SummaryAs global temperatures rise, improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalysing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. We explored the effects of temperature on the activity of the photorespiratory enzyme glycerate kinase (GLYK) from various organisms and the homologue from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand enzyme features underlying the thermotolerance of C. merolae GLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold‐predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, hybrid proteins were produced and analysed. These hybrid enzymes contained loop regions from CmGLYK replacing the most mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 °C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. Whilst this hybrid enzyme exhibited enhanced thermostability and a similar Km for ATP compared to AtGLYK, its Km for glycerate increased threefold. This study demonstrates that molecular dynamics simulation‐guided structure‐based recombination offers a promising strategy for enhancing the thermostability of other plant enzymes with possible application to increasing the thermotolerance of plants under warming climates.

中文翻译：

---

通过基于结构的重组提高关键光呼吸酶甘油酸 3-激酶的热稳定性


摘要随着全球气温上升，提高作物产量需要提高作物的耐热性。提高耐热性的一种方法是使用生物工程来提高催化基本生物过程的酶的热稳定性。光呼吸是植物中必不可少的回收过程，是光合作用和作物生长不可或缺的一部分。光呼吸酶是增强植物耐热性的靶标，因为该途径限制了高温下的碳固定。我们探讨了温度对各种生物体光呼吸酶甘油酸激酶 （GLYK） 活性的影响，嗜热藻类 Cyanidioschyzon merolae 的同系物比嗜温植物（包括拟南芥）的同系物更耐热。为了了解梅罗拉 Glyk （CmGLYK） 耐热性的酶特征，我们使用 AlphaFold 预测结构进行了分子动力学模拟，结果显示与 CmGLYK 相比，嗜温植物 GLYKs 的环区域在较高温度下的运动更大。基于这些模拟，生产和分析了杂交蛋白。这些杂交酶包含来自 CmGLYK 的环区域，取代了 AtGLYK 最具流动性的相应环。其中两种杂交酶具有增强的热稳定性，熔解温度升高了 6 °C。 一个具有三个接枝环的杂交体在高温下保持更高的活性。虽然与 AtGLYK 相比，这种杂交酶表现出增强的热稳定性和相似的 ATP Km，但其甘油酸盐的 Km 增加了三倍。 这项研究表明，分子动力学模拟引导的基于结构的重组为增强其他植物酶的热稳定性提供了一种很有前途的策略，并可能应用于提高植物在变暖气候下的耐热性。