Structural Elucidation and Engineering of a Bacterial Carbohydrate Oxidase

• Published in: Biochemistry (Easton) 2023-01, Vol.62 (2), p.429-436
• Main Authors: Boverio, Alessandro, Widodo, Wahyu S., Santema, Lars L., Rozeboom, Henriëtte J., Xiang, Ruite, Guallar, Víctor, Mattevi, Andrea, Fraaije, Marco W.
• Format: Article
• Language: English
• Subjects: Catalytic Domain; Disaccharides - chemistry; Flavin-Adenine Dinucleotide - metabolism; Monosaccharides; Oxidation-Reduction; Oxidoreductases - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.2c00307

Flavin-dependent carbohydrate oxidases are valuable tools in biotechnological applications due to their high selectivity in the oxidation of carbohydrates. In this study, we report the biochemical and structural characterization of a recently discovered carbohydrate oxidase from the bacterium Ralstonia solanacearum, which is a member of the vanillyl alcohol oxidase flavoprotein family. Due to its exceptionally high activity toward N-acetyl-d-galactosamine and N-acetyl-d-glucosamine, the enzyme was named N-acetyl-glucosamine oxidase (NagOx). In contrast to most known (fungal) carbohydrate oxidases, NagOx could be overexpressed in a bacterial host, which facilitated detailed biochemical and enzyme engineering studies. Steady state kinetic analyses revealed that non-acetylated hexoses were also accepted as substrates albeit with lower efficiency. Upon determination of the crystal structure, structural insights into NagOx were obtained. A large cavity containing a bicovalently bound FAD, tethered via histidyl and cysteinyl linkages, was observed. Substrate docking highlighted how a single residue (Leu251) plays a key role in the accommodation of N-acetylated sugars in the active site. Upon replacement of Leu251 (L251R mutant), an enzyme variant was generated with a drastically 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 of being overexpressed in a bacterial host, NagOx can be considered a promising alternative engineerable biocatalyst for selective oxidation of monosaccharides and oligosaccharides.