Identification of catalytic residues in glyoxal oxidase by targeted mutagenesis

Mei M. Whittaker, Philip J. Kersten, Daniel Cullen, James W. Whittaker

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97 Scopus citations


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

Original languageEnglish (US)
Pages (from-to)36226-36232
Number of pages7
JournalJournal of Biological Chemistry
Issue number51
StatePublished - Dec 17 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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