Unknown

Dataset Information

0

Structural Elucidation and Engineering of a Bacterial Carbohydrate Oxidase.


ABSTRACT: 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.

SUBMITTER: Boverio A 

PROVIDER: S-EPMC9850908 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

altmetric image

Publications

Structural Elucidation and Engineering of a Bacterial Carbohydrate Oxidase.

Boverio Alessandro A   Widodo Wahyu S WS   Santema Lars L LL   Rozeboom Henriëtte J HJ   Xiang Ruite R   Guallar Víctor V   Mattevi Andrea A   Fraaije Marco W MW  

Biochemistry 20220726 2


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 <i>Ralstonia solanacearum</i>, which is a member of the vanillyl alcohol oxidase flavoprotein family. Due to its exceptionally high activity toward <i>N</i>-acetyl-d-galactosamine and <i>N</i>-acetyl-d-gluc  ...[more]

Similar Datasets

| S-EPMC9397409 | biostudies-literature
| S-EPMC2794033 | biostudies-literature
| S-EPMC4224326 | biostudies-literature
| S-EPMC5764748 | biostudies-literature
| S-EPMC2688431 | biostudies-literature
| S-EPMC9056321 | biostudies-literature
| PRJEB47515 | ENA
| S-EPMC4276719 | biostudies-literature
| S-EPMC5577056 | biostudies-literature
| S-EPMC4190269 | biostudies-literature