Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, India; DBT PAN-IIT Center for Bioenergy, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, India.
Int J Biol Macromol. 2020 Apr 1;148:364-377. doi: 10.1016/j.ijbiomac.2020.01.116. Epub 2020 Jan 13.
Chimera (CtGH1-L1-CtGH5-F194A) developed by fusing β-glucosidase (CtGH1) at N-terminal and endoglucanase (CtGH5-F194A) at C-terminal was structurally characterized. Its secondary structure analysis by CD showed 38% α-helix, 9.3% β-sheets and 52.7% random coils corroborating with prediction. In-silico modeled structure of Chimera comprised two modules, CtGH1 and CtGH5-F194A displaying (α/β) fold. Ramachandran plot of Chimera showed 99.9% residues in allowed region. Binding interaction of Chimera with cello-oligosaccharides suggested active forms of CtGH1 and CtGH5-F194A and their involvement in catalysis. MD simulation of cellohexaose bound endoglucanase module of Chimera showed favourable flexibility in loops, LA with H-bond formation with Asn510 and in loop LC relocation of Tyr687 away from active site efficiently releasing the product after catalysis. Higher short range interaction energy of Chimera, -383 kJ/mol than the individual endoglucanase, 254 kJ/mol against cellohexaose suggested higher efficient catalysis by Chimera. β-Glucosidase module of Chimera showed fluctuations in outer loops suggesting conformational changes that might be contributing to improved hydrolysis. SAXS analysis of Chimera displayed monodispersed state. Guinier analysis of Chimera showed globular shape (R= 3.15 ± 0.10 nm). Kratky plot confirmed fully folded and flexible behaviour in solution. Gasbor modeled structure of Chimera displayed an elongated structure with two modules having shape similar to bean-bag contour.
融合了β-葡萄糖苷酶（CtGH1）的 N 端和内切葡聚糖酶（CtGH5-F194A）的 C 端的嵌合体（CtGH1-L1-CtGH5-F194A）被结构表征。CD 法的二级结构分析表明，38%的α-螺旋、9.3%的β-折叠和 52.7%的无规卷曲与预测相符。嵌合体的计算机建模结构由两个模块组成，CtGH1 和 CtGH5-F194A 呈现（α/β）折叠。嵌合体的 Ramachandran 图显示 99.9%的残基处于允许区域。嵌合体与纤维寡糖的结合相互作用表明 CtGH1 和 CtGH5-F194A 的活性形式及其在催化中的参与。与纤维六糖结合的嵌合体内切葡聚糖酶模块的 MD 模拟表明，在环 LA 中与 Asn510 形成氢键，在 LC 环中 Tyr687 远离活性部位的重新定位，有效地在催化后释放产物，具有良好的灵活性。嵌合体的短程相互作用能较高，-383 kJ/mol，比单个内切葡聚糖酶对纤维六糖的-254 kJ/mol更高，表明嵌合体的催化效率更高。嵌合体的β-葡萄糖苷酶模块的外环波动表明构象变化，这可能有助于提高水解效率。嵌合体的 SAXS 分析显示出单分散状态。嵌合体的 Guiniér 分析显示出球形（R=3.15±0.10nm）。Kratky 图证实了在溶液中完全折叠和灵活的行为。Gasbor 建模的嵌合体结构显示出两个模块的拉长结构，形状类似于豆袋轮廓。
