7DKT

Crystal structure of TxGH116 E441A nucleophile mutant from Thermoanaerobacterium xylanolyticum with alpha-glucosyl fluoride

7DKT の概要

• エントリーDOI: 10.2210/pdb7dkt/pdb
• 関連するPDBエントリー: 5BVU 5BX2 5BX3 5BX4 5BX5
• 分子名称: beta-glucosidase, alpha-D-glucopyranosyl fluoride, GLYCEROL, ... (5 entities in total)
• 機能のキーワード: beta-glucosidase, nucleophile mutant, hydrolase
• 由来する生物種: Thermoanaerobacterium xylanolyticum LX-11
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 93155.59

構造登録者

Pengthaisong, S.,Ketudat Cairns, J.R. (登録日: 2020-11-25, 公開日: 2021-06-23, 最終更新日: 2023-11-29)

主引用文献

Pengthaisong, S.,Hua, Y.,Ketudat Cairns, J.R.
Structural basis for transglycosylation in glycoside hydrolase family GH116 glycosynthases.
Arch.Biochem.Biophys., 706:108924-108924, 2021

PubMed Abstract: Glycosynthases are glycoside hydrolase mutants that can synthesize oligosaccharides or glycosides from an inverted donor without hydrolysis of the products. Although glycosynthases have been characterized from a variety of glycoside hydrolase (GH) families, family GH116 glycosynthases have yet to be reported. We produced the Thermoanaerobacterium xylanolyticum TxGH116 nucleophile mutants E441D, E441G, E441Q and E441S and compared their glycosynthase activities to the previously generated E441A mutant. The TxGH116 E441G and E441S mutants exhibited highest glycosynthase activity to transfer glucose from α-fluoroglucoside (α-GlcF) to cellobiose acceptor, while E441D had low but significant activity as well. The E441G, E441S and E441A variants showed broad specificity for α-glycosyl fluoride donors and p-nitrophenyl glycoside acceptors. The structure of the TxGH116 E441A mutant with α-GlcF provided the donor substrate complex, while soaking of the TxGH116 E441G mutant with α-GlcF resulted in cellooligosaccharides extending from the +1 subsite out of the active site, with glycerol in the -1 subsite. Soaking of E441A or E441G with cellobiose or cellotriose gave similar acceptor substrate complexes with the nonreducing glucosyl residue in the +1 subsite. Combining structures with the ligands from the TxGH116 E441A with α-GlcF crystals with that of E441A or E441G with cellobiose provides a plausible structure of the catalytic ternary complex, which places the nonreducing glucosyl residue O4 2.5 Å from the anomeric carbon of α-GlcF, thereby explaining its apparent preference for production of β-1,4-linked oligosaccharides. This functional and structural characterization provides the background for development of GH116 glycosynthases for synthesis of oligosaccharides and glycosides of interest.

PubMed: 34019851
DOI: 10.1016/j.abb.2021.108924

実験手法

X-RAY DIFFRACTION (1.65 Å)