Identification of catalytic residues in glyoxal oxidase by targeted mutagenesis

Glyoxal oxidase is a copper metalloenzyme produced by the wood-rot fungus P hanerochaete chrysosporium as an essential component of its extracellular l ignin degradation pathways. Previous spectroscopic studies on glyoxal oxida se have demonstrated that it contains a free radical-coupled copper active site remarkably similar to that found in another fungal metalloenzyme, gala ctose oxidase. Alignment of primary structures has allowed four catalytic r esidues of glyoxal oxidase to be targeted for site-directed mutagenesis in the recombinant protein. Three glyoxal oxidase mutants have been heterologo usly expressed in both a filamentous fungus (Aspergillus nidulans) and in a methylotrophic yeast (Pichia pastoris), the latter expression system produ cing as much as 2 g of protein per liter of culture medium under conditions of high density methanol-induced fermentation. Biochemical and spectroscop ic characterization of the mutant enzymes supports structural correlation i ons between galactose oxidase and glyoxal oxidase, clearly identifying the catalytically important residues in glyoxal oxidase and demonstrating the f unctions of each of these residues.