ABSTRACT: Flavin-dependent carbohydrate oxidases are valuable tools in biotechnological applications due to their highselectivity in the oxidation of carbohydrates. In this study, we report the biochemical and structural characterization of a recentlydiscovered carbohydrate oxidase from the bacterium Ralstonia solanacearum, which is a member of the vanillyl alcohol oxidaseflavoprotein family. Due to its exceptionally high activity toward N-acetyl-D-galactosamine and N-acetyl-D-glucosamine, the enzymewas named N-acetyl-glucosamine oxidase (NagOx). In contrast to most known (fungal) carbohydrate oxidases, NagOx could beoverexpressed in a bacterial host, which facilitated detailed biochemical and enzyme engineering studies. Steady state kinetic analysesrevealed that non-acetylated hexoses were also accepted as substrates albeit with lower efficiency. Upon determination of the crystalstructure, structural insights into NagOx were obtained. A large cavity containing a bicovalently bound FAD, tethered via histidyl andcysteinyl linkages, was observed. Substrate docking highlighted how a single residue (Leu251) plays a key role in the accommodationof N-acetylated sugars in the active site. Upon replacement of Leu251 (L251R mutant), an enzyme variant was generated with adrastically modified substrate acceptance profile, tuned toward non-N-acetylated monosaccharides and disaccharides. Furthermore,the activity toward bulkier substrates such as the trisaccharide maltotriose was introduced by this mutation. Due to its advantage ofbeing overexpressed in a bacterial host, NagOx can be considered a promising alternative engineerable biocatalyst for selectiveoxidation of monosaccharides and oligosaccharides.