Biochemical and Structural Characterization of a Uronic Acid Oxidase from Citrus sinensis

Aldaric acids are attractive diacids that can be prepared by selective oxidation of carbohydrates. For this, effective biocatalysts are in demand. This work reports on the discovery, biochemical and structural characterization of a VAO-type flavin-containing carbohydrate oxidase from Citrus sinensis: URAO_Cs3. URAO_Cs3 could be overexpressed using prokaryotic and eukaryotic expression systems. Extensive biochemical characterization revealed that the enzyme displays a high thermostability and an exquisite selectivity for uronic acids, galacturonic acid and glucuronic acid. The enzyme was further investigated by determining the crystal structure. The selective oxidation of D-galacturonic acid in a complex mixture was demonstrated, showing how URAO_Cs3 was found to be highly effective in selectively producing galactaric acid while leaving other carbohydrates untouched. In addition to the specific discovery of URAO_Cs3, these findings suggest that plant proteomes can be an interesting source for new biocatalysts.