Study of the cyclodextrin hydrolyzing mechanism of Thermoactinomyces vulgaris R47 α-amylases based on X-ray structures

基于X射线结构研究嗜热放线菌R47α-淀粉酶环糊精水解机制

• 批准号: 14580621
• 负责人: KAMITORI Shigehiro
• 金额: $ 2.24万
• 依托单位: Tokyo University Agriculture Arid Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580621/
• 关键词: X-ray structure; α-amylase; cyclodextrin; hydrolysis; mutant enzyme; Thermoactinomyces vulgaris R47; glucoside linkage; kinetic parameters; 放射光

Thermoactinomyces vulgaris R-47 produces two α-amylases. TVAI and TVAII. In addition to acting on starch, they have unique hydrolyzing activities for pullulan with the structure [α-(1,6)-Glc-α-(1,4)-Glc-α-(1,4)-Glc], and cyclic-oligosaccharides (cyclodextrins. CDs)which are scarcely hydrolyzed by other α-amylases. TVAI as an extracellular enzyme favors high molecular weight substrates like starch and pullulan. and can hydrolyze γ-CD (eight glucose units), but can not hydrolyze α-and β-CDs (six and seven glucose units) with a small cavity. On the other hand, TVAII as an intracellular enzyme favors low molecular weight substrates and can efficiently hydrolyze small malto-oligosaccharides including α-and β-CDs, by what is called cyclodextrinase activity. To investigate this interesting enzyme property of TVAI and TVAII. we carroed out the following studiesX-ray structures of a TVAII/acarbose complex and inactive mutant TVAII (D325N-D421N)/α-, β-and γ-CDs complexes e determined at resoluti … More ons of 2.9Å, 2.9Å, 2.8Å and 3.1Å, respectively. In all complexes, the interactions between ligands and enzymes at subsites -1 -2 and -3 were almost the same. but striking differences in the catalytic site structure were found at subsites +1 and +2. where Trp356 and Tyr374 changed the conformation of the side chain depending on the structure and size of the ligands. Trp356 and Tyr374 are thought to be responsible for the multiple substrate-recognition mechanism of TVAII. providin~ the unique substrate specificity.The X-ray structures of complexes of TVAI with an inhibitor acarbose and an inactive mutant TVAI with malto-hexaose and malto-tridecaose have been determined at 2.6. 2.0 and 1.8Å resolutions. Besides the catalytic site. four sugar binding sites on the molecular surface are found in these X-ray structures. Two sugar binding sites in domain N hold the oligosaccharides with a regular helical structure of amylose, which suggests the domain N is a starch binding domain acting as an anchor to starch in the catalytic reaction of the enzyme. An assay of hydrolyzing activity for the raw starches confirmed that TVAI can efficiently hydrolyze raw starch.According to X-ray structures; it is expected that Phe313 located in the active center inhibited α.-β-cyclodextrin to bind the active site. while Trp398 located at the subsite +1 in the active site play in the recognizing for starch. The replacement of Phe313 by Ala (F313A ) increased the enzymatic activity for α.-β-cyclodextrin. and the replacement of Trp398 by Ala(W398A ) and Val (W398V) decreased that for starch. The enzymatic activity of TVA2 mutant AB76, which has an active cleft of TVA I -type. changed to TVA1 -type activity. These results showed that the difference of substrate specificity between TVA I and TVA2 is related to the shape of active cleft and dimerization. Less

普通高温放线菌R-47产生两种α-淀粉酶。TVAI和TVAII。除了作用于淀粉外，它们还对具有结构[α-（1，6）-Glc-α-（1，4）-Glc-α-（1，4）-Glc]的普鲁兰多糖和环状寡糖（环糊精）具有独特的水解活性。CD），其几乎不被其它α-淀粉酶水解。TVAI作为胞外酶有利于高分子量底物，如淀粉和支链淀粉。能水解γ-CD（8个葡萄糖单元），但不能水解α-CD和β-CD（6个和7个葡萄糖单元）。另一方面，TVA II作为细胞内酶有利于低分子量底物，并且可以通过所谓的环糊精酶活性有效地水解包括α-CD和β-CD的小麦芽寡糖。为了研究TVAI和TVAII的这种有趣的酶性质。我们进行了下列研究：测定了TVA Ⅱ/阿卡波糖复合物和失活突变体TVA Ⅱ（D325 N-D421 N）/α-、β-和γ-CD复合物的X-射线结构。 ...更多信息 分别为2.9 μ m、2.9 μ m、2.8 μ m和3.1 μ m。在所有配合物中，配体与酶在亚位点-1 -2和-3处的相互作用几乎相同。但在亚位点+1和+2处发现催化位点结构的显著差异。其中Trp 356和Tyr 374根据配体的结构和大小改变侧链的构象。Trp 356和Tyr 374被认为负责TVAII的多底物识别机制。TVAI与抑制剂阿卡波糖和失活突变体TVAI与麦芽六糖和麦芽十三糖的复合物的X射线结构已在2.6处确定。2.0 1.8分辨率除了催化位点。在这些X射线结构中发现分子表面上的四个糖结合位点。结构域N中的两个糖结合位点将寡糖与直链淀粉的规则螺旋结构结合，这表明结构域N是淀粉结合结构域，在酶的催化反应中充当淀粉的锚。对生淀粉的水解活性测定证实，TVAI可以有效地水解生淀粉。根据X射线结构，预计位于活性中心的Phe 313抑制α.-β-环糊精结合活性位点。而Trp 398则位于活性位点+1亚位点上，参与对淀粉的识别。用Ala（F313 A）取代Phe 313可提高α.β-环糊精。Trp 398被Ala（W398 A）和瓦尔（W398 V）取代后，淀粉的转化率降低。TVA 2突变体AB 76的酶活性，其具有TVA I型活性裂。改变为TVA 1型活动。这些结果表明，TVA 1和TVA 2的底物特异性差异与活性裂的形状和二聚化有关。少

项目成果

Mizuno, M., Tonozuka, T., Uechi, A., Ohtaki, A., Ichikawa, K., Kamitori, S., Nishikawa, A., Sakano, Y.: "The Crystal Structure of Thermoactinomyces vulgaris R-47 α-Amylase II (TVA II) Complexed with Transglycosylated Product"European Journal of Biochemist

Mizuno, M.、Tonozuka, T.、Uechi, A.、Ohtaki, A.、Ichikawa, K.、Kamitori, S.、Nishikawa, A.、Sakano, Y.：“普通热放线菌 R-47 的晶体结构α-淀粉酶 II (TVA II) 与转糖基化产物复合”《欧洲生物化学家杂志》

Ohtaki, A., Mizuno, M., Tonozuka, T., Sakano, Y., Kamitori, S.: "Complex Structures of Thermoactinomyces vulgaris R-47 α-Amylase 2 with acarbose and cyclodextrins demonstrate the multiple substrate recognition mechanism."J.Biol.Chem.. (2004/05/12受理).

Ohtaki, A.、Mizuno, M.、Tonozuka, T.、Sakano, Y.、Kamitori, S.：“Thermoactinomyces vulgaris R-47 α-淀粉酶 2 与阿卡波糖和环糊精的复杂结构证明了多底物识别机制。” J.Biol.Chem..（2004/05/12 收稿）。

Abe.A., Tonozuka, T., Sakano.Y., Kamitori.S.: "Complex structures of Thermoactinomyces R-47 α-amylase 1 with malto-oligosaccharides demonstrate the role of domain N acting as a starch binding domain."J. Mol. Biol.. 335. 811-822 (2004)

Abe.A.、Tonozuka, T.、Sakano.Y.、Kamitori.S.：“Thermoactinomyces R-47 α-淀粉酶 1 与麦芽寡糖的复合结构证明了结构域 N 作为淀粉结合结构域的作用。”分子生物学杂志 335. 811-822 (2004)

Tonozuka, T., Yokota, T., Ichikawa, K., Mizino, M., Kondo, S., Nishikawa, A., Kamitori, S., Sakano, Y.: "Crystal structures and substrate specificities of two α-amylases hydrolyzing cyclodextrins and pullulan from Thermoactinomyces vulgaris R-47"Biologia

Tonozuka, T.、Yokota, T.、Ichikawa, K.、Mizino, M.、Kondo, S.、Nishikawa, A.、Kamitori, S.、Sakano, Y.：“两种 α- 的晶体结构和底物特异性淀粉酶水解普通嗜热放线菌 R-47 中的环糊精和普鲁兰多糖”Biologia

Mizuno.M., Tonozuka.T., Uechi.A., Ohtaki.A., Ichikawa.K., Kamitori, S., Nishikawa, A., Sakano, Y: "The Crystal Structure of The Thermoactinomyces vulgaris R-47-Amylase II (TX/A II) Complexed with Transglvcosvlated Product"European Journal of Biochemistry.

Mizuno.M.、Tonozuka.T.、Uechi.A.、Ohtaki.A.、Ichikawa.K.、Kamitori, S.、Nishikawa, A.、Sakano, Y：“普通热放线菌 R-47 的晶体结构