2QPU

Sugar tongs mutant S378P in complex with acarbose

2QPU の概要

• エントリーDOI: 10.2210/pdb2qpu/pdb
• 関連するPDBエントリー: 1AMY 1HT6 1P6W 1RP8 1RP9 1RPK 2QPS
• 関連するBIRD辞書のPRD_ID: PRD_900022
• 分子名称: Alpha-amylase type A isozyme, 4,6-dideoxy-4-{[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)cyclohex-2-en-1-yl]amino}-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-beta-D-glucopyranose, CALCIUM ION, ... (7 entities in total)
• 機能のキーワード: alpha beta 8 barrel, sugar tongs mutant complex, carbohydrate metabolism, germination, glycosidase, hydrolase, metal-binding, secreted
• 由来する生物種: Hordeum vulgare (Barley)
• 細胞内の位置: Secreted, extracellular space: P00693
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 137259.03

構造登録者

Aghajari, N.,Jensen, M.H.,Tranier, S.,Haser, R. (登録日: 2007-07-25, 公開日: 2008-07-01, 最終更新日: 2023-08-30)

主引用文献

Bozonnet, S.,Jensen, M.T.,Nielsen, M.M.,Aghajari, N.,Jensen, M.H.,Kramhoft, B.,Willemoes, M.,Tranier, S.,Haser, R.,Svensson, B.
The 'pair of sugar tongs' site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity
Febs J., 274:5055-5067, 2007

PubMed Abstract: Some starch-degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley alpha-amylase 1, oligosaccharide is thus bound to the 'sugar tongs' site. This site on the non-catalytic domain C in the C-terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to beta-cyclodextrin-Sepharose, a starch-mimic resin used for alpha-amylase affinity purification. The K(d) for beta-cyclodextrin binding to Y380A and Y380M was 1.4 mm compared to 0.20-0.25 mm for the wild-type, S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley alpha-amylase 2 isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at the 'sugar tongs' in the structure. Crystal structures of both wild-type and S378P enzymes, but not the Y380A enzyme, showed binding of the pseudotetrasaccharide acarbose at the 'sugar tongs' site. The 'sugar tongs' site also contributed importantly to the adsorption to starch granules, as Kd = 0.47 mg.mL(-1) for the wild-type enzyme increased to 5.9 mg.mL(-1) for Y380A, which moreover catalyzed the release of soluble oligosaccharides from starch granules with only 10% of the wild-type activity. beta-cyclodextrin both inhibited binding to and suppressed activity on starch granules for wild-type and S378P enzymes, but did not affect these properties of Y380A, reflecting the functional role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced multiple attack, emphasizing that the 'sugar tongs' participates in multivalent binding of polysaccharide substrates.

PubMed: 17803687
DOI: 10.1111/j.1742-4658.2007.06024.x

実験手法

X-RAY DIFFRACTION (1.7 Å)