Restore the Activity and Stability of Nicotinamide Riboside Kinase by α‐Helix Reconstruction and Semi‐Rational Design

Nicotinamide riboside kinase (NRK) is an important enzyme in the nicotinamide riboside (NR) metabolic pathway, converting NR to nicotinamide mononucleotide (NMN), which has promising industrial applications. However, structural collapse in many NRKs reduces their activity and thermal stability, limiting their industrial potential. Herein, we developed an α-helix reconstruction method on a Kag-NRK from Kluyveromyces marxianus for restore its activity and stability together. First, we constructed an α-helix library and performed in silico screening using both dynamic and static evaluations. An α-helix containing 29 residues was then inserted between residues 55 and 56 of Kag-NRK, effectively repairing structural defects and significantly enhancing both enzyme activity and stability. Furthermore, semi-rational design was performed, leading to the identification of two beneficial substitutions, W171V and N214V. The resulting variant NRK^W171V enhanced local flexibility around the active site, which facilitated substrate access and increased catalytic activity by 155%, while NRK^N214V reduced surface polar exposure, increasing thermal stability by 15.7°C and extending half-life at 40°C by 9 h. Molecular dynamics simulations showed that the α-helix enhanced rigidity, and the substitutions stabilized local structures through hydrophobic interactions. This study highlights the importance of structural integrity in enzyme design and provides a new approach for improving enzyme performance.