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

普通热放线菌R-47产生两种α-淀粉酶。TVAI和TVAI。除了作用于淀粉外，它们对结构为[α-(1,6)- glc -α-(1,4)- glc -α-(1,4)- glc]的普鲁兰和环低聚糖（环糊精）具有独特的水解活性。CDs)，几乎不被其他α-淀粉酶水解。作为一种胞外酶，tai倾向于淀粉和普鲁兰等高分子量底物。能水解γ-CD（8个葡萄糖单位），但不能水解α- cd和β- cd（6和7个葡萄糖单位），且空腔较小。另一方面，作为一种细胞内酶，TVAII倾向于低分子量底物，可以通过环糊精酶活性有效地水解小的麦芽低聚糖，包括α和β- cd。探讨了TVAI和TVAII的酶学特性。我们进行了以下研究：TVAII/阿卡波糖配合物和失活突变体TVAII (D325N-D421N)/α-、β和γ-CDs配合物的x射线结构，分别在2.9Å、2.9Å、2.8Å和3.1Å的分辨率上进行了测定。在所有配合物中，-1 -2和-3亚位上的配体与酶的相互作用几乎相同。但在+1和+2亚位上发现了催化位点结构的显著差异。其中Trp356和Tyr374根据配体的结构和大小改变侧链的构象。Trp356和Tyr374被认为与TVAII的多底物识别机制有关。提供独特的底物特异性。与抑制剂阿卡波糖和与麦芽-己糖和麦芽-三糖的失活突变体TVAI配合物的x射线结构已在2.6测定。2.0和1.8Å分辨率。除了催化位点。在这些x射线结构中发现了分子表面的四个糖结合位点。结构域N的两个糖结合位点含有直链淀粉的规则螺旋结构的低聚糖，这表明结构域N是淀粉结合结构域，在酶的催化反应中起淀粉锚定作用。对原料淀粉的水解活性测定证实了tai能有效地水解原料淀粉。根据x射线结构；推测位于活性中心的Phe313抑制α。-β-环糊精结合活性位点。而位于活性位点+1亚位的Trp398则参与淀粉的识别。用Ala （F313A）取代Phe313后，α -β-环糊精的酶活性增加。Ala（W398A）和Val （W398V）替代Trp398降低了淀粉的Trp398。TVA2突变体AB76的酶活性，该突变体具有tva1型的活性裂缝。改成了TVA1类型的活动。这些结果表明，TVA I和TVA2的底物特异性差异与活性间隙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