Roze, L. V.; Antoniak, A.; Sarkar, D.; Liepman, A. H.; Tejera-Nieves, M.; Vermaas, J. V.; Walker, B. J.

As global temperatures rise, maintaining and improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalyzing 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. Exploring inter-specific variation of the key photorespiratory enzyme glycerate kinase (GLYK) from various photosynthetic organisms, we found that the homolog from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand factors influencing 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, a series of hybrid proteins were produced and analyzed. These hybrid enzymes contained selected loop regions from CmGLYK replacing the most highly 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. While 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 thermostability of other plant enzymes.