Laboratory of Enzyme Technology, Department of Biotechnology, School of Applied Biology and Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, 11855 Athens, Greece.
Turku Bioscience Centre, University of Turku and Åbo Akademi University, 20520 Turku, Finland.
Int J Mol Sci. 2022 Jul 5;23(13):7469. doi: 10.3390/ijms23137469.
The extensive application of herbicides in crop cultivation has indisputably led to the emergence of weed populations characterized by multiple herbicide resistance (MHR). This phenomenon is associated with the enhanced metabolism and detoxifying ability of endogenous enzymes, such as phi class glutathione transferases (GSTFs). In the present work, a library of mutant GSTFs was created by in vitro directed evolution via DNA shuffling. Selected genes from the weeds and , and the cereal crops and were recombined to forge a library of novel chimeric GSTFs. The library was activity screened and the best-performing enzyme variants were purified and characterized. The work allowed the identification of enzyme variants that exhibit an eight-fold improvement in their catalytic efficiency, higher thermal stability (8.3 °C) and three-times higher inhibition sensitivity towards the herbicide butachlor. The crystal structures of the best-performing enzyme variants were determined by X-ray crystallography. Structural analysis allowed the identification of specific structural elements that are responsible for k regulation, thermal stability and inhibition potency. These improved novel enzymes hold the potential for utilization in biocatalysis and green biotechnology applications. The results of the present work contribute significantly to our knowledge of the structure and function of phi class plant GSTs and shed light on their involvement in the mechanisms of MHR.
除草剂在作物种植中的广泛应用，不可避免地导致了具有多种除草剂抗性（MHR）的杂草种群的出现。这种现象与内源性酶如 phi 类谷胱甘肽转移酶（GSTFs）代谢和解毒能力的增强有关。在本工作中，通过体外定向进化的 DNA 改组创建了突变 GSTFs 文库。从杂草 和 以及谷类作物 和 中选择基因进行重组，形成了新型嵌合 GSTFs 文库。对文库进行了活性筛选，并对表现最佳的酶变体进行了纯化和表征。这项工作鉴定出了酶变体，其催化效率提高了 8 倍，热稳定性（8.3°C）提高了 1.5 倍，对除草剂丁草胺的抑制敏感性提高了 3 倍。通过 X 射线晶体学确定了表现最佳的酶变体的晶体结构。结构分析确定了负责 k 调节、热稳定性和抑制效力的特定结构元素。这些改进后的新型酶在生物催化和绿色生物技术应用中具有潜在的用途。本工作的结果极大地丰富了我们对 phi 类植物 GSTs 的结构和功能的认识，并阐明了它们在 MHR 机制中的作用。
