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Specificity analysis of a glycosyl hydrolase

糖基水解酶的特异性分析

基本信息

批准号：
10680622
负责人：
NAKAYAMA Toru
金额：
$ 0.83万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680622/
关键词：
Glycosyl hydrolase Glucosidase Substrate specificity Site-derected mutagenesis 糖質加水分解酵素

项目摘要

The Bacillus sp. SAM1606 α-glucosidase catalyzes transglucosylation of sucrose to produce three regioisomers of the glucosylsucroses, with theanderose (6-OィイD1GィエD1-glucosylsucrose) as the most abundant transfer product. To find the active-site amino acid residues which can affect the reactivity and regiospecificity of the glucosyl transfer, 16 mutants with amino acid substitutions near the active site were reacted with 1.75M sucrose at 60℃, pH 6.0, and the course of transglucosylation as well as the product specificity were analyzed. The sites of the amino acid substitutions were selected by comparing the conserved amino acid sequences located near the active site of the SAM1606 enzyme with those of the Bacillus oligo-1,6-glucosidases (O16G), which have very high amino acid sequence similarities near the active site but have a distinct substrate specificity. The results showed that, among the mutated SAM1606 enzymes examined, only the mutants with the substitution of Gly273 with Pro showed an altered reactivity and specificity of transglucosylation; these mutants exhibited a significantly enhanced initial velocity of glucosyl transfer, yielding isomelezitose (6-OィイD1FィエD1-glucosylsucrose) instead of theanderose as the major transfer product. These results indicate that the substitution of Gly273 with Pro critically governs the enhanced reactivity and altered specificity of the transglucosylation. The notion that amino acid residue at this position is the determinant of the glucosyl-transfer specificity was further confirmed by observation that the B. cereus O16G, which has a proline at the corresponding position, produced isomelezitose as the major transfer product during transglucosylation with sucrose.

芽孢杆菌属 sp. SAM1606 α-葡萄糖苷酶催化蔗糖的转葡萄糖基化，产生葡萄糖基蔗糖的三种区域异构体，其中茶山糖（6-OiiD1GィエD1-葡萄糖基蔗糖）是最丰富的转移产物。为了寻找影响糖基转移反应活性和区域特异性的活性位点氨基酸残基，将16个活性位点附近有氨基酸取代的突变体与1.75M蔗糖在60℃、pH 6.0下反应，分析转糖基化过程和产物特异性。通过比较 SAM1606 酶活性位点附近的保守氨基酸序列与芽孢杆菌寡-1,6-葡萄糖苷酶 (O16G) 的保守氨基酸序列来选择氨基酸取代位点，O16G 在活性位点附近具有非常高的氨基酸序列相似性，但具有独特的底物特异性。结果表明，在检测的突变型 SAM1606 酶中，只有 Gly273 被 Pro 取代的突变体表现出改变的反应性和转糖基化特异性；这些突变体表现出显着增强的葡萄糖基转移初始速度，产生异麦三糖（6-OィイD1FィエD1-葡萄糖基蔗糖）而不是山茶糖作为主要转移产物。这些结果表明，用 Pro 取代 Gly273 关键性地控制了转糖基化反应性的增强和特异性的改变。通过观察在相应位置具有脯氨酸的蜡样芽胞杆菌O16G在蔗糖转葡萄糖过程中产生作为主要转移产物的异松三糖，进一步证实了该位置的氨基酸残基是葡萄糖基转移特异性的决定因素的观点。