Study on Substrates-Recognition Mechanism of Thermoactinomyces vulgaris R-47 α-Amylase 2 (TVAII) based on X-ray Structures.

基于X射线结构的热放线菌R-47 α-淀粉酶2（TVAII）底物识别机制研究。

• 批准号: 12680606
• 负责人: KAMITORI Shigehiro
• 金额: $ 1.66万
• 依托单位: Tokyo University of Agriculture & Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680606/
• 关键词: X-ray structure; α-amvlase; substrate-recognition; cyclodextrin; hydrolysis; glucoside linkage; mutant enzyme; Thermoactinomvces vulgaris

Thermoactinomyces vulgaris R-47 produces two α-amylases TVAI (637 amino acid residues, MW=71,OOODa) and TVAII (585 amino acid residues, MW = 67,500Da), and they can hydrolyze not only starch but also cyclodextrins (CDs), which are scarcely hydrolyzed by other a-amylases. The X-ray structures of inactive mutant TVAIIs (Glu354->Ala or Asp325→Asn) complexed with β-CD (E354A/ β-CD) methyl-β-CD (D325N/methyl-β-CD) and maltohexose (D325N/G6), have been determined, showing that Phe286 is an important residue for TVAII to recognize CDs, and that the binding mode ofG6 as a substrate is very similar to that observed in the other a-amylase and a-amylase family enzymes. Kinetic analyzes of the mutant TVAIIs, F286A, F286L, F286Y and F286W (Phe286→Ala, Leu, Tyr, Trp) have also been done, supporting results from X-ray structures. The X-ray structure of TVAI has been determined at 1.6 Å, showing that there are some differences in the structures between TVAI and TVAII, related to the substrate specificity and thermostability of the enzyme.

Thermoactinomyces vulgaris R-47产生两种α-淀粉酶TVAI（637个氨基酸残基，MW=71,OOODa）和TVAII（585个氨基酸残基，MW=67,500Da），它们不仅可以水解淀粉，还可以水解环糊精（CD） ，几乎不被其他α-淀粉酶水解。失活突变体的X射线结构。已测定与 β-CD (E354A/ β-CD) 甲基-β-CD (D325N/甲基-β-CD) 和麦芽己糖 (D325N/G6) 复合的 TVAII（Glu354->Ala 或 Asp325→Asn），显示Phe286是TVAII识别CD的重要残基，G6作为底物的结合模式与观察到的非常相似在其他α-淀粉酶和α-淀粉酶家族酶中，突变体TVAII、F286A、F286L、F286Y和F286W（Phe286→Ala、Leu、Tyr、Trp）的动力学缝合也已完成，支持X射线结构的结果。 TVAI的X射线结构已确定为1.6 Å，表明TVAI和TVAI之间的结构存在一些差异TVAII，与酶的底物特异性和热稳定性有关。

项目成果

Yokota, T., Tonozuka, T., Shimura, Y., Ichikawa, K., Kamitori, S., Sakano, Y.: "Structures of Thermoaclinomyces vulgaris R-47 α-Amylase II complexed with substrate analogues"Biosci. Biotecimol. Biochem.. 65. 619-626 (2001)

Yokota, T.、Tonozuka, T.、Shimura, Y.、Ichikawa, K.、Kamitori, S.、Sakano, Y.：“与类似底物复合的嗜热酸霉菌 R-47 α-淀粉酶 II 的结构”Biotecimol。生物化学.. 65. 619-626 (2001)

Kondo, S., Kaji, A., Yuguchi, K., Tonozuka, T., Sakano, Y., Kamitori, S.: "Crystallization and Preliminary X-ray Analysis of Thermoactinomyces vulgaris R-47 α-Amylase 1."Protein and Peptide letters. 7. 197-200 (2000)

Kondo, S.、Kaji, A.、Yuguchi, K.、Tonozuka, T.、Sakano, Y.、Kamitori, S.：“普通嗜热放线菌 R-47 α-淀粉酶 1 的结晶和初步 X 射线分析。”蛋白质和肽字母。7. 197-200 (2000)

Kamitori, S., Abe, A., Ohtaki, A., Kaji, A., Tonozuka, T., Sakano, Y.: "Crystal Structures and Structural Comparison of Thermoactinomyces vulgaris R-47 α-Amylase 1 (TVAI) at 1.6 Å Resolution and α-Amylase 2 (TVAII) at 2.3 Å Resolution"J. Mol. Bid.. (印刷中).

Kamitori, S.、Abe, A.、Ohtaki, A.、Kaji, A.、Tonozuka, T.、Sakano, Y.：“普通嗜热放线菌 R-47 α-淀粉酶 1 (TVAI) 的晶体结构和结构比较1.6 Å 分辨率和 α-淀粉酶 2 (TVAII) 2.3 Å 分辨率”J. Mol. Bid..（印刷中）。

Ohtaki, A, Kondo, S., Shimura, Y., Tonozuka, T., Sakano, Y., Kamitori, S.: "Role of Phe286 in the recognition mechanism of cyclomaltooligosaccharides (cyclodextrins) by Thermoactinomyces vulgaris R-47 a-amylase 2 (TVAII). X-ray structures of the mutant TV

Ohtaki, A、Kondo, S.、Shimura, Y.、Tonozuka, T.、Sakano, Y.、Kamitori, S.：“Phe286 在普通嗜热放线菌 R-47 a- 环麦芽低聚糖（环糊精）识别机制中的作用

Kondo, S., Kaji, A., Yuguchi, K., Tonozuka, T., Sakano, Y., Kamitori, S.: "Crystallization and Preliminary X-ray Analysis of Thermoactinomyces vulgaris R-47 α-Amylase 1"Protein and Peptide letters. 7. 197-200 (2000)

Kondo, S.、Kaji, A.、Yuguchi, K.、Tonozuka, T.、Sakano, Y.、Kamitori, S.：“普通嗜热放线菌 R-47 α-淀粉酶 1 蛋白质的结晶和初步 X 射线分析”和肽字母。7. 197-200 (2000)