広範な基質特異性を有するα-グルコシダーゼとそのトレハロース認識に関わるアミノ酸残基
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- 中山 亨
- Department of Nutritional Physiology, Faculty of Nutrition, Kobe Gakuin University
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- 落合 美佐
- Suntory Research Center
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- 中尾 正宏
- Suntory Research Center
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- 後藤 理枝子
- Department of Nutritional Physiology, Faculty of Nutrition, Kobe Gakuin University
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- 上田 隆史
- Department of Nutritional Physiology, Faculty of Nutrition, Kobe Gakuin University
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- 柴野 裕次
- Biomolecular Engineering Research Institute
書誌事項
- タイトル別名
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- SAM1606 α-Glucosidase with a Broad Substrate Specificity and Its Amino Acid Residues Responsible for Trehalose Recognition
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説明
Thermostable α-glucosidase with a broad substrate specificity from Bacillus sp. SAM1606 can hydrolyze various 1-O-α-D-glucopyranosides includingg maltose, isomaltose and sucrose, and is the only known α-glucosidase that can efficiently hydrolyze a, α-trehalose. The SAM1606 α-glucosidase has several short conserved regions (CR) which are detected in the α-amylase family' enzymes. The putative catalytic residues conserved among the α-amylase family enzymes are also identified in the CR of the SAM1606 enzyme; suggesting that the enzyme can be categorized into the family. The enzyme exhibits a very high sequence similarity to two oligo-1, 6-glucosidases (016G) of Bacilli that cannot act on trehalose. To identify the critical residues which determine the broad substrate specificity of the SAM1606 enzyme by site-specific mutagenesis, we selected five residues to be mutagenized in the SAM1606 a-glucosidase by comparison of the CR sequences of these three glucosidases. These 5 residues have been specifically replaced by in vitro mutagenesis with the residues as in Bacillus 016G. The twelve mutant enzymes with one through five substitutions were expressed and kinetically characterized. None of the single and multiple mutations caused a significant reduction in Vmax for all of the substrates tested; all mutant retained Vmax values more than 20% of those of the wild-type enzyme. There was no significant variation in Km detected with maltose, sucrose or isomaltose upon each mutation. For trehalose, however, G1y273Pro as well as all multiple mutations containing the G1y273Pro caused appreciable increases in the Km value for this substrate, indicating that the loss in affinity for trehalose is critically governed by G1y273Pro, whose effect is specifically enhanced by Thr342Asn. From recent X-ray crystallographic studies of the porcine pancreatic α-amylase (PPA) complexed with acarbose, G1y273 which corresponds to I1e235 in PPA appears to be in close contact with the bound inhibitors.
収録刊行物
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- 応用糖質科学
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応用糖質科学 45 (2), 185-197, 1998
日本応用糖質科学会
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詳細情報 詳細情報について
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- CRID
- 1390001205170330368
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- NII論文ID
- 130004091130
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- COI
- 1:CAS:528:DyaK1cXksFOmtrw%3D
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- ISSN
- 18844898
- 13403494
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- データソース種別
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- JaLC
- CiNii Articles
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- 抄録ライセンスフラグ
- 使用不可