Analyses of the crystal structure of 4-α-glucanotransferase and its production mechanism of new cycloamylose

4-α-葡聚糖转移酶晶体结构分析及其新型环直链淀粉生产机制

• 批准号: 12460047
• 负责人: MATSUZAWA Hiroshi
• 金额: $ 9.15万
• 依托单位: Aomori University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12460047/
• 关键词: 4-α-Glucanotransferase; Cycloamylose; X-ray crystal analysis; Crystal structure; Glycoside hydrolase family; Acarbose; Substrate-enzyme complex structure; Archaeon; 4-α-グルカン転位酵素; 基質類似阻害剤; グリコシドロラーゼ・ファミリー; 多波長異常分散法; TIMバレル構造; Thermococcus litr

4-α-Glucanotransferase of Thermococcus litoralis, a hyperthermophilic archeon, consists of 659 amino acid residues. The enzyme catalyzes not only intermolecular transglycosylation (so-called disproportionation) to produce linearα-1,4-glucans with various length of glucose units, but also intramolecular transglycosylation to produce cyclicα-1,4-glucans (cycloamyloses) with 16 to several hundred glucose units from linear amylose. Following results were obtained in this research.(1) The enzyme was composed of two domains; an N-terminal domain (residues 1-381), which contained a (β/α)_7 barrel fold, and a C-terminal domain (residues 389-659), which had a twistedβ-sandwich fold.(2) Glu123 and Asp214 were found to be the catalytic nucleophile and acid/base catalyst, respectively, through biochemical and crystal structure analyses. The catalytic residues were located in the cleft of the N-terminal domain, indicating that the N-terminal domain is a catalytic domain of the enzyme.(3) On the structure of a complex between the enzyme and acarbose (an enzyme inhibitor), the acarbose molecule bound to subsites -1 to +3. A maltose molecule was also found to bind at the edge of the active site cleft; the binding site corresponds to subsites -5 and -6. The enzyme was revealed to possess at least nine subsites, -6 to +3.(4) The active site cleft of the enzyme was tunnel-like in shape, as evidenced by the three lids that covered the cleft. The first lid (residues 220-224) protruded from the (β/α)^7 barrel. The second (residues 358-363) and third (627-630) lids protruded from a three-helix bundle and the C-terminal domain, respectively. Upon binding of acarbose, the conformation of lids 2 and 3 changed significantly.(5) It seemed that the enzyme produces large cyclic glucans by preventing the production of small cyclic glucans by steric hindrance, which is achieved by three lids protruding into the active site cleft, as well as an extended active site cleft.

嗜热菌Thermococcus litoralis的4-α-葡萄糖基转移酶（4-α-Glucanotransferase，4-α-glucanotransferase）是一种由659个氨基酸残基组成的酶。该酶不仅催化分子间转糖基化（所谓的反硝化）以产生具有各种长度的葡萄糖单元的线性α-1，4-葡聚糖，而且催化分子内转糖基化以从线性直链淀粉产生具有16至几百个葡萄糖单元的环状α-1，4-葡聚糖（环淀粉）。本研究取得了以下成果。(1)该酶由两个结构域组成：N端结构域（残基1-381）具有（β/α）_7桶形折叠，C端结构域（残基389-659）具有扭曲的β夹心折叠。(2)通过生化和晶体结构分析，发现Glu 123和Asp 214分别是催化亲核试剂和酸/碱催化剂。催化残基位于N端结构域的裂缝中，表明N端结构域是酶的催化结构域。(3)在酶和阿卡波糖（一种酶抑制剂）之间的复合物的结构上，阿卡波糖分子结合到亚位点-1至+3。还发现麦芽糖分子在活性位点裂缝的边缘结合;结合位点对应于亚位点-5和-6。该酶被发现至少有9个亚位点，-6到+3。(4)酶的活性位点裂缝是隧道状的形状，如由覆盖裂缝的三个盖子所证明的。第一个盖子（残基220-224）从（β/α）^[7]桶中伸出。第二个（残基358-363）和第三个（627-630）盖子分别从三螺旋束和C-末端结构域突出。在阿卡波糖结合后，盖子2和3的构象发生显著变化。(5)似乎该酶通过空间位阻阻止小环葡聚糖的产生来产生大环葡聚糖，这是通过三个盖子突出到活性位点裂缝以及扩展的活性位点裂缝来实现的。