Characterization and redesign of galactonolactone dehydrogenase, a flavoprotein producing vitamin C

N.G.H. Leferink

Research output: Thesisinternal PhD, WU


Keywords: aldonolactone oxidoreductases, Arabidopsis thaliana, flavoprotein, galactonolactone dehydrogenase, molecular gatekeeper, oxidase, protein engineering, vanillyl-alcohol oxidase family, vitamin C

Redox enzymes are attractive biocatalysts because of their intrinsic (enantio-)selectivity and catalytic efficiency, which are often difficult to achieve by conventional chemical approaches. The discovery of new redox enzymes together with novel insights into their catalytic mechanism will increase the biocatalytic potential for application of these enzymes. Carbohydrate oxidases are valuable enzymes that can be applied in diagnostics and the food industry. Such enzymes often contain a flavin cofactor as redox-active group. Most carbohydrate oxidase are up to now isolated from fungi, but an extensive genome analysis revealed that also plants are a rich source of these enzymes. Carbohydrate oxidases are, for example, involved in maintenance of the plant cell wall, de protection against pathogens, and the production of vitamin C.
In this research the vitamin C producing enzyme galactonolactone dehydrogenase from the model plant Arabidopsis thaliana was studied. This enzyme is a so-called aldonolactone oxidoreductases that belongs to the vanillyl-alcohol oxidase family of flavoproteins. Most members of this family are oxidases with a covalently bound FAD cofactor. Galactonolactone dehydrogenase differs in some crucial properties from aldonolactone oxidoreductases from animals, yeasts and fungi. The plant enzyme contains a non-covalently bound FAD cofactor, has a different substrate specificity and hardly reacts with molecular oxygen. Several critical amino acid residues involved in cofactor and substrate binding were identified and the enzyme was redesigned into variants with altered substrate and electron acceptor specificities. One of the major results was the identification of a gatekeeper residue in galactonolactone dehydrogenase that prevents molecular oxygen from reacting with the flavin cofactor. Removal of this residue resulted in a catalytically competent galactonolactone oxidase that can efficiently react with oxygen.
The knowledge obtained with this research provides a firm basis for the design of suitable biocatalysts that can be used for the biotechnological production of vitamin C or related carbohydrates, as an alternative for the currently applied chemical methods.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • de Vries, Sacco, Promotor
  • van Berkel, Willem, Co-promotor
Award date8 Apr 2009
Place of Publication[S.l.]
Print ISBNs9789085853527
Publication statusPublished - 2009


  • oxidoreductases
  • arabidopsis thaliana
  • enzymology

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