Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India; Department of Newborn Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.
Int J Biol Macromol. 2022 Nov 30;221:751-762. doi: 10.1016/j.ijbiomac.2022.09.072. Epub 2022 Sep 11.
Structural and conformational insights of a putative β-1,4-xylosidase (PsGH43F) of glycoside hydrolase family 43 from Pseudopedobacter saltans were investigated by computational and Circular Dichroism (CD) analyses. PsGH43F was cloned and expressed in E. coli BL21 (DE3) cells and the purified enzyme gave the size ~50 kDa on SDS-PAGE analysis. Multiple Sequence Alignment of PsGH43F sequence followed by superposition of modeled structure with homologous structures displayed the presence of three conserved catalytic amino acid residues, Asp33, Asp149 and Glu212. The secondary structure analysis by CD showed 2.72 % α-helix and 36.06 % β-strands. The homology modeled structure of PsGH43F displayed a 5-bladed β-propeller fold for catalytic module at N-terminal and a β-sandwich structure for CBM6 at the C-terminal. Ramachandran plot displayed 99.5 % of residues in the allowed regions. MD simulation of PsGH43F revealed the compactness and stability of the structure. Molecular docking studies of PsGH43F with xylo-oligosaccharides revealed its maximum binding affinity for xylobiose. MD simulation of PsGH43F-xylobiose complex confirmed the increased structural and conformational stability in presence of substrate. The Hydrodynamic diameter analysis of PsGH43F by DLS was in the range, 0.25-0.28 μm.
通过计算和圆二色性 (CD) 分析研究了糖苷水解酶家族 43 中的推定 β-1,4-木糖苷酶 (PsGH43F) 的结构和构象。PsGH43F 在大肠杆菌 BL21 (DE3) 细胞中克隆和表达，SDS-PAGE 分析表明纯化的酶大小约为 50 kDa。PsGH43F 序列的多重序列比对，然后将建模结构与同源结构叠加，显示存在三个保守的催化氨基酸残基，Asp33、Asp149 和 Glu212。CD 的二级结构分析显示 2.72%的α-螺旋和 36.06%的β-折叠。PsGH43F 的同源建模结构在 N 端显示出催化模块的 5 叶 β-三叶桨折叠，在 C 端显示出 CBM6 的 β-夹心结构。Ramachandran 图显示 99.5%的残基位于允许区域。PsGH43F 的 MD 模拟揭示了结构的紧凑性和稳定性。PsGH43F 与木寡糖的分子对接研究表明，它与木二糖的结合亲和力最大。PsGH43F-木二糖复合物的 MD 模拟证实了在存在底物时结构和构象稳定性的增加。DLS 对 PsGH43F 的水动力直径分析范围在 0.25-0.28 μm 之间。
