Structural basis for branching-enzyme activity of glycoside hydrolase family 57: Structure and stability studies of a novel branching enzyme from the hyperthermophilic archaeon Thermococcus Kodakaraensis KOD1
Structural basis for branching-enzyme activity of glycoside hydrolase family 57: Structure and stability studies of a novel branching enzyme from the hyperthermophilic archaeon Thermococcus Kodakaraensis KOD1
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DOI：
10.1002/prot.22902
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发表时间：
2011-02-01
影响因子：
2.9
通讯作者：
Murakami, Mario T.
中科院分区：
文献类型：
--
作者：
Santos, Camila R.;Tonoli, Celisa C. C.;Murakami, Mario T.
分支酶 (BE) 通过裂解 α-1,4 糖苷键并随后转移到新的 α-1,6 位置来催化糖原和支链淀粉中分支点的形成。 BE 通常属于糖苷水解酶家族 13 (GH13)；然而，从超嗜热古菌Thermococcus kodakaraensis KOD1中分离出来的TK1436是第一个具有BE活性的GH57成员。迄今为止，唯一已确定的 BE 结构是来自大肠杆菌的 GH13 型。在此，我们确定了天然状态下以及与葡萄糖和底物模拟物复合的 TK1436 的晶体结构，从而可以绘制底物结合通道图并鉴定葡聚糖转移酶活性的关键残基。其结构包含与 C 端 α 螺旋结构域并列的扭曲 (β/α)(7) 桶，该结构也参与活性位点裂缝的形成。活性位点包含两个酸性催化残基（Glu183 和 Asp354）、偏振器 His 10、芳香族守门者（Trp28、Trp270、Trp407 和 Trp416）以及残基 Tyr233（在 GH13 型和 GH57 型 BE 中完全保守）。尽管与大肠杆菌 BE 相比，TK1436 显示出完全不同的折叠和结构域组织，但它们具有相同的 BE 活性结构决定因素。与 AmyC（一种缺乏 BE 活性的 GH57 α-淀粉酶）进行结构比较表明，AmyC 结构中参与底物识别和结合的催化环被缩短，这已被视为其无法发挥葡聚糖转移酶活性的一个关键特征。寡聚化也被指出是 GH57 成员功能分化的可能决定因素。蛋白质 2011; 79：547-557。 (C) 2010 Wiley-Liss, Inc.
Branching enzymes (BEs) catalyze the formation of branch points in glycogen and amylopectin by cleavage of alpha-1,4 glycosidic bonds and subsequent transfer to a new alpha-1,6 position. BEs generally belong to glycoside hydrolase family 13 (GH13); however TK1436, isolated from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, is the first GH57 member, which possesses BE activity. To date, the only BE structure that had been determined is a GH13-type from Escherichia coli. Herein, we have determined the crystal structure of TK1436 in the native state and in complex with glucose and substrate mimetics that permitted mapping of the substrate-binding channel and identification of key residues for glucanotransferase activity. Its structure encompasses a distorted (beta/alpha)(7)-barrel juxtaposed to a C-terminal alpha-helical domain, which also participates in the formation of the active-site cleft. The active site comprises two acidic catalytic residues (Glu183 and Asp354), the polarizer His 10, aromatic gate-keepers (Trp28, Trp270, Trp407, and Trp416) and the residue Tyr233, which is fully conserved among GH13- and GH57-type BEs. Despite TK1436 displaying a completely different fold and domain organization when compared to E. coli BE, they share the same structural determinants for BE activity. Structural comparison with AmyC, a GH57 alpha-amylase devoid of BE activity, revealed that the catalytic loop involved in substrate recognition and binding, is shortened in AmyC structure and it has been addressed as a key feature for its inability for glucanotransferase activity. The oligomerization has also been pointed out as a possible determinant for functional differentiation among GH57 members. Proteins 2011; 79:547-557. (C) 2010 Wiley-Liss, Inc.