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

普通嗜热放线菌R-47产生两种α-淀粉酶。TVAI和TVAII。除了作用于淀粉外，它们还对结构为[α-(1，6)-Glc-α-(1，4)-Glc-α-(1，4)-Glc]的普鲁兰多糖和环寡糖(环糊精)具有独特的水解活性。Cd)，几乎不被其他α-淀粉酶所降解。TVAI作为一种胞外酶，有利于淀粉、普鲁兰等大分子底物的合成。能降解γ-CD(8个葡萄糖单位)，但不能水解α-和β-Cd(6个和7个葡萄糖单位)，空腔较小。另一方面，TVAII作为一种胞内酶，有利于低分子底物，可以通过环糊精酶活性有效地降解包括α-和β-CDS在内的小麦芽寡糖。研究TVAI和TVAII这一有趣的酶的性质。我们对TVAII/阿卡波糖络合物和失活突变体TVAII(D325N-D421N)/α-，β-和γ-CDS络合物的X-射线结构进行了研究。…测定分别为2.9Å、2.9Å、2.8Å和3.1Å。在所有的配合物中，配体与-1-2和-3亚基上的酶的相互作用几乎是相同的。但Trp356和Tyr374在+1和+2亚位的催化中心结构有显著差异，它们的侧链构象随配体的结构和大小而变化。Trp356和Tyr374被认为与TVAII的多底物识别机制有关。TVAI与抑制剂阿卡波糖以及失活突变体TVAI与麦芽六糖和麦芽十三糖的络合物的X射线结构已测定为2.6。2.0%和1.8%Å分辨率。除了催化点。在这些X射线结构中发现了分子表面的四个糖结合部位。结构域N中的两个糖结合部位含有规则的直链淀粉螺旋结构的低聚糖，这表明结构域N是一个淀粉结合域，在酶的催化反应中充当淀粉的锚。通过对粗淀粉的水解性测定，证实了TVAI能有效地水解生淀粉。根据X-射线结构，推测位于活性中心的Phe313抑制了α.-β-环糊精与活性中心的结合。而位于活性位点+1亚位的Trp398则参与了对淀粉的识别。用丙氨酸(F313A)取代Phe313提高了α.-β-环糊精的酶活。Ala(W398A)和Val(W398V)取代Trp398降低了淀粉的产量。TVA2突变体AB76的酶活性测定，该突变体具有TVA I型活性裂隙。已更改为TVA1类型的活动。这些结果表明，TVA1和TVA2底物专一性的差异与活性裂解和二聚化的形状有关。较少

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