Identification of catalytic residues in glyoxal oxidase by targeted mutagenesis

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

Research output: Contribution to journalArticle

84 Citations (Scopus)

Abstract

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
Volume274
Issue number51
DOIs
StatePublished - Dec 17 1999

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Mutagenesis
Galactose Oxidase
Fungi
Copper
Phanerochaete
Aspergillus nidulans
Pichia
Lignin
Aspergillus
Site-Directed Mutagenesis
Recombinant Proteins
Yeast
Fermentation
Free Radicals
Methanol
Culture Media
glyoxal oxidase
Catalytic Domain
Wood
Yeasts

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of catalytic residues in glyoxal oxidase by targeted mutagenesis. / Whittaker, Mei M.; Kersten, Philip J.; Cullen, Daniel; Whittaker, James.

In: Journal of Biological Chemistry, Vol. 274, No. 51, 17.12.1999, p. 36226-36232.

Research output: Contribution to journalArticle

Whittaker, MM, Kersten, PJ, Cullen, D & Whittaker, J 1999, 'Identification of catalytic residues in glyoxal oxidase by targeted mutagenesis', Journal of Biological Chemistry, vol. 274, no. 51, pp. 36226-36232. https://doi.org/10.1074/jbc.274.51.36226
Whittaker, Mei M. ; Kersten, Philip J. ; Cullen, Daniel ; Whittaker, James. / Identification of catalytic residues in glyoxal oxidase by targeted mutagenesis. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 51. pp. 36226-36232.
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