A Versatile Protein Scaffold Engineered for the Hierarchical Assembly of Robust and Highly Active Enzymes

Abstract Scaffold proteins play immense roles in bringing enzymes together to enhance their properties. However, the direct fusion of scaffold with bulky guest enzymes may disrupt the assembly process or diminish catalytic efficiency. Most self‐assembling protein scaffolds are engineered to form structures beforehand, and then carry guest proteins via different conjugation strategies in vitro. Here, a robust self‐assembling scaffold is presented, engineered from Methanococcus jannaschii using disulfide bonds, which efficiently assembles bulky enzymes into higher‐order helices without additional chemistry or bio‐conjugation in vitro. When fused directly with monomeric Endo‐1,4‐beta‐xylanase A, the catalytic efficiency of the guest enzyme increased by 2.5 times with enhanced thermostability. Additionally, integrating the scaffold with the multimeric metalloenzyme nitrile hydratase overcame the typical stability‐activity trade‐off of such industrial enzyme, yielding three‐fold higher activity and 28‐fold higher thermostability. Structural analyses suggest that the artificially made helical twist structures create new interface interactions and provide a concentration of active sites of guest enzymes. Further fusion of fluorescent protein pairs with the scaffold exhibited a 12‐fold higher FRET efficiency, suggesting its potential for dual‐enzyme cascade applications. Overall, this study showcases a simple yet powerful protein scaffold that organizes guest enzymes into hierarchical structures with enhanced catalytic performance.