active pro tein had an apparent molecular mass of 190 kDa and yielded subunits with mo lecular masses of 60 kDa when denatured and deglycosylated, This laccase ha d a pI of 5.6 and an optimal pH of 4.5 with 2,6-dimethoxyphenol as its subs trate. Like other, previously purified laccases, this one contained several copper atoms in each subunit, as determined by inductively coupled plasma spectroscopy. The active enzyme catalyzed the oxidation of 2,6-dimethoxyphe nol (K-m = 2.6 x 10(-5) +/- 7 x 10(-6) M), catechol (K-m = 2.5 x 10(-4) +/- 1 x 10(-5) M), pyrogallol (K-m = 3.1 x 10(-4) +/- 4 x 10(-5) M), and guaia col (K-m = 5.1 x 10-4 a 2 x 10-5 M), In addition, the laccase catalyzed the polymerization of 1,8-dihydroxynaphthalene, a natural fungal melanin precu rsor, into a high-molecular-weight melanin and catalyzed the oxidation, or decolorization, of the dye poly B-411, a lignin-like polymer. These finding s indicate that this laccase may be involved in melanin polymerization in t his phytopathogen's hyphae and/or in lignin depolymerization in its infecte d plant host.