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

4-α-葡聚糖转移酶是一种超嗜热细菌，由659个氨基酸残基组成。该酶不仅催化分子间转糖基化（歧化）产生具有不同长度葡萄糖单位的直链α-1,4-葡聚糖，而且催化分子内转糖基化产生具有16至数百个葡萄糖单位的环状α-1,4-葡聚糖（环直链淀粉）。本研究得到以下结果：(1)该酶由两个结构域组成；一个n端结构域（残基1-381）包含一个（β/α）_7桶状褶皱，一个c端结构域（残基389-659）包含一个扭曲的β-三明治状褶皱。(2)通过生化和晶体结构分析，发现Glu123和Asp214分别是催化亲核试剂和酸碱催化剂。催化残基位于n端结构域的间隙中，说明n端结构域是酶的催化结构域。(3)在酶与阿卡波糖（一种酶抑制剂）复合物的结构上，阿卡波糖分子结合在-1至+3亚位上。一个麦芽糖分子也被发现结合在活性位点的边缘；结合位点对应于子位点-5和-6。该酶具有至少9个亚位，从-6到+3。(4)酶活性位点的裂口呈隧道状，裂口上有三个盖子。第一个盖子（残留物220-224）从（β/α）^7桶中突出。第二个（残基358-363）和第三个（残基627-630）分别从三螺旋束和c端结构域突出。与阿卡波糖结合后，盖子2和3的构象发生了显著变化。(5)该酶产生大环葡聚糖似乎是通过空间位阻阻止小环葡聚糖的产生，这是通过三个盖子突出到活性位点间隙，以及一个延伸的活性位点间隙来实现的。

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.