Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, United Kingdom.
Sci Rep. 2017 Feb 1;7:41212. doi: 10.1038/srep41212.
Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. In this study, two parallel strategies were applied to identify mutation candidates within the flexible loops of Escherichia coli transketolase (TK). The first was a "back to consensus mutations" approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterised experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wild-type TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved k and a T increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzymes.
柔性位点是工程化酶稳定性的潜在目标。然而，由于对如何确定最佳突变候选物的理解有限，刚性化柔性位点（RFS）策略的成功率仍然较低。在这项研究中，应用了两种平行策略来确定大肠杆菌转酮醇酶（TK）柔性环中的突变候选物。第一种是“回到共识突变”方法，第二种是基于 Rosetta 中 ΔΔG 计算的计算设计。生成并实验表征了 49 个单变体。其中，三个单变体 I189H、A282P 和 D143K 比野生型 TK 更耐热。将 A282P 与之前构建的变体 H192P 组合，得到了最佳的全能变体，在 60°C 时半衰期提高了 3 倍，在 65°C 时比野生型提高了 5 倍，k 值提高了 1.3 倍，T 提高了 5°C。基于对所有变体稳定性变化的统计分析，Rosetta 程序的定性预测准确性达到了 65.3%。研究中考察的两种策略都有助于指导突变候选物进入柔性环，并且有可能用于其他酶。
