Kumar Krishan, Singh Shubha, Sharma Kedar, Goyal Arun
Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India.
Division of Biological Sciences and Engineering, Netaji Subhas University of Technology, Delhi, 110078, India.
J Mol Graph Model. 2021 Mar;103:107808. doi: 10.1016/j.jmgm.2020.107808. Epub 2020 Nov 17.
The cellulases of family 9 glycoside hydrolase with subtle difference in amino acid sequence have shown different types of catalytic activities such as endo-, exo- or processive endocellulase. However, the reason behind the different types of catalytic activities still unclear. In this study, the processive endocellulase, HtGH9 of family 9 GH from Hungateiclostridium thermocellum was modeled by homology modeling. The catalytic module (HtGH9t) of HtGH9 modeled structure displayed the (α/α) barrel topology and associated family 3 carbohydrate binding module (HtCBM3c) displayed β-sandwich fold. Ramachandran plot of HtGH9 modeled structure displayed all the amino acid residues in allowed region except Asn225 and Asp317. Secondary structure analysis of modeled HtGH9 showed the presence of 41.3% α-helices and 11.0% β-strands which was validated through circular dichroism analysis that showed the presence of 42.6% α-helices and 14.5% β-strands. Molecular Dynamic (MD) simulation of HtGH9 structure for 50 ns showed Root Mean Square Deviation (RMSD), 0.84 nm and radius of gyration (Rg) 3.1 nm. The Small-angle X-ray scattering of HtGH9 confirmed the monodisperse state. The radius of gyration for globular shape (Rg) was 5.50 ± 0.15 nm and for rod shape (Rc) by Guinier plot was 2.0 nm. The loop formed by amino acid residues, 264-276 towards one end of the catalytic site of HtGH9 forms a barrier, that blocks the non-reducing end of the cellulose chain causing the processive cleavage resulting in the release of cellotetraose. The position of the corresponding loop in cellulases of family 9 GH is responsible for different types of cleavage patterns.
第9家族糖苷水解酶的纤维素酶，其氨基酸序列存在细微差异，已表现出不同类型的催化活性，如内切、外切或连续内切纤维素酶。然而，不同类型催化活性背后的原因仍不清楚。在本研究中，通过同源建模对来自嗜热栖热放线菌的第9家族GH的连续内切纤维素酶HtGH9进行了建模。HtGH9建模结构的催化模块（HtGH9t）呈现出（α/α）桶状拓扑结构，相关的第3家族碳水化合物结合模块（HtCBM3c）呈现出β-三明治折叠结构。HtGH9建模结构的拉氏图显示，除Asn225和Asp317外，所有氨基酸残基均在允许区域内。对建模的HtGH9进行二级结构分析，结果显示α-螺旋占41.3%，β-链占11.0%，通过圆二色性分析得到验证，该分析显示α-螺旋占42.6%，β-链占14.5%。对HtGH9结构进行50 ns的分子动力学（MD）模拟，结果显示均方根偏差（RMSD）为0.84 nm，回转半径（Rg）为3.1 nm。HtGH9的小角X射线散射证实了其单分散状态。通过Guinier图得到的球状形状的回转半径（Rg）为5.50±0.15 nm，棒状形状的回转半径（Rc）为2.0 nm。由HtGH9催化位点一端的氨基酸残基264 - 276形成的环形成了一个屏障，该屏障阻断了纤维素链的非还原端，导致连续切割，从而释放出纤维四糖。第9家族GH纤维素酶中相应环的位置决定了不同类型的切割模式。
