Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
Laboratory of Small Molecules & Macro Molecular Crystallography at Department of Bioengineering, Indian Institute of Technology Gandhinagar, Gandhinagar, India.
J Biomol Struct Dyn. 2022 Nov;40(18):8437-8454. doi: 10.1080/07391102.2021.1911858. Epub 2021 Apr 16.
Pectin is a complex form of polysaccharide and is composed of several structural components that require the concerted action of several pectinases for its complete degradation. In this study, and solution structure of a pectin acetyl esterase (Pae12B) of family 12 carbohydrate esterase (CE12) from was determined. The Pae12B modelled structure, showed a new α/β hydrolase fold, similar to the fold found in the crystal structures of its nearest homologues from CE12 family, which differed from α/β hydrolase fold found in glycoside hydrolases. In the active site of Pae12B, two loops (loop1 and loop6) play an important role in the formation of a catalytic triad Ser15-Asp187-His190, where Ser15 acts as a nucleophile. The structural stability of Pae12B and its catalytic site was detected by performing molecular dynamic (MD) simulation which showed stable and compact conformation of the structure. Molecular docking method was employed to analyse the conformations of various suitable ligands docked at the active site of Pae12B. The stability and structural specificity of the catalytic residues with the ligand, 4-nitrophenyl acetate (4-NPA) was confirmed by MD simulation of Pae12B-4NPA docked complex. Moreover, it was found that the nucleophile Ser15, forms hydrophobic interaction with 4-NPA in the active site to complete covalent catalysis. Small angle X-ray scattering analysis of Pae12B at 3 mg/mL displayed elongated, compact and monodispersed nature in solution. The derived dummy model showed that Pae12B exists as a homotrimer at 3 mg/mL which was also confirmed by dynamic light scattering.Communicated by Ramaswamy H. Sarma.
果胶是一种复杂的多糖形式，由几个结构组成，需要几种果胶酶的协同作用才能完全降解。在这项研究中，测定了来自 的果胶乙酰酯酶（Pae12B）的结构和溶液结构。Pae12B 模型结构显示出一种新的 α/β 水解酶折叠，类似于其最近的 CE12 家族同源物的晶体结构中发现的折叠，与糖苷水解酶中发现的 α/β 水解酶折叠不同。在 Pae12B 的活性部位，两个环（loop1 和 loop6）在形成催化三联 Ser15-Asp187-His190 中发挥重要作用，其中 Ser15 起亲核作用。通过进行分子动力学（MD）模拟来检测 Pae12B 的结构稳定性及其催化部位，结果表明结构稳定且紧凑。采用分子对接方法分析各种合适的配体在 Pae12B 活性部位的构象。通过 Pae12B-4NPA 对接复合物的 MD 模拟，证实了催化残基与配体 4-硝基苯乙酸酯（4-NPA）的稳定性和结构特异性。此外，发现活性部位的亲核试剂 Ser15 与 4-NPA 形成疏水相互作用，以完成共价催化。Pae12B 在 3 mg/mL 时的小角度 X 射线散射分析显示在溶液中呈拉长、紧凑和单分散的性质。衍生的虚拟模型表明 Pae12B 在 3 mg/mL 时以同源三聚体存在，这也通过动态光散射得到证实。由 Ramaswamy H. Sarma 传达。
