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

4-α-葡萄糖转移酶是一种高度嗜热的嗜热菌，由659个氨基酸残基组成。该酶不仅催化分子间转糖基化(所谓的歧化)生成不同长度葡萄糖单元的直链α-1，4-葡聚糖，还催化分子内转糖基化从直链直链淀粉生成16~数百个葡萄糖单元的环状α-1，4-葡聚糖(环淀粉酶)。本研究得到以下结果：(1)该酶由两个结构域组成：N-末端结构域(残基1-381)和C-末端结构域(残基389-659)，分别含有一个(β/α)_7桶状折叠和一个扭曲的β-夹心折叠。在酶与阿卡波糖(一种酶抑制剂)的复合体结构上，阿卡波糖分子结合在-1到+3亚基上，麦芽糖分子也结合在活性中心裂隙的边缘，结合位置对应于-5和-6亚基。该酶至少含有9个亚基，从-6到+3。(4)该酶的活性部位裂隙呈隧道状，裂隙上有三个盖子。第一个盖子(残留物220-224)从(β/α)^7桶伸出。第二个(残基358-363)和第三个(627-630)盖分别从三螺旋束和C-末端结构域伸出。阿卡波糖结合后，LID2和LID3的构象发生了显著的变化。(5)该酶似乎是通过空间位阻阻止小环状葡聚糖的产生而产生大的环状葡聚糖，这是通过三个盖子突出到活性中心裂隙以及延伸的活性中心裂隙来实现的。

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.