School of Biological Sciences, University of the Punjab, Lahore, Pakistan.
Department of Chemistry, University of Balochistan, Quetta, Pakistan.
Protein Expr Purif. 2021 Mar;179:105799. doi: 10.1016/j.pep.2020.105799. Epub 2020 Nov 26.
TK1646 is a highly thermostable single strand specific 3'-5' exonuclease. Exonucleases play important role in maintaining the genome integrity at elevated temperatures. Therefore, it is important to examine the factors contributing to thermostability of these exonucleases. In this study we report on production, purification and characterization of S187G and Y50H mutants of TK1646, focusing on the factors leading to thermostability of TK1646. Characterization of the recombinant proteins indicated that these substitutions did not drastically affect the catalysis of single stranded DNA. However, both of these substitutions reduced the thermostability of the recombinant proteins. Half-lives of Y50H and S187G mutants were 95 and 155 min, respectively, at 100 °C in comparison to 180 min of the wild type. Bioinformatics analysis indicated an increase in solvent accessibility of the mutated residues and disruption of hydrogens bonds. Molecular modelling and superimposition of the 3D structures of the mutants and the wild type demonstrated that one of the active site residues, Glu145, was shifted away from the metal ion in both the mutants which may be responsible for the decrease in catalytic activity. Compact secondary structure, hydrophobicity and hydrogen bonding might be the major factors contributing to the thermostability of TK1646.
TK1646 是一种高度热稳定的单链特异性 3'-5'外切核酸酶。外切核酸酶在维持高温下基因组完整性方面发挥着重要作用。因此，研究导致这些外切核酸酶热稳定性的因素非常重要。在这项研究中，我们报告了 TK1646 的 S187G 和 Y50H 突变体的生产、纯化和特性，重点研究了导致 TK1646 热稳定性的因素。重组蛋白的特性表明，这些取代并没有极大地影响单链 DNA 的催化作用。然而，这两种取代都降低了重组蛋白的热稳定性。与野生型相比，Y50H 和 S187G 突变体的半衰期分别为 95 和 155 分钟，在 100°C 下。生物信息学分析表明，突变残基的溶剂可及性增加，氢键被破坏。突变体和野生型的三维结构的分子建模和叠加表明，两个突变体中的一个活性位点残基 Glu145 都从金属离子上移位，这可能是催化活性降低的原因。紧凑的二级结构、疏水性和氢键可能是导致 TK1646 热稳定性的主要因素。
