Structure, activity, and stability of metagenome‐derived glycoside hydrolase family 9 endoglucanase with an N‐terminal Ig‐like domain
Structure, activity, and stability of metagenome‐derived glycoside hydrolase family 9 endoglucanase with an N‐terminal Ig‐like domain
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DOI：
10.1002/pro.2632
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发表时间：
2015-03
期刊：
影响因子：
8
通讯作者：
H. Okano;E. Kanaya;M. Ozaki;C. Angkawidjaja;S. Kanaya
中科院分区：
文献类型：
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作者：
H. Okano;E. Kanaya;M. Ozaki;C. Angkawidjaja;S. Kanaya
对具有 N 末端免疫球蛋白样（Ig 样）结构域的宏基因组衍生糖苷水解酶家族 9 叶枝堆肥 (LC)-CelG 进行了表征，并确定了其晶体结构。 LC-CelG 不水解对硝基苯基纤维二糖苷，但水解 CM-纤维素，表明它是内切葡聚糖酶。 LC-CelG 在 70°C 时表现出最高活性，在 5-9 的广泛 pH 范围内表现出 >80% 的最大活性。其变性温度为81.4℃，表明LC-CelG是一种热稳定性酶。 LC-CelG 的结构类似于来自热纤梭菌 (CtCelD) 的 CelD、来自酸热脂环酸芽孢杆菌 (AaCel9A) 的 Cel9A 和来自热纤梭菌 (CtCbhA) 的纤维二糖水解酶 CbhA，它们与 LC-CelG 的氨基酸序列同一性相对较低 (29-31%)。 LC-CelG 中三个酸性活性位点残基被保守为 Asp194、Asp197 和 Glu558。形成 AaCel9A 底物结合袋的 13 个残基中有 10 个在 LC-CelG 中是保守的。去除 Ig 样结构域使 LC-CelG 的活性和稳定性分别降低 100 倍和 6.3°C。去除 Ig 样结构域和催化结构域之间的 Gln40 和 Asp99 介导的相互作用使 LC-CelG 的稳定性降低 5.0°C，但不会显着影响其活性。这些结果表明，Ig 样结构域有助于 LC-CelG 的稳定，主要是由于 Gln40 和 Asp99 介导的相互作用。由于缺乏Ig样结构域的LC-CelG衍生物在大肠杆菌细胞中主要以不溶性形式积累，而这种以可溶性形式积累的衍生物表现出非常弱的活性，因此可能需要Ig样结构域来使活性位点的构象发挥作用并防止催化结构域的聚集。
A metagenome‐derived glycoside hydrolase family 9 enzyme with an N‐terminal immunoglobulin‐like (Ig‐like) domain, leaf‐branch compost (LC)‐CelG, was characterized and its crystal structure was determined. LC‐CelG did not hydrolyze p‐nitrophenyl cellobioside but hydrolyzed CM‐cellulose, indicating that it is endoglucanase. LC‐CelG exhibited the highest activity at 70°C and >80% of the maximal activity at a broad pH range of 5–9. Its denaturation temperature was 81.4°C, indicating that LC‐CelG is a thermostable enzyme. The structure of LC‐CelG resembles those of CelD from Clostridium thermocellum (CtCelD), Cel9A from Alicyclobacillus acidocaldarius (AaCel9A), and cellobiohydrolase CbhA from C. thermocellum (CtCbhA), which show relatively low (29–31%) amino acid sequence identities to LC‐CelG. Three acidic active site residues are conserved as Asp194, Asp197, and Glu558 in LC‐CelG. Ten of the thirteen residues that form the substrate binding pocket of AaCel9A are conserved in LC‐CelG. Removal of the Ig‐like domain reduced the activity and stability of LC‐CelG by 100‐fold and 6.3°C, respectively. Removal of the Gln40‐ and Asp99‐mediated interactions between the Ig‐like and catalytic domains destabilized LC‐CelG by 5.0°C without significantly affecting its activity. These results suggest that the Ig‐like domain contributes to the stabilization of LC‐CelG mainly due to the Gln40‐ and Asp99‐mediated interactions. Because the LC‐CelG derivative lacking the Ig‐like domain accumulated in Escherichia coli cells mostly in an insoluble form and this derivative accumulated in a soluble form exhibited very weak activity, the Ig‐like domain may be required to make the conformation of the active site functional and prevent aggregation of the catalytic domain.