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 是一种高度耐热的单链特异性 3'-5' 核酸外切酶。核酸外切酶在高温下维持基因组完整性方面发挥重要作用。因此，重要的是检查有助于这些外切核酸酶的热稳定性的因素。在本研究中，我们报告了 TK1646 的 S187G 和 Y50H 突变体的生产、纯化和表征，重点关注导致 TK1646 热稳定性的因素。重组蛋白的表征表明，这些取代不会显着影响单链 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。与野生型的 180 分钟相比，Y50H 和 S187G 突变体在 100°C 下的半衰期分别为 95 和 155 分钟。生物信息学分析表明突变残基的溶剂可及性增加和氢键的破坏。突变体和野生型的 3D 结构的分子建模和叠加表明，活性位点残基之一 Glu145 在两个突变体中都从金属离子转移，这可能是催化活性降低的原因。致密的二级结构、疏水性和氢键可能是影响 TK1646 热稳定性的主要因素。在两个突变体中都从金属离子转移，这可能是催化活性降低的原因。致密的二级结构、疏水性和氢键可能是影响 TK1646 热稳定性的主要因素。在两个突变体中都从金属离子转移，这可能是催化活性降低的原因。致密的二级结构、疏水性和氢键可能是影响 TK1646 热稳定性的主要因素。