Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.
Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.
Int J Biol Macromol. 2020 Nov 15;163:582-592. doi: 10.1016/j.ijbiomac.2020.07.007. Epub 2020 Jul 5.
The structure and molecular dynamics of α-L-arabinofuranosidase (PsGH43_12) of family 43 glycoside hydrolase, subfamily 12 from Pseudopedobacter saltans were studied. The modeled PsGH43_12 structure displayed 5-bladed β-propeller fold at N-terminal and β-sandwich fold at C terminal. Ramachandran plot showed 95.7% residues in favored and 3.3% in the generously allowed region and only 1% residues in the disallowed region. The secondary structure analysis of PsGH43_12 by circular dichroism revealed 2.7% α-helices, 30.33% β-strands and 66.97% random coils. Protein melting study of PsGH43_12 showed complete unfolding at 65°C and did not require any metal ion for its stability. Molecular docking analysis confirmed the involvement of active site residues Asp71, Asp180 and Glu247 in the catalysis, which was also confirmed by the site-directed mutagenesis of these residues. SAXS analysis displayed that PsGH43_12 is monomeric and a fully folded state in solution form. Guinier analysis gave the radius of gyration (Rg) 2.8 ± 0.09 nm. The maximum dimension and Rg of PsGH43_12 estimated from P(R) plot were 9.7 nm and 2.81 nm, respectively. The ab initio derived dummy model of PsGH43_12 displayed a bell-like shape. The ab initio derived dummy model superposed well with its comparative modeled structure except the N-terminal His-tag region.
研究了嗜盐假单胞菌（Pseudopedobacter saltans）家族 43 糖苷水解酶、亚家族 12 的 α-L-阿拉伯呋喃糖苷酶（PsGH43_12）的结构和分子动力学。建模的 PsGH43_12 结构在 N 端显示 5 叶 β-螺旋桨折叠，在 C 端显示 β-夹心折叠。Ramachandran 图谱显示 95.7%的残基处于有利区域，3.3%的残基处于宽松允许区域，只有 1%的残基处于不允许区域。圆二色性分析表明，PsGH43_12 的二级结构中含有 2.7%的α-螺旋、30.33%的β-折叠和 66.97%的无规卷曲。PsGH43_12 的蛋白质熔融研究表明，其在 65°C 时完全展开，且不需要任何金属离子来稳定其结构。分子对接分析证实了活性位点残基 Asp71、Asp180 和 Glu247 参与了催化作用，这也得到了这些残基的定点突变的证实。小角 X 射线散射（SAXS）分析表明，PsGH43_12 在溶液中呈单体和完全折叠状态。Guinier 分析得出回转半径（Rg）为 2.8±0.09nm。从 P(R) 图谱估计的 PsGH43_12 的最大尺寸和 Rg 分别为 9.7nm 和 2.81nm。从头计算得到的 PsGH43_12 的虚拟模型呈钟形。从头计算得到的虚拟模型与比较建模结构除了 N 端 His 标签区域外，都很好地叠加。
