2D20

Crystal structure of michaelis complex of catalytic-site mutant xylanase from Streptomyces olivaceoviridis E-86

2D20 の概要

• エントリーDOI: 10.2210/pdb2d20/pdb
• 関連するPDBエントリー: 1ISW 1ISX 1XYF 2D1Z 2D22 2D23 2D24
• 分子名称: ENDO-1,4-BETA-D-XYLANASE, alpha-D-xylopyranose-(1-4)-alpha-D-xylopyranose, P-NITROPHENOL, ... (5 entities in total)
• 機能のキーワード: tim-barrel, retaining enzyme, catalytic-site mutant, chemical rescue, michaelis complex, hydrolase
• 由来する生物種: Streptomyces olivaceoviridis
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 95033.83

構造登録者

Suzuki, R.,Kuno, A.,Fujimoto, Z.,Ito, S.,Kawahara, S.I.,Kaneko, S.,Hasegawa, T.,Taira, K. (登録日: 2005-09-02, 公開日: 2006-10-10, 最終更新日: 2024-10-30)

主引用文献

Suzuki, R.,Fujimoto, Z.,Ito, S.,Kawahara, S.,Kaneko, S.,Taira, K.,Hasegawa, T.,Kuno, A.
Crystallographic snapshots of an entire reaction cycle for a retaining xylanase from Streptomyces olivaceoviridis E-86
J.Biochem., 146:61-70, 2009

PubMed Abstract: Retaining glycosyl hydrolases, which catalyse both glycosylation and deglycosylation in a concerted manner, are the most abundant hydrolases. To date, their visualization has tended to be focused on glycosylation because glycosylation reactions can be visualized by inactivating deglycosylation step and/or using substrate analogues to isolate covalent intermediates. Furthermore, during structural analyses of glycosyl hydrolases with hydrolytic reaction products by the conventional soaking method, mutarotation of an anomeric carbon in the reaction products promptly and certainly occurs. This undesirable structural alteration hinders visualization of the second step in the reaction. Here, we investigated X-ray crystallographic visualization as a possible method for visualizing the conformational itinerary of a retaining xylanase from Streptomyces olivaceoviridis E-86. To clearly define the stereochemistry at the anomeric carbon during the deglycosylation step, extraneous nucleophiles, such as azide, were adopted to substitute for the missing base catalyst in an appropriate mutant. The X-ray crystallographic visualization provided snapshots of the components of the entire reaction, including the E*S complex, the covalent intermediate, breakdown of the intermediate and the enzyme-product (E*P)complex.

PubMed: 19279191
DOI: 10.1093/jb/mvp047

実験手法

X-RAY DIFFRACTION (1.85 Å)