9LPY

X-ray structure of GH1 beta-glucosidase Td2F2 2F-Glc complex at cryogenic temperature

9LPY の概要

• エントリーDOI: 10.2210/pdb9lpy/pdb
• 関連するPDBエントリー: 9LPH 9LPI 9LPV 9LPX
• 分子名称: BETA-GLUCOSIDASE, 2-deoxy-2-fluoro-alpha-D-glucopyranose, SODIUM ION, ... (6 entities in total)
• 機能のキーワード: tim barrel, hydrolase, cryogenic-temperature
• 由来する生物種: metagenome
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 51535.56

構造登録者

Yano, N.,Arakawa, H.,Lin, C.C.,Ishiwata, A.,Tanaka, K.,Kusaka, K.,Fushinobu, S. (登録日: 2025-01-26, 公開日: 2025-10-29, 最終更新日: 2025-11-19)

主引用文献

Yano, N.,Kashima, T.,Arakawa, H.,Lin, C.C.,Ishiwata, A.,Namiki, H.,Takaya, N.,Tanaka, K.,Kusaka, K.,Fushinobu, S.
Neutron crystallography of the covalent intermediate of beta-glucosidase reveals remodeling of the catalytic center.
Proc.Natl.Acad.Sci.USA, 122:e2502828122-e2502828122, 2025

PubMed Abstract: Anomer-retaining glycoside hydrolases (GHs) generally catalyze a double displacement reaction via a covalent intermediate. However, neutron crystallography of glycoside ligand-bound states has not been performed. In this study, we investigated β-glucosidase Td2F2 from GH family 1 as a model enzyme for anomer-retaining GHs. We determined joint X-ray/neutron structures of Td2F2 in ligand-free form, covalent intermediate with a 2-deoxy-2-fluoro glucoside (2F-Glc) inhibitor, and glucose product complex using hydrogen/deuterium-exchanged crystals at room temperature, with neutron diffraction resolutions of 1.80-1.70 Å. Extensive hydrogen bonds recognizing the hydroxy groups of 2F-Glc were identified, along with the positions of deuterium atoms. The acid/base catalyst residue Glu166 was anchored by a hydrogen bond network pivoted by Asn293. Tyr295 forms a hydrogen bond with the catalytic nucleophile residue Glu352 in the ligand-free and glucose complex forms, while the active center undergoes significant reorganization, including side chain displacements of Glu352 and Tyr295, as well as the incorporation of a water molecule. An alternative conformation of Tyr295 was observed in the 2F-Glc structure at room temperature, suggesting its role in positioning the nucleophilic water during the deglycosylation step. Steady-state and pre-steady-state kinetic analyses of Y295F mutant supported the functional involvement of Tyr295 in both glycosylation and deglycosylation steps. The tyrosine hydrogen bonded to the nucleophile is also conserved in many other anomer-retaining GH families, underscoring its importance in catalysis. Based on the deuterium/hydrogen positions determined from neutron structures, we proposed a detailed reaction mechanism for Td2F2.

PubMed: 41166426
DOI: 10.1073/pnas.2502828122

実験手法

X-RAY DIFFRACTION (1.11 Å)