5DGR

Crystal structure of GH9 exo-beta-D-glucosaminidase PBPRA0520, glucosamine complex

5DGR の概要

• エントリーDOI: 10.2210/pdb5dgr/pdb
• 関連するPDBエントリー: 5DGQ
• 分子名称: Putative endoglucanase-related protein, SODIUM ION, 2-amino-2-deoxy-beta-D-glucopyranose, ... (4 entities in total)
• 機能のキーワード: glycoside hydrolase family 9, alpha-alpha-6 barrel, exo-d-beta-glucosaminidase, hydrolase
• 由来する生物種: Photobacterium profundum
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 133677.59

構造登録者

Suzuki, K.,Honda, Y.,Fushinobu, S. (登録日: 2015-08-28, 公開日: 2015-12-09, 最終更新日: 2024-10-16)

主引用文献

Honda, Y.,Arai, S.,Suzuki, K.,Kitaoka, M.,Fushinobu, S.
The crystal structure of an inverting glycoside hydrolase family 9 exo-beta-D-glucosaminidase and the design of glycosynthase.
Biochem.J., 473:463-472, 2016

PubMed Abstract: Exo-β-D-glucosaminidase (EC 3.2.1.165) from Photobacterium profundum (PpGlcNase) is an inverting GH (glycoside hydrolase) belonging to family 9. We have determined the three-dimensional structure of PpGlcNase to describe the first structure-function relationship of an exo-type GH9 glycosidase. PpGlcNase has a narrow and straight active-site pocket, in contrast with the long glycan-binding cleft of a GH9 endoglucanase. This is because PpGlcNase has a long loop, which blocks the position corresponding to subsites -4 to -2 of the endoglucanase. The pocket shape of PpGlcNase explains its substrate preference for a β1,4-linkage at the non-reducing terminus. Asp(139), Asp(143) and Glu(555) in the active site were located near the β-O1 hydroxy group of GlcN (D-glucosamine), with Asp(139) and Asp(143) holding a nucleophilic water molecule for hydrolysis. The D139A, D143A and E555A mutants significantly decreased hydrolytic activity, indicating their essential role. Of these mutants, D139A exclusively exhibited glycosynthase activity using α-GlcN-F (α-D-glucosaminyl fluoride) and GlcN as substrates, to produce (GlcN)2. Using saturation mutagenesis at Asp(139), we obtained D139E as the best glycosynthase. Compared with the wild-type, the hydrolytic activity of D139E was significantly suppressed (<0.1%), and the F(-)-release activity also decreased (<3%). Therefore the glycosynthase activity of D139E was lower than that of glycosynthases created previously from other inverting GHs. Mutation at the nucleophilic water holder is a general strategy for creating an effective glycosynthase from inverting GHs. However, for GH9, where two acidic residues seem to share the catalytic base role, mutation of Asp(139) might inevitably reduce F(-)-release activity.

PubMed: 26621872
DOI: 10.1042/BJ20150966

実験手法

X-RAY DIFFRACTION (1.9 Å)