Computational design of coevolutionary residues for improved stability and activity of nitrile hydratase

Nitrile Hydratase (NHase), an industrially significant enzyme, catalyzes the conversion of nitriles into amides. High activity and thermostability are crucial for its broad applications. Compared with classical evaluation and subsequent combination of single-point mutations, redesigning coevolutionary residues offers a more precise approach by targeting key functional sites and facilitating efficient computational design and iteration. Here, we proposed an optimized strategy for redesigning coevolutionary residues to enhance the robustness of NHase, a heterotetrameric protein. We conducted an extensive analysis of 80 coevolutionary residue pairs in NHase from Pseudonocardia thermophila JCM3095 (PtNHase) and identified 21 hotspot designable residue pairs lacking explicit interactions. Virtual saturating combinatorial mutations were applied to these pairs, yielding 27 positive candidates from 8379 theoretical mutations based on changes in folding free energy. After screening and iterative combinations, the optimal mutant A3 (αG86Y/αK57L/αE183F) was obtained, whose specific activity toward acrylonitrile and half-life at 65 °C were increased from 1656.8 ± 21.2 U/mg and 20.1 min in WT to 2370.1 ± 102.7 U/mg and 62.3 min, respectively. Benefiting from higher activity and thermostability, the whole-cell catalyst of A3 significantly facilitated the bioconversion of acrylonitrile to acrylamide. Molecular dynamics simulations further revealed that the newly formed inter-residue interactions stabilized the active site and enhanced the flexibility of the substrate channel, thereby improving both activity and thermostability. This study not only developed a highly robust NHase, but also established a framework for the design of other industrial enzymes.