7ZZK

Structure of the N-acetyl-D-glucosamine oxidase from Ralstonia Solanacearum

7ZZK の概要

• エントリーDOI: 10.2210/pdb7zzk/pdb
• 分子名称: N-acetyl-D-hexosamine oxidase, ALANINE, FLAVIN-ADENINE DINUCLEOTIDE, ... (6 entities in total)
• 機能のキーワード: oxidase, carbohydrate modification, flavoprotein
• 由来する生物種: Ralstonia solanacearum
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 116018.67

構造登録者

Boverio, A.,Rozeboom, H.J.,Fraaije, M.W. (登録日: 2022-05-25, 公開日: 2022-08-03, 最終更新日: 2024-10-23)

主引用文献

Boverio, A.,Widodo, W.S.,Santema, L.L.,Rozeboom, H.J.,Xiang, R.,Guallar, V.,Mattevi, A.,Fraaije, M.W.
Structural Elucidation and Engineering of a Bacterial Carbohydrate Oxidase.
Biochemistry, 62:429-436, 2023

PubMed 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 , which is a member of the vanillyl alcohol oxidase flavoprotein family. Due to its exceptionally high activity toward -acetyl-d-galactosamine and -acetyl-d-glucosamine, the enzyme was named -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.

PubMed: 35881507
DOI: 10.1021/acs.biochem.2c00307

実験手法

X-RAY DIFFRACTION (1.5 Å)